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For other uses, see ELISA (disambiguation). ELISA A 96-well microtiter plate being used for ELISA MeSH D004797 The enzyme-linked immunosorbent assay (ELISA) (/ɪˈlaɪzə/, /ˌiːˈlaɪzə/) is a test that uses antibodies and color change to identify a substance. ELISA is a popular format of "wet-lab" type analytic biochemistry assay that uses a solid-phase enzyme immunoassay (EIA) to detect the presence of a substance, usually an antigen, in a liquid sample or wet sample.
The ELISA has been used as a diagnostic tool in medicine and plant pathology, as well as a quality-control check in various industries. Antigens from the sample are attached to a surface. Then, a further specific antibody is applied over the surface so it can bind to the antigen. This antibody is linked to an enzyme, and, in the final step, a substance containing the enzyme's substrate is added. The subsequent reaction produces a detectable signal, most commonly a color change in the substrate.
Performing an ELISA involves at least one antibody with specificity for a particular antigen. The sample with an unknown amount of antigen is immobilized on a solid support (usually a polystyrene microtiter plate) either non-specifically (via adsorption to the surface) or specifically (via capture by another antibody specific to the same antigen, in a "sandwich" ELISA). After the antigen is immobilized, the detection antibody is added, forming a complex with the antigen.
The detection antibody can be covalently linked to an enzyme, or can itself be detected by a secondary antibody that is linked to an enzyme through bioconjugation. Between each step, the plate is typically washed with a mild detergent solution to remove any proteins or antibodies that are non-specifically bound. After the final wash step, the plate is developed by adding an enzymatic substrate to produce a visible signal, which indicates the quantity of antigen in the sample.
Of note, ELISA can perform other forms of ligand binding assays instead of strictly "immuno" assays, though the name carried the original "immuno" because of the common use and history of development of this method. The technique essentially requires any ligating reagent that can be immobilized on the solid phase along with a detection reagent that will bind specifically and use an enzyme to generate a signal that can be properly quantified.
In between the washes, only the ligand and its specific binding counterparts remain specifically bound or "immunosorbed" by antigen-antibody interactions to the solid phase, while the nonspecific or unbound components are washed away. Unlike other spectrophotometric wet lab assay formats where the same reaction well (e.g. a cuvette) can be reused after washing, the ELISA plates have the reaction products immunosorbed on the solid phase which is part of the plate, and so are not easily reusable.
Principle As an analytic biochemistry assay, ELISA involves detection of an "analyte" (i.e. the specific substance whose presence is being quantitatively or qualitatively analyzed) in a liquid sample by a method that continues to use liquid reagents during the "analysis" (i.e. controlled sequence of biochemical reactions that will generate a signal which can be easily quantified and interpreted as a measure of the amount of analyte in the sample) that stays liquid and remains inside a reaction chamber or well needed to keep the reactants contained; It is opposed to "dry lab" that can use dry strips – and even if the sample is liquid (e.
g. a measured small drop), the final detection step in "dry" analysis involves reading of a dried strip by methods such as reflectometry and does not need a reaction containment chamber to prevent spillover or mixing between samples. As a heterogenous assay, ELISA separates some component of the analytical reaction mixture by adsorbing certain components onto a solid phase which is physically immobilized.
In ELISA, a liquid sample is added onto a stationary solid phase with special binding properties and is followed by multiple liquid reagents that are sequentially added, incubated and washed followed by some optical change (e.g. color development by the product of an enzymatic reaction) in the final liquid in the well from which the quantity of the analyte is measured. The qualitative "reading" usually based on detection of intensity of transmitted light by spectrophotometry, which involves quantitation of transmission of some specific wavelength of light through the liquid (as well as the transparent bottom of the well in the multiple-well plate format).
The sensitivity of detection depends on amplification of the signal during the analytic reactions. Since enzyme reactions are very well known amplification processes, the signal is generated by enzymes which are linked to the detection reagents in fixed proportions to allow accurate quantification – thus the name "enzyme linked". The analyte is also called the ligand because it will specifically bind or ligate to a detection reagent, thus ELISA falls under the bigger category of ligand binding assays.
The ligand-specific binding reagent is "immobilized", i.e., usually coated and dried onto the transparent bottom and sometimes also side wall of a well (the stationary "solid phase'/"solid substrate" here as opposed to solid microparticle/beads that can be washed away), which is usually constructed as a multiple-well plate known as the "ELISA plate". Conventionally, like other forms of immunoassays, the specificity of antigen-antibody type reaction is used because it is easy to raise an antibody specifically against an antigen in bulk as a reagent.
Alternatively, if the analyte itself is an antibody, its target antigen can be used as the binding reagent. History Before the development of the ELISA, the only option for conducting an immunoassay was radioimmunoassay, a technique using radioactively labeled antigens or antibodies. In radioimmunoassay, the radioactivity provides the signal, which indicates whether a specific antigen or antibody is present in the sample.
Radioimmunoassay was first described in a scientific paper by Rosalyn Sussman Yalow and Solomon Berson published in 1960. Because radioactivity poses a potential health threat, a safer alternative was sought. A suitable alternative to radioimmunoassay would substitute a nonradioactive signal in place of the radioactive signal. When enzymes (such as horseradish peroxidase) react with appropriate substrates (such as ABTS or TMB), a change in color occurs, which is used as a signal.
However, the signal has to be associated with the presence of antibody or antigen, which is why the enzyme has to be linked to an appropriate antibody. This linking process was independently developed by Stratis Avrameas and G. B. Pierce. Since it is necessary to remove any unbound antibody or antigen by washing, the antibody or antigen has to be fixed to the surface of the container; i.e., the immunosorbent must be prepared.
A technique to accomplish this was published by Wide and Jerker Porath in 1966. A paramedic assistant prepares analyses in an ELISA laboratory In 1971, Peter Perlmann and Eva Engvall at Stockholm University in Sweden, and Anton Schuurs and Bauke van Weemen in the Netherlands independently published papers that synthesized this knowledge into methods to perform EIA/ELISA. Traditional ELISA typically involves chromogenic reporters and substrates that produce some kind of observable color change to indicate the presence of antigen or analyte.
Newer ELISA-like techniques use fluorogenic, electrochemiluminescent, and quantitative PCR reporters to create quantifiable signals. These new reporters can have various advantages, including higher sensitivities and multiplexing. In technical terms, newer assays of this type are not strictly ELISAs, as they are not "enzyme-linked", but are instead linked to some nonenzymatic reporter. However, given that the general principles in these assays are largely similar, they are often grouped in the same category as ELISAs.
In 2012, an ultrasensitive, enzyme-based ELISA test using nanoparticles as a chromogenic reporter was able to give a naked-eye colour signal, from the detection of mere attograms of analyte. A blue color appears for positive results and red color for negative. Note that this detection only can confirm the presence or the absence of analyte not the actual concentration. Types Direct ELISA Direct ELISA diagram The steps of direct ELISA follows the mechanism below: A buffered solution of the antigen to be tested for is added to each well of a microtiter plate, where it is given time to adhere to the plastic through charge interactions.
A solution of nonreacting protein, such as bovine serum albumin or casein, is added to well (usually 96-well plates) in order to cover any plastic surface in the well which remains uncoated by the antigen. The primary antibody with an attached (conjugated) enzyme is added, which binds specifically to the test antigen coating the well. A substrate for this enzyme is then added. Often, this substrate changes color upon reaction with the enzyme.
The higher the concentration of the primary antibody present in the serum, the stronger the color change. Often, a spectrometer is used to give quantitative values for color strength. The enzyme acts as an amplifier; even if only few enzyme-linked antibodies remain bound, the enzyme molecules will produce many signal molecules. Within common-sense limitations, the enzyme can go on producing color indefinitely, but the more antibody is bound, the faster the color will develop.
A major disadvantage of the direct ELISA is the method of antigen immobilization is not specific; when serum is used as the source of test antigen, all proteins in the sample may stick to the microtiter plate well, so small concentrations of analyte in serum must compete with other serum proteins when binding to the well surface. The sandwich or indirect ELISA provides a solution to this problem, by using a "capture" antibody specific for the test antigen to pull it out of the serum's molecular mixture.
ELISA may be run in a qualitative or quantitative format. Qualitative results provide a simple positive or negative result (yes or no) for a sample. The cutoff between positive and negative is determined by the analyst and may be statistical. Two or three times the standard deviation (error inherent in a test) is often used to distinguish positive from negative samples. In quantitative ELISA, the optical density (OD) of the sample is compared to a standard curve, which is typically a serial dilution of a known-concentration solution of the target molecule.
For example, if a test sample returns an OD of 1.0, the point on the standard curve that gave OD = 1.0 must be of the same analyte concentration as the sample. The use and meaning of the names "direct ELISA" and "indirect ELISA" differs in the literature and on web sites depending on the context of the experiment. When the presence of an antigen is analyzed, the name "direct ELISA" refers to an ELISA in which only a labelled primary antibody is used, and the term "indirect ELISA" refers to an ELISA in which the antigen is bound by the primary antibody which then is detected by a labeled secondary antibody.
In the latter case a sandwich ELISA is clearly distinct from an indirect ELISA. When the "primary" antibody is of interest, e.g. in the case of immunization analyses, this antibody is directly detected by the secondary antibody and the term "indirect ELISA" applies to a setting with two antibodies. Sandwich ELISA A sandwich ELISA. (1) Plate is coated with a capture antibody; (2) sample is added, and any antigen present binds to capture antibody; (3) detecting antibody is added, and binds to antigen; (4) enzyme-linked secondary antibody is added, and binds to detecting antibody; (5) substrate is added, and is converted by enzyme to detectable form.
A "sandwich" ELISA is used to detect sample antigen. The steps are: A surface is prepared to which a known quantity of capture antibody is bound. Any nonspecific binding sites on the surface are blocked. The antigen-containing sample is applied to the plate, and captured by antibody. The plate is washed to remove unbound antigen. A specific antibody is added, and binds to antigen (hence the 'sandwich': the antigen is stuck between two antibodies).
This primary antibody could also be in the serum of a donor to be tested for reactivity towards the antigen. Enzyme-linked secondary antibodies are applied as detection antibodies that also bind specifically to the antibody's Fc region (nonspecific). The plate is washed to remove the unbound antibody-enzyme conjugates. A chemical is added to be converted by the enzyme into a color or fluorescent or electrochemical signal.
The absorbance or fluorescence or electrochemical signal (e.g., current) of the plate wells is measured to determine the presence and quantity of antigen. The image to the right includes the use of a secondary antibody conjugated to an enzyme, though, in the technical sense, this is not necessary if the primary antibody is conjugated to an enzyme (which would be direct ELISA). However, the use of a secondary-antibody conjugate avoids the expensive process of creating enzyme-linked antibodies for every antigen one might want to detect.
By using an enzyme-linked antibody that binds the Fc region of other antibodies, this same enzyme-linked antibody can be used in a variety of situations. Without the first layer of "capture" antibody, any proteins in the sample (including serum proteins) may competitively adsorb to the plate surface, lowering the quantity of antigen immobilized. Use of the purified specific antibody to attach the antigen to the plastic eliminates a need to purify the antigen from complicated mixtures before the measurement, simplifying the assay, and increasing the specificity and the sensitivity of the assay.
A sandwich ELISA used for research often need validation because of the risk of false positive results. Competitive ELISA A third use of ELISA is through competitive binding. The steps for this ELISA are somewhat different from the first two examples: Unlabeled antibody is incubated in the presence of its antigen (sample). These bound antibody/antigen complexes are then added to an antigen-coated well.
The plate is washed, so unbound antibodies are removed. (The more antigen in the sample, the more Ag-Ab complexes are formed and so there are less unbound antibodies available to bind to the antigen in the well, hence "competition".) The secondary antibody, specific to the primary antibody, is added. This second antibody is coupled to the enzyme. A substrate is added, and remaining enzymes elicit a chromogenic or fluorescent signal.
The reaction is stopped to prevent eventual saturation of the signal. Some competitive ELISA kits include enzyme-linked antigen rather than enzyme-linked antibody. The labeled antigen competes for primary antibody binding sites with the sample antigen (unlabeled). The less antigen in the sample, the more labeled antigen is retained in the well and the stronger the signal. Commonly, the antigen is not first positioned in the well.
For the detection of HIV antibodies, the wells of microtiter plate are coated with the HIV antigen. Two specific antibodies are used, one conjugated with enzyme and the other present in serum (if serum is positive for the antibody). Cumulative competition occurs between the two antibodies for the same antigen, causing a stronger signal to be seen. Sera to be tested are added to these wells and incubated at 37 °C, and then washed.
If antibodies are present, the antigen-antibody reaction occurs. No antigen is left for the enzyme-labelled specific HIV antibodies. These antibodies remain free upon addition and are washed off during washing. Substrate is added, but there is no enzyme to act on it, so a positive result shows no color change. Applications Human anti-IgG, double antibody sandwich ELISA Because the ELISA can be performed to evaluate either the presence of antigen or the presence of antibody in a sample, it is a useful tool for determining serum antibody concentrations (such as with the HIV test or West Nile virus).
It has also found applications in the food industry in detecting potential food allergens, such as milk, peanuts, walnuts, almonds, and eggs and as serological blood test for coeliac disease. ELISA can also be used in toxicology as a rapid presumptive screen for certain classes of drugs. Enzyme-linked immunosorbent assay plate The ELISA was the first screening test widely used for HIV because of its high sensitivity.
In an ELISA, a person's serum is diluted 400 times and applied to a plate to which HIV antigens are attached. If antibodies to HIV are present in the serum, they may bind to these HIV antigens. The plate is then washed to remove all other components of the serum. A specially prepared "secondary antibody" — an antibody that binds to other antibodies — is then applied to the plate, followed by another wash.
This secondary antibody is chemically linked in advance to an enzyme. Thus, the plate will contain enzyme in proportion to the amount of secondary antibody bound to the plate. A substrate for the enzyme is applied, and catalysis by the enzyme leads to a change in color or fluorescence. ELISA results are reported as a number; the most controversial aspect of this test is determining the "cut-off" point between a positive and a negative result.
A cut-off point may be determined by comparing it with a known standard. If an ELISA test is used for drug screening at workplace, a cut-off concentration, 50 ng/ml, for example, is established, and a sample containing the standard concentration of analyte will be prepared. Unknowns that generate a stronger signal than the known sample are "positive." Those that generate weaker signal are "negative".
Dr Dennis E Bidwell and Alister Voller created the ELISA test to detect various kind of diseases, such as malaria, Chagas disease, and Johne's disease. ELISA tests also are used as in in vitro diagnostics in medical laboratories. The other uses of ELISA include: detection of Mycobacterium antibodies in tuberculosis detection of rotavirus in feces detection of hepatitis B markers in serum detection of enterotoxin of E.
coli in feces detection of HIV antibodies in blood samples See also Immunoscreening Lateral flow test Magnetic immunoassay microtitre plate Plaque reduction neutralization test Plate reader Secretion assay Agglutination-PCR Notes and references ^ Yalow, Rosalyn S.; Berson, Solomon A. (1960). "Immunoassay of endogenous plasma insulin in man". The Journal of Clinical Investigation. 39: 1157–75. doi:10.
1172/JCI104130. PMC 441860 . PMID 13846364. ^ Lequin, R. M. (2005). "Enzyme Immunoassay (EIA)/Enzyme-Linked Immunosorbent Assay (ELISA)". Clinical Chemistry. 51 (12): 2415–8. doi:10.1373/clinchem.2005.051532. PMID 16179424. ^ Wide, Leif; Porath, Jerker (1966). "Radioimmunoassay of proteins with the use of Sephadex-coupled antibodies". Biochimica et Biophysica Acta. 130 (1): 257–60. doi:10.
1016/0304-4165(66)90032-8. ^ Engvall, Eva; Perlmann, Peter (1971). "Enzyme-linked immunosorbent assay (ELISA) quantitative assay of immunoglobulin G". Immunochemistry. 8 (9): 871–4. doi:10.1016/0019-2791(71)90454-X. PMID 5135623. ^ Van Weemen, B.K.; Schuurs, A.H.W.M. (1971). "Immunoassay using antigen—enzyme conjugates". FEBS Letters. 15 (3): 232–236. doi:10.1016/0014-5793(71)80319-8. PMID 11945853.
^ Leng, S. X.; McElhaney, J. E.; Walston, J. D.; Xie, D.; Fedarko, N. S.; Kuchel, G. A. (2008). "ELISA and Multiplex Technologies for Cytokine Measurement in Inflammation and Aging Research". The Journals of Gerontology Series A: Biological Sciences and Medical Sciences. 63 (8): 879–84. doi:10.1093/gerona/63.8.879. PMC 2562869 . PMID 18772478. ^ Adler, Michael; Schulz, Sven; Spengler, Mark (2009).
"Cytokine Quantification in Drug Development: A comparison of sensitive immunoassay platforms". Chimera Biotech. ^ de la Rica, Roberto; Stevens, Molly M. (2012). "Plasmonic ELISA for the ultrasensitive detection of disease biomarkers with the naked eye". Nature Nanotechnology. 7 (12): 821–4. doi:10.1038/nnano.2012.186. hdl:10044/1/21938. PMID 23103935. ^ Schmidt, SD; Mazzella, MJ; Nixon, RA; Mathews, PM (2012).
"Aβ measurement by enzyme-linked immunosorbent assay". Methods in Molecular Biology. 849: 507–27. doi:10.1007/978-1-61779-551-0_34. PMID 22528112. ^ Kragstrup, Tue W; Vorup-Jensen, Thomas; Deleuran, Bent; Hvid, Malene (2013). "A simple set of validation steps identifies and removes false results in a sandwich enzyme-linked immunosorbent assay caused by anti-animal IgG antibodies in plasma from arthritis patients".
SpringerPlus. 2 (1): 263. doi:10.1186/2193-1801-2-263. ^ MedlinePlus Encyclopedia ELISA/Western blot tests for HIV ^ "Food Allergen Partnership" (Press release). FDA. January 2001. Retrieved August 20, 2015. ^ Sblattero, D.; Berti, I.; Trevisiol, C.; Marzari, R.; Tommasini, A.; Bradbury, A.; Fasano, A.; Ventura, A.; Not, T. (2000). "Human recombinant tissue transglutaminase ELISA: an innovative diagnostic assay for celiac disease".
The American Journal of Gastroenterology. 95 (5): 1253–7. doi:10.1111/j.1572-0241.2000.02018.x. PMID 10811336. ^ Porcelli, Brunetta; Ferretti, Fabio; Vindigni, Carla; Terzuoli, Lucia (2014). "Assessment of a Test for the Screening and Diagnosis of Celiac Disease". Journal of Clinical Laboratory Analysis. doi:10.1002/jcla.21816. PMID 25385391. ^ Griffin, J. F. T.; Spittle, E.; Rodgers, C. R.; Liggett, S.
; Cooper, M.; Bakker, D.; Bannantine, J. P. (2005). "Immunoglobulin G1 Enzyme-Linked Immunosorbent Assay for Diagnosis of Johne's Disease in Red Deer (Cervus elaphus)". Clinical and Vaccine Immunology. 12 (12): 1401–9. doi:10.1128/CDLI.12.12.1401-1409.2005. PMC 1317074 . PMID 16339063. External links More publications about ELISA kits An animated illustration of an ELISA assay The ELISA technique illustrated An animated tutorial comparing the direct and indirect ELISA methods "Introduction to ELISA Activity – beginner walkthrough of ELISA used for detecting HIV, including animations at University of Arizona A descriptive animation of the application of sandwich ELISA to Pregnancy test Animated video overview of competitive ELISA formats ELISA at the US National Library of Medicine Medical Subject Headings (MeSH) v t e Medical tests: Immunologic techniques and tests (CPT 86000–86849) Immunologic techniques and tests serology/diagnostic immunology Immunoprecipitation Chromatin immunoprecipitation Immunodiffusion Ouchterlony double immunodiffusion Radial immunodiffusion Immunoelectrophoresis Counterimmunoelectrophoresis Immunoassay ELISA ELISPOT Enzyme Multiplied Immunoassay Technique RAST test Radioimmunoassay Radiobinding assay Immunofluorescence Agglutination Hemagglutination/Hemagglutinin Coombs test Latex fixation test Other Nephelometry Complement fixation test Immunocytochemistry Immunohistochemistry Direct fluorescent antibody Epitope mapping Skin allergy test Patch test Inflammation C-reactive protein Procalcitonin Total complement activity MELISA CBC lymphocyte count Retrieved from "https://en.
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In-Vivo Diagnostic tests of immunoglobulin E (IgE)-dependent reactions Epicutaneous (Scratch, Prick or Puncture) and Intracutaneous In-Vivo Diagnostic Skin Tests Skin tests for IgE-mediated disease with allergenic extracts have been shown to be effective aids in the assessment of allergic patients. These tests involve the introduction of small quantities of test allergens below the epidermis. Within 15 to 20 mins, a characteristic wheal and flare reaction occurs in patients sensitive to one or more of the test allergens.
The majority of allergists use prick or puncture and/or intracutaneous skin tests, since the amount of allergen delivered by these methods is better controlled than by scratch tests. Although skin testing is considered to be a safe procedure, adverse events, such as large local reactions and systemic symptoms may occur in extremely sensitive individuals. Deaths from anaphylaxis after skin testing have been reported.
These extremely rare systemic symptoms are less likely to occur with prick or puncture than with intracutaneous tests. Prick or puncture tests are generally considered to be the most convenient, least expensive and most specific screening method for detecting the presence of IgE antibodies in patients with appropriate exposure histories. Prick or puncture tests are generally less sensitive than intracutaneous tests.
For inhalant allergens, prick or puncture tests are generally felt to correlate better with the presence of clinical allergy. However, intracutaneous (within the skin) testing may detect relevant sensitivity and should be considered when the prick or puncture test is negative or equivocal to allergens strongly suggested by the patient's history or exposure, or when skin sensitivity may be decreased such as in infants or older patients.
Intracutaneous tests permit identification of a larger number of clinically reactive patients, especially those with lower skin test sensitivity. Skin testing to drugs is generally unreliable, except for the penicillins and macromolecular agents, such as foreign antisera, hormone (e.g., insulin), enzymes (e.g., L-asparaginase, streptokinase, chymopapain), and egg-containing vaccines. In January 2003, the Board of Directors of the American Academy of Otolaryngic Allergy (AAOA) endorsed strategies for testing for inhalant allergy (Krouse and Mabry, 2003), stating that “[m]embers should practice in ethical and fiscally responsible ways.
” The AAOA provided the following guidelines on the necessary number of tests for inhalant allergy (e.g., prick testing, intradermal testing, intradermal dilutional testing (IDT), and in vitro testing): Screening: Screen with no more than 14 relevant antigens plus appropriate controls. Antigen survey: If screening is positive and immunotherapy is contemplated, use no more than 40 antigens. More extensive testing may be justified in special circumstances.
Quantification for safe starting point: Use no more than 80 IDT tests routinely. More extensive testing may be justified in special circumstances. Skin Endpoint Titration (SET) Skin endpoint titration (SET) (also known as intradermal dilutional testing (IDT)) is intradermal testing of sequential and incremental dilutions of a single antigen. SET involves serial testing with several dilutions of a single treatment allergen or mixture of allergens to identify the lowest dilution that produces a positive skin reaction.
In performing the test, wheals of identical size are made in the most superficial layers of the skin and measured for uniformity. The first wheal is made with approximately 0.1 ml of a dilution estimated to be too weak to produce symptoms. Successive wheals are made with serial dilutions, each generally five times stronger than the previous one, until negative responses are replaced by positive responses of increasing size.
The "endpoint" is the weakest dilution that produces a positive skin reaction and initiates progressive increase in the diameter of wheals with each stronger dilution. Proponents of SET emphasize that it quantifies skin testing and replaces a single equivocal reaction with a progressive pattern easily identified. When immunotherapy is initiated, starting with too strong an extract may precipitate dangerous allergic reactions, while starting with one too weak may delay treatment results.
Skin endpoint titration allows the initiation of immunotherapy with a safe but relatively potent dose, and allows the beginning dosage for each positive responding allergen to be varied depending on its specific “endpoint.” Although traditional allergists often rely on single dilution “classical” testing, they have accepted SET over the last decade as effective for quantifying patient sensitivity and for providing a guide for a safe starting dose for immunotherapy noting that studies have not shown it to be an effective guide to a final therapeutic dose.
The AAOA also has advised that costly, repetitive endpoint titrations are usually unnecessary because, regardless of what the titration indicates, the dose will be advanced either until the patient can tolerate no more or until a dose is reached that produces satisfactory results. Skin endpoint titration is considered the gold standard of skin testing by the AAOA; the American Medical Association’s Council of Scientific Affairs also is on record that SET is helpful for the delineation of patient-specific sensitivity to various antigens as well as to evaluate a patient’s response to various forms of immunotherapy.
They note that controlled studies have shown that the intradermal method of SET is effective for quantifying sensitivity to ragweed pollen extract and for identifying patients highly sensitive to ragweed. While allowing that SET is a valid method for obtaining semi-quantitative information about a person's sensitivity and for determining a safe beginning dose for immunotherapy, the American College of Physicians (ACP) advises that the primary use of SET is to identify hymenoptera venom (yellow jacket, honey bee, hornet, wasp, fire ant) sensitivity and to determine the safe starting dose for venom immunotherapy.
In a guideline, revised in 2003, the AAOA recommends screening prick tests with relevant antigens to determine which to use in subsequent SET (Krouse and Mabry, 2003). The literature on screening supports, and the AAOA recommends, usually screening and billing for no more than 14 antigens (plus the appropriate controls) for an initial allergy evaluation. In most geographic regions, a range of up to 14 allergens is sufficient to check the most prevalent molds, dust components, grasses, trees, animals, and weeds.
If screening is positive and immunotherapy is contemplated, the AAOA recommends no more than 40 antigen be tested unless indicated by unusual clinical circumstances. For SET, the AAOA says that up to 80 injections are usually necessary to identify the offending antigen and find a safe starting point for immunotherapy. Provocation (Challenge) Testing In provocation or challenge testing, a suspected allergen in a clinically relevant exposure is administered in an attempt to reproduce symptoms.
Challenge tests have been broadly applied under research conditions for many years, but there also may be clinical situations in which they can be useful for confirmation of clinical disease. Considerable experience with these methods is required for proper interpretation and analysis. Patch Testing Patch testing is an accepted method of differentiating allergic contact dermatitis and irritant contact dermatitis.
Twenty to 30 antigens are used in the usual routine screening panel of patch tests. The patches are removed after 48 hours and an initial reading is taken 1 hour later. The final reading is taken a further 48 hours later. Photo Patch Testing Some chemicals or medications (e.g., lomefloxacin, ofloxacin, ciprofloxacin and norfloxacin) produce an allergic reaction only when exposed to light (usually ultraviolet type A, UVA).
Patients who are over-sensitive to light and those with a rash that appears on parts of the body normally exposed to light but that does not appear in areas shielded from the light should have a photo-patch test. With photo patch testing, 2 identical sets of allergens are placed onto the patient's back on day-1. One of the sets is exposed to UVA light, and the sites are then examined as described above for patch testing.
A positive photo patch test is recorded when an allergic reaction appears only on the light-exposed site. Photo Tests Photo testing is skin irradiation with a specific range of ultraviolet light. Photo tests are performed for the evaluation of photosensitivity disorders. Bronchial Challenge Testing Bronchial challenge testing with methacholine, histamine, or allergens is an accepted method of defining asthma or airway hyperactivity when skin testing results are not consistent with the patient's medical history.
Results of these tests are ordinarily evaluated by objective measures of pulmonary function and occasionally by characterization of bronchoalveolar lavage samples. Recommended dosage is an incremental increase of pharmacologic dose until a response is produced. Exercise Challenge Testing Exercise challenge testing is an accepted method of diagnosing exercise induced bronchospasm in asthmatic and non-asthmatic patients.
Ingestion (Oral) Challenge Testing Ingestion (oral) challenge testing is an accepted method of diagnosing allergies to food, drug or other substances (i.e., metabisulfite). Drug challenge testing should not be confused with cutaneous or sublingual provocation and neutralization therapy, which is a non-covered modality. Nasal or Conjunctival Provocative or Challenge Tests Nasal or conjunctival provocative or challenge tests employed for the diagnosis of either food or inhalant allergies, involve the direct administration of the allergen to the mucosa.
The patient is then observed for signs and symptoms and the presence of symptoms is interpreted as a positive indication of allergies. These tests are time consuming, only 1 antigen may be administered per session, a non-standardized quantity of allergen is administered and they have the potential of inducing severe symptoms. There is currently no standard of techniques for nasal or conjunctival challenge tests that can be applied to clinical practice.
Prausnitz-Kustner or P-K Testing Prausnitz-Kustner testing has been used in patients with dermatographia or generalized skin eruptions. A control site on the forearm of a non-allergic recipient is selected. This site is injected intradermally with allergy serum from a patient on whom direct skin tests cannot be done. Allergenic extract is later injected intradermally into the initial injection site of the recipient and observed for the development of a wheal and flare.
Because of the risk of transmitting hepatitis or AIDS, this test is contraindicated. Provocation-Neutralization (Rinkel Test) Provocation-neutralization is a method of testing for the presence of food, inhalant or environmental chemical allergies by exposing the individual to test doses of these substances intradermally, subcutaneously, or sublingually with the purpose of either producing or preventing subjective symptoms.
Provocation-neutralization evolved from the serial end-point titration skin testing procedure (a covered modality), and is based on the concept that extremely small quantities of allergens can cause immediate disappearance (“neutralization”) of ongoing symptoms. Once a test is considered positive (results are interpreted either by subjective symptom provocation or objective skin whealing), a progressive series of lower concentrations are administered under the tongue or skin until a dose is reached at which the patient reports no sensations.
This amount of the test substance is considered the “neutralizing dose”, which is then used for future treatment. Sublingual testing has been used mainly in diagnosing food allergy, although extracts of chemicals, inhalant allergens, drugs, and hormones have been administered by the sublingual route. Published literature frequently combines the discussion of testing and treatment as a single entity.
Provocation-neutralization is used by those physicians who subscribe to the concept of multiple food and chemical sensitivities (also known as idiopathic environmental intolerance's (IEI), clinical ecological illness, clinical ecology, environmental illness, chemical AIDS, environmental/chemical hypersensitivity disease, total allergy syndrome, cerebral allergy, 20th century disease) and “delayed food allergy”.
When used for the latter, provocative testing may be identified as the intracutaneous progressive dilution food test (IPDFT). Since provocation-neutralization requires the provoking and neutralizing of symptoms to a single item at a time, the patient could be required to undergo hundreds of individual tests requiring weeks or months of full-day testing. Traditional allergists believe that food hypersensitivities are primarily IgE-mediated and treat with avoidance diet and/or drug therapy.
Diagnosis is by history, elimination diets, skin tests, or food challenge. Non IgE-mediated food intolerance is classified as non-immune adverse reactions to food of a pharmacologic (caffeine, histamine, tyramine, serotonin, dopamine, etc.); metabolic (lactose intolerance); or idiosyncratic nature, e.g., food dyes, preservatives (sulfites), flavor enhancers (MSG). The AAOA indicates that provocation-neutralization techniques were developed primarily for these delayed, less obvious, non-IgE-mediated food hypersensitivities and not for confirmation of immediate food allergy obvious by history.
Test substances have also included chemicals such as formaldehyde and alcohol, histamine, tobacco, newsprint and inhalant allergens. Sublingual provocative neutralization with hormones utilizes the same principles as noted above and involves preliminary extensive blood testing for allergies to hormones and the subsequent administration of small doses of hormones suspected of causing the allergic symptoms.
There have been no well-controlled studies that have shown this procedure to be effective in the diagnosis and treatment of symptoms thought to be caused by allergy to hormones. Both the ACP and the American Academy of Allergy and Immunology (AAAI) consider provocation-neutralization therapy an unproven modality. In a Training Program Directors' Committee Report on Controversial Practices published by the AAAI, provocation-neutralization testing and neutralization therapy are listed as unproven.
The AMA's Council on Scientific Affairs, based on the reports in the peer-reviewed scientific literature, stated that there are no well-controlled studies establishing a clear mechanism or cause for multiple chemical sensitivity syndrome. More importantly, there are no well-controlled studies that have demonstrated either diagnostic or therapeutic value for provocation-neutralization therapy. Provocation-neutralization must not be confused with the recognized forms of target-organ challenge testing (bronchial, ingestion, patch testing), which are covered modalities.
According to the National Asthma Education and Prevention Program Guidelines for the Diagnosis and Management of Asthma, advantages of RAST and other in vitro tests over skin tests include the fact that they do not require knowledge of skin testing technique, they do not require availability of allergen extracts, they can be performed on patients who are taking medications that suppress the immediate skin test (e.
g., antihistamines, antidepressants), they carry no risk of systemic reactions, and they can be done on patients with extensive eczema. Despite the advantages, there are 2 major concerns limiting the use of in-vitro tests for allergen-specific IgE in the United States. The first limitation is the rather consistent finding that in-vitro tests are not as sensitive as skin tests for detecting allergen-specific IgE.
The second limitation is that on a per test basis skin tests have lower time and reagent costs. Other advantages of skin tests are that they are faster (results are available within an hour), and the results are visible to the patient (this may enhance patient compliance). A variety of modifications have been made to tests related to RAST (such as MAST, PRIST, RIST, FAST, MRT, VAST, ELISA, and ImmunoCAP).
ImmunoCAP (Pharmacia Diagnostics, Clayton, N.C.) is an in vitro-specific immunoglobulin E test that uses a three-dimensional cellulose solid allergen phase; by contrast, the older modified Phadezym-Rast (Pharmacia Diagnostics) uses a 2-dimensional solid phase. The ImmunoCAP provides more rapid results (available in 6 hours) compared to traditional RAST tests (Phadezym-RAST results take 3 days to obtain).
With the ImmunoCAP, solid-phase bound allergens are allowed to react with IgE antibodies in the sample; the IgE antibodies are detected by labeled anti-IgE. To minimize handling and increase safety, the system includes instrumentation and computer software that handles the technical manipulations, the measurements and the data management. The assay is calibrated against the WHO standard for IgE and includes 2 sets of calibrators, 1 for specific IgE Ab and low-range total IgE, and the other for wide-range total IgE.
Results from published studies report the overall sensitivity and specificity of different allergens compared to expert clinical diagnosis range from 78 to 94 % and 77 to 94 %, respectively. Total Serum IgE Total serum IgE concentrations (paper radioimmunosorbent test [PRIST], radioimmunosorbent test [RIST]) – This type of testing is less useful in assessing the risk of allergic disease, but may be indicated for those patients suspected of having allergic bronchopulmonary aspergillosis, eczema, hyper-IgE syndrome, certain stages of human immunodeficiency virus (HIV), IgE myeloma, graft versus host disease or immune deficiency diseases characterized by increased IgE levels (eg, Wiskott-Aldrich syndrome).
An elevated serum IgE level is one of the diagnostic criteria of allergic bronchopulmonary aspergillosis (ABPA). IgE levels can be used to follow the course of the disease. Serum IgE levels will fall when the disease is successfully treated with corticosteroids; rising IgE levels indicate disease exacerbations. Total serum level of IgE is correlated with allergic disease in only a general way. Elevated levels are associated with the presence of allergy, while normal levels are not.
However there are many individuals with clinical symptoms and allergen-specific IgE who have serum IgE levels within the normal range. Because of this, routine measurement of serum IgE is not a useful screening test for allergy. IgG RAST/ELISA Testing There is no evidence that IgG antibodies are responsible for delayed allergic symptoms or intolerance to foods. In their Choosing Wisely Campaign, the American Academy of Allergy, Asthma and Immunology recommends against immunoglobulin G (IgG) testing in the evaluation of allergy.
The American Academy of Allergy, Asthma & Immunology (AAAAI) states that appropriate diagnosis and treatment of allergies requires specific IgE testing (either skin or blood tests) based on the patient’s clinical history. ALCAT ALCAT food allergy testing utilizes an indirect method of measuring mediator releases and the effects of other pathogenic mechanisms of allergy and delayed hypersensitivity.
It employs semi-automated Coulter Electronics and fully automated computer analysis. This automated testing has not been validated and has not been established as a useful allergy test in clinical practice. Cytotoxic Testing (Bryans Test) Cytotoxic testing is based on the theory that the addition of a specific allergen to either whole blood or a serum leukocyte suspension from a suspected allergic patient will result in reduction of the white blood cell count or death of the leukocytes, thereby indicating the presence of an immune response.
Controlled studies have failed to substantiate the value of cytotoxic testing for the diagnosis of allergies, whether they are airborne, foods, or chemicals. ELISA/ACT ELISA/ACT tests lymphocytes in a laboratory culture for their reaction to up to 300 purified foods, preservatives, chemicals and minerals. The test is offered by Serammune Physicians Laboratory. This test is not FDA approved and is not established as a useful test in clinical practice.
Food Immune Complex Assays (FICA) FICA are based on the standard solid phase radioimmunoassay methodology. These assays have not yet been subjected to rigorous study of potential false-negative and false-positive results. Clinical studies to date indicate that circulating immune complexes can be found in a normal population of people having no food allergy. The value of the measurement of FICA toward the diagnosis of food allergy remains unproven and does not have a place in current clinical practice.
Rebuck Skin Window Test Rebuck skin window test is an immunologic test in which the skin is abraded with a scalpel. Laboratory cover slips are placed over the abraded areas for 24 hours. The cover slips are then stained and analyzed. An immune deficiency may be present if there is an abnormality of monocytes displayed either by their absence or their inability to migrate to intracellular sites of antigen within 12 hours.
This test is not useful in documenting allergies since other immunodeficiencies can be found in patients with allergic conditions. Leukocyte Histamine Release Test The leukocyte histamine release test is a measurement of the amount of histamine released in-vitro. Varying concentrations of an allergen extract are added to the patient's peripheral blood leukocytes. Histamine is normally released as a consequence of the interaction of allergen with cell-bound IgE antibodies.
If an individual is atopic to a specific antigen, the leukocytes will not release the histamine in-vitro. Only a limited number of allergens can be tested from a single aliquot of blood and quality control studies have shown considerable variability in the measurement of histamine results. Mediator Release Test The mediator release test (MRT) (Signet Diagnostic Corporation) has primarily been used to detect intolerance to foods and additives in patients with irritable bowel syndrome.
The MRT measures the aggregate release of inflammatory mediators from the patient's immunocytes in vitro after exposure to specific foods and food additives. The results of the mediator release test have been used to design a patient-specific diet to treat IBS by avoiding foods and additives that trigger significant inflammatory mediator release. For the mediator release test, the patient's blood sample is incubated with various extracts of foods and food additives and then analyzed for the presence and aggregate amount of release of inflammatory mediators from the patient's leukocytes.
Results are compared to control samples of the patient's blood that have not been exposed to food extracts or additives. The MRT-directed patient-specific diet is one component of the Lifestyle Eating and Performance (LEAP) Disease Management Program (Don Self & Associates, Inc., Whitehouse, TX). The LEAP program is based on the theory that symptoms irritable bowel syndrome and other certain conditions are caused by the physiological effects of non-IgE mediated immune reactions in response to sensitivities to specific foods and food additives.
The LEAP program also includes patient selection tools, a self-directed stress reduction program, and outcomes assessment tools. According to the manufacturer, the LEAP program has been successful in reducing or eliminating symptoms in 84 % of patients with irritable bowel syndrome, functional diarrhea, and related conditions. However, there is no evidence in the peer-reviewed published medical literature to substantiate these claims.
The mediator release test has also been promoted for use in patients with chronic fatigue syndrome, metabolic conditions (e.g., diabetes, obesity), gastrointestinal disorders (e.g., gastroesophageal reflux disease, chronic ulcerative colitis, and Crohn's disease), neurologic disorders (e.g., migraine headaches, cluster headaches), rheumatologic disorders (inflammatory arthritis, arthralgias, fibromyalgia), otolaryngologic disorders (e.
g., perennial rhinitis, chronic sinusitis, chronic otitis media with effusion), dermatologic conditions (e.g., eczema, urticaria, dermatitis), and in patients with behavioral conditions (e.g., attention deficit disorder, hyperactivity, frequent mood swings, inability to concentrate). There are, however, no studies of the mediator release test reported in the peer-reviewed published medical literature that demonstrate improvements in clinical outcomes by incorporating the mediator release test and associated dietary modifications into the clinical management of patients with these conditions.
Thus, the mediator release test is considered experimental and investigational. Eosinophil Cationic Protein Eosinophil cationic protein (ECP) is an eosinophil-specific mediator that can be measured in bodily fluids to estimate the extent of eosinophil activation, although it provides no information about the presence of IgE-mediated allergy. This test requires further characterization before it can be recommended for routine clinical use.
Anti-IgE and Anti-Fc Epsilon Receptor Antibodies Anti-Fc epsilon receptor antibodies are natural antibodies against the alpha chain of the high-affinity receptor for IgE. Guidelines on urticaria from the British Association of Dermatologists (Grattan et al, 2007) stated that the presence of anti-Fc epsilon receptor antibodies indicates an autoimmune urticaria, but make no recommendation for testing for anti-Fc epsilon antibody in the work-up of patients with urticaria.
Saini (2010) stated that tests used in investigations of pathogenesis of chronic urticaria include the autologous serum and plasma skin tests, assays for autoantibodies directed against IgE or the FcepsilonRI receptor, and in vitro assessments of basophil function. However, these tests lack specificity and prognostic value for chronic urticaria, are not standardized, and can not be recommended for routine clinical use.
Clifford Materials Reactivity Testing According to Clifford Consulting Research Laboratories, Clifford materials reactivity testing (CMRT) is a laboratory screening process used to help identify sensitivity to various chemicals and compounds used in dental, orthopedic, or surgical implants, in order to select a product to which the patient exhibits the least sensitivity. The laboratory states that they report on more than 11,300 trade-named dental products and 94 chemical groups and families.
They state that they have also added an Orthopedic panel reporting on over 4,000 trade-named products for surgical applications. However, there is a lack of peer-reviewed published evidence of the clinical effectiveness of CMRT. Complement Antigen TestComplement Antigen Testing (Sage Medical Laboratories) has been used to identify delayed food allergies. However, there is insufficient evidence in the peer-reviewed published medical literuture for this approach.
Allergy ImmunotherapyThe treatment of allergy is approached 3 ways: (i) avoidance therapy, (ii) pharmacologic therapy, and (iii) immunotherapy. Complete avoidance of the known allergen responsible for inducing the signs and symptoms of the allergy is the most effective treatment for any allergic condition and results in a cure. When avoidance of a specific allergen such as house dust, molds or pollens is impossible, pharmacologic therapy is used (e.
g., antihistamines, adrenergic agonists, anticholinergics, beta-adrenergic agonists, corticosteroids, cromolyn sodium and methylxanthines). It has been advocated that the utilization of air cleaners, humidifiers, or dehumidifiers is helpful in reducing allergic irritant substances in the environment; however, research indicates that the use of these mechanical devices was ineffective in reducing clinical symptoms.
Allergy immunotherapy (also known as desensitization, hyposensitization, allergy injection therapy, or "allergy shots"), is indicated in patients whose triggering allergens are not readily avoidable, the allergy is IgE-mediated as documented by skin testing or RAST, the symptoms are not easily controlled with medication, the symptoms encompass more than one season and the patients are likely to cooperate in the program.
The severity, duration and frequency of episodes should be explored. Patients with life-threatening allergy (severe anaphylactic reaction) to hymenoptera (venom from bees, hornets, wasps or fire ants) have been shown to respond well to allergy immunotherapy, as well as patients with severe seasonal allergic rhinitis or conjunctivitis, perennial allergic rhinitis, allergic (extrinsic) asthma and mold induced allergic rhinitis.
Allergy immunotherapy will help desensitize the patient to the effects of the allergen. The documented allergy should correspond to the allergen planned for immunotherapy. A trial of systemic medications or avoidance of the allergens should be attempted. Two or more medications (antihistamines, steroids, bronchodilators, intranasal cromolyn) if not contraindicated should have been prescribed during the past year or the patient should be currently receiving immunotherapy.
Allergy immunotherapy is defined as the repeated administration of specific allergens to patients with IgE-mediated conditions, for the purpose of providing protection against the allergic symptoms and inflammatory reactions associated with natural exposure to these allergens. The exact mechanism of action is not known but may involve an increase in allergen-specific IgG antibodies, a decrease in IgE synthesis, and alteration in T-lymphocyte activity.
The principal and most effective route of allergen application is by subcutaneous injection. Oral/sublingual application of allergen extracts is discussed controversially in the literature (see provocation-neutralization therapy). There is a great assortment of different allergen extracts available, but only standardized extracts should be used. In the United States, the Food and Drug Administration (FDA) determined that the intracutaneous technique should be used for assigning standardized unitage (i.
e., bioequivalency allergy units [BAU]). Patients with allergic rhinitis and/or asthma from tree and grass pollens in the spring, ragweed pollen in the fall and year-round dust-mite sensitivity who have had inadequate response to acceptable symptomatic medication and allergen avoidance are excellent candidates for immunotherapy. Immunotherapy is recommended for patients with allergic asthma unresponsive to allergen avoidance, even when symptomatic relief can be achieved with drug therapy.
Treatment plans vary, but generally follow an initial dosing of short intervals (2 to 7 days) and should be increased 1.5 to 2 times with each injection if no reaction occurs. This dosing is followed by a maintenance dosage regimen at 3- or 4-week intervals and is determined by patient tolerance and relief of symptoms. Length of therapy varies from 3 to 5 years. The progress of the patient should be reviewed at regular intervals by the physician.
Progressive improvement may be observed over the first 2 to 3 years of treatment. Discontinuation of therapy may be considered any time after a 2 to 3 year trial. The risk of relapse must be weighed against patient preference for continuation of therapy. Examples of potential allergens for which immunotherapy is effective include: animal dander, animal feathers, animal fur, dust, grasses, insects, mites, molds, mushrooms, orris root, plants, pyrethrum, seeds, trees, vegetable gums, weeds, hymenoptera or stinging insects (bees, hornets, wasps, fire ants).
According to guidelines from the American Academy of Asthma, Allergy and Immunotherapy (Cox, et al., 2011), allergen immunotherapy should be administered in a medical facility with trained staff and medical equipment capable of recognizing and treating anaphylaxis. Under rare circumstances, when the benefit of allergen immunotherapy clearly outweighs the risk of withholding immunotherapy (eg, patients with a history of venom-induced anaphylaxis living in a remote region), at-home administration of allergen immunotherapy can be considered on an individual basis.
There are a limited number of studies of home-based allergy immunotherapy. The largest is a prospective study by Hurst, et al. (1999). During a 1-year period, 27 otolaryngic allergy practices recorded all systemic reactions to immunotherapy resulting from 635,600 patient visits and 1,144,000 injections. Sixty percent of injections were given at home. Major systemic reactions were observed after 0.005% of injections.
There were no hospitalizations or deaths. Eighty-seven percent of major reactions began within 20 minutes of injection. Frequently observed risk factors for major reactions were buildup phase of immunotherapy, active asthma, and first injection from a treatment vial. The authors reported that home and office injections had similar rates of total systemic reactions, but home-based immunotherapy had far fewer major reactions.
A major limitation of the study is that it was limited to otolaryngic allergy practices; the generalizability of the results to primary care practices is uncertain.There is no evidence that immunotherapy is beneficial for food allergy, migraine headaches, vasomotor rhinitis, intrinsic (non-allergic) asthma, or chronic urticaria. In addition, there is little evidence that immunotherapy benefits atopic dermatitis and angioedema.
The major risk factor of allergy immunotherapy is anaphylaxis. Immunotherapy should be administered under the supervision of an appropriately trained physician who can recognize early signs and symptoms of anaphylaxis and administer emergency medications if needed.A structured evidence-based assessment of sublingual immunotherapy for adults conducted by the BlueCross BlueShield Association Technology Evaluation Center (2003) concluded that “[w]hether [sublingual immunotherapy] improves health outcomes when compared with injection [allergen-specific immunotherapy] has not yet been demonstrated in the investigational setting.
It is uncertain whether FDA-licensed allergen preparations manufactured for allergy testing and injection [allergen-specific immunotherapy] are suitable for sublingual administration. Based on the above, use of sublingual immunotherapy for patients with allergies does not meet the TEC criteria.” Cox and colleagues (2006) stated that sublingual immunotherapy (SLIT) has been utilized with increasing frequency in Europe and is viewed with increasing interest by allergists in the United States.
To address this interest, a Joint Task Force of the American College of Allergy, Asthma and Immunology and the American Academy of Allergy, Asthma and Immunology's Immunotherapy and Allergy Diagnostic Committees evaluated the evidence on the effectiveness of SLIT. The task force concluded that despite clear evidence that SLIT is an effective treatment, there are still many unanswered questions, including effective dosage, treatment schedules, and overall duration of treatment.
Until these questions have been answered, an assessment of the cost/benefit ratio of the treatment cannot be made. Sublingual immunotherapy does seem to be associated with few severe side effects, but it has not been used in high-risk asthmatic patients, nor in the studies reviewed has it been used as a mixture of non-cross-reacting allergens. Furthermore, there is currently no allergy extract approved for this use in the United States, nor is there a Current Procedural Terminology code for billing purposes.
All of these factors should be considered before contemplating initiation of SLIT treatment for allergic patients. Nelson (2009) reviewed the literature on allergen immunotherapy for studies simultaneously using 2 or more distinct allergen extracts in either subcutaneous or sublingual immunotherapy. A total of 13 studies were identified, subcutaneous injections (n = 11), sublingual administration (n = 1), and both (n = 1).
In studies with adequate information, administration of 2 extracts by means of either subcutaneous immunotherapy or sublingual immunotherapy was effective. In studies using multiple allergens, 3 studies showed clear efficacy, whereas in the other 2 studies, lack of efficacy might have been due to inadequate doses of extract or omission of clinically relevant allergens in the treatment regimen.
The author concluded that simultaneous administration of more than 1 allergen extract is clinically effective. However, more studies are needed, particularly with more than 2 allergen extracts and with sublingual administration.Hoeks et al (2008) examined the evidence of the safety and effectiveness of SLIT as a curative therapy for allergies in children. All randomized, double-blind and placebo-controlled studies (DBRPCT's) on SLIT in asthma or rhinoconjunctivitis in children were selected from Medline, Embase and Cochrane Central Register of Controlled Trials.
Also references of the found articles were used. The selected studies were assessed for quality and the different outcomes were evaluated. A total of 13 DBRPCT's on SLIT in children were selected, 5 studies on children with house dust mite allergy and 8 studies on children with grass pollen allergy. There was considerable heterogeneity among the different studies with respect to the choice and definition of outcome criteria.
The quality of the included studies was moderate. After treatment with SLIT, especially reported symptoms decreased without improvement of objective parameters. Positive results originated especially from significant differences within the intervention group before and after treatment. These investigators concluded that it was impossible to substantiate the claim of authors of the studies regarding the favorable effects of SLIT in children with asthma or rhinoconjunctivitis, since all studies had serious methodological flaws.
However, the studies showed that SLIT seems to be safe in children in the doses applied. This is in agreement with the findings of Roder et al (2008) who reported that there is currently insufficient evidence that immunotherapy in any administration form has a positive effect on symptoms and/or medication use in children and adolescents with allergic rhinoconjunctivitis.In a randomized, double-blind, placebo-controlled study, Severino et al (2008) evaluated if SLIT might potentially be beneficial in hymenoptera (honeybee)allergy.
The sting challenge in large local reactions (LLRs) was used to test this hypothesis. After the baseline sting challenge, subjects were randomized to either SLIT or placebo for 6 months. The treatment involved a 6-week build-up period, followed by maintenance with 525 microg of venom monthly. The sting challenge was repeated after 6 months. A total of 30 patients (18 males; mean age of 44.
5 years) were enrolled, and 26 completed the study, with 1 dropout in the active group and 3 dropouts in the placebo group. In the active group the median of the peak maximal diameter of the LLRs decreased from 20.5 to 8.5 cm (p = 0.014), whereas no change was seen in the placebo group (23.0 versus 20.5 cm, p = not significant). The diameter was reduced more than 50 % in 57 % of patients. One case of generalized urticaria occurred in a placebo-treated patient at sting challenge.
No adverse event caused by SLIT was reported. The authors concluded that honeybee SLIT significantly reduced the extent of LLRs, and its safety profile was good. Although LLRs are not an indication for immunotherapy, this proof-of-concept study suggested that SLIT in hymenoptera allergy deserves further investigation. Trials involving systemic reactions and dose-ranging studies are needed.
Skoner and colleagues (2010) examined the maintenance dose range of sublingual standardized glycerinated short ragweed pollen extract in adults with ragweed-induced rhinoconjunctivitis. A total of 115 patients with ragweed-induced rhino-conjunctivitis were randomly allocated to placebo (n = 40), medium-dose extract (4.8 microg Amb a 1/d; n = 39), or high-dose extract (48 microg Amb a 1/d; n = 36).
In a 1-day (rush) dose-escalation regimen, ragweed pollen extract was administered sublingually in incremental doses until maximum tolerable or scheduled dose was reached and then maintained during the ragweed pollen season. Patient diaries were used to monitor nasal and ocular symptoms and medication. The primary endpoint was symptom score. Both active treatment groups achieved a 15 % reduction in total rhino-conjunctivitis symptom scores compared with placebo during the entire ragweed pollen season, but the difference was not statistically significant (p > 0.
10). However, in an analysis of co-variance correcting for pre-seasonal symptoms, both mean daily symptom scores (0.19 +/- 1.16 versus 1.00 +/- 2.30) and medication scores (0.0003 +/- 1.64 versus 0.63 +/- 1.06) for the entire pollen season were significantly reduced in the high-dose versus placebo groups, respectively (p < or = 0.05). Ragweed-specific IgG, IgG(4), and IgA antibodies were increased after treatment in the medium- and high-dose groups and not the placebo group.
Frequency of adverse events was similar between the placebo and treatment groups, but oral-mucosal adverse events occurred more often with treatment. The authors concluded that standardized glycerinated short ragweed pollen extract administered sublingually at maintenance doses of 4.8 to 48 microg Amb a 1/d was safe and can induce favorable clinical and immunologic changes in ragweed-sensitive subjects.
However, the authors noted that additional trials are needed to establish efficacy.Sieber et al (2010) compared the effectiveness of perennial and co-seasonal high-dose SLIT treatments as well as ultra-rush and classical titrations in a real-world setting for pollen allergens. An individual patient data (IPD) meta-analysis was performed of 3 open, prospective observational studies on high-dose SLIT using IR-standardized allergen extracts in patients with allergic rhinitis with and without asthma.
A total of 1,052 patients aged 24.9 years (mean) were treated with SLIT and included in this IPD meta-analysis. Individual studies and total data pool analyses revealed consistent improvements in rhino-conjunctivitis symptom scores. Stratified analyses revealed consistent improvements in symptomatic score and medication score regardless of the type of sensitization and type of treatment. Ultra-rush titration resulted in considerably more pronounced improvement in symptom scores than classical titration, possibly due to better compliance of patients receiving that supervised titration.
Adverse events occurred in 24 % of patients during titration and in 18 % of patients during maintenance treatment. The vast majority of events (89 % and 87 %) were mild-to-moderate, predominantly local symptoms in the oral cavity. There were no differences detected between the study titration or treatment schedules. No serious adverse reactions were reported. Nearly all patients (88 %) decided to continue SLIT after completion of the studies.
High-dose SLIT with seasonal allergens given as co-seasonal or perennial treatment appears to be effective and well-tolerated in daily medical practice. Improved compliance under ultra-rush titration and seasonal SLIT treatment may further enhance effectiveness. The authors stated that randomized controlled trials are needed for the further evaluation of these findings. Lin and colleagues (2013) systematically reviewed the safety and effectiveness of aqueous sublingual immunotherapy for allergic rhino-conjunctivitis and asthma.
The databases of MEDLINE, EMBASE, LILACS, and the Cochrane Central Register of Controlled Trials were searched through December 22, 2012. English-language RCTs were included if they compared sublingual immunotherapy with placebo, pharmacotherapy, or other sublingual immunotherapy regimens and reported clinical outcomes. Studies of sublingual immunotherapy that are unavailable in the U.S. and for which a related immunotherapy is unavailable in the U.
S. were excluded. Paired reviewers selected articles and extracted the data. The strength of the evidence for each comparison and outcome was graded based on the risk of bias (scored on allocation, concealment of intervention, incomplete data, sponsor company involvement, and other bias), consistency, magnitude of effect, and the directness of the evidence. A total of 63 studies with 5,131 participants met the inclusion criteria.
Participants' ages ranged from 4 to 74 years; 20 studies (n = 1,814 patients) enrolled only children. The risk of bias was medium in 43 studies (68 %). Strong evidence supports that sublingual immunotherapy improves asthma symptoms, with 8 of 13 studies reporting greater than 40 % improvement versus the comparator. Moderate evidence supports that sublingual immunotherapy use decreases rhinitis or rhino-conjunctivitis symptoms, with 9 of 36 studies demonstrating greater than 40 % improvement versus the comparator.
Medication use for asthma and allergies decreased by more than 40 % in 16 of 41 studies of sublingual immunotherapy with moderate grade evidence. Moderate evidence supports that sublingual immunotherapy improves conjunctivitis symptoms (13 studies), combined symptom and medication scores (20 studies), and disease-specific quality of life (8 studies). Local reactions were frequent, but anaphylaxis was not reported.
The authors concluded that the overall evidence provided a moderate grade level of evidence to support the effectiveness of sublingual immunotherapy for the treatment of allergic rhinitis and asthma, but high-quality studies are still needed to answer questions regarding optimal dosing strategies. There were limitations in the standardization of adverse events reporting, but no life-threatening adverse events were noted in this review.
In an editorial that accompanied the afore-mentioned study, Nelson (2013) stated that “[A]lthough patients may prefer a therapy that is relatively safe and can be administered at home, FDA approval has not been granted yet, and many unanswered questions remain about the use of sublingual immunotherapy”. The National Institute of Allergy and Infectious Diseases' guidelines for the diagnosis and management of food allergy (Boyce et al, 2010) stated that (i) the expert panel does not recommend using allergen-specific immunotherapy to treat IgE-mediated food allergy (Rationale: Allergen-specific immunotherapy improves clinical symptoms of FA while on treatment.
However, it is currently difficult to draw conclusions on the safety of such an approach and whether clinical tolerance [i.e., improvement in clinical symptoms that persists even after allergen-specific immunotherapy is discontinued] will develop with long-term treatment). Allergen-specific immunotherapy can improve clinical symptoms of food allergy for some patients. However, additional safety and efficacy data are needed before such treatment can be recommended.
Because of the risk of severe reactions, the approach should only be used in highly controlled settings, and (ii) the expert panel does not recommend immunotherapy with cross-reactive allergens for treating IgE-mediated food allergy (Rationale: Although some evidence exists to suggest that specific immunotherapy with cross-reactive allergens is beneficial in treating food allergy, additional safety and efficacy data are needed before such treatment can be recommended).
It has been hypothesized that immunotherapy with cross-reactive antigens could benefit patients with food allergy, yet the safety of this approach has been evaluated in a highly controlled setting in only 1 study to date. Replication of these findings with additional safety and efficacy data in clinical practice settings is needed. de Bot et al (2011) evaluated the quality of systematic reviews and meta-analyses of SLIT for allergic rhinitis in children, published since 2000.
Eligible reviews were identified by searching Medline/PubMed, Embase, and the Cochrane Library, from 2000 through 2008. Methodological quality was assessed using the assessment of multiple systematic reviews instrument. A total of 10 systematic reviews were included, 1 of which was published in the Cochrane Library. Eight reviews gave some details about the search strategy. None of the reviews included measures to avoid selection bias.
In 60 % of the reviews, the methodological quality of the included studies was (partly) assessed. Four reviews pooled the results of individual studies, neglecting clinical heterogeneity. Three of the 10 reviews provided information about sources of funding or grants from industry. Of the 10 reviews, the 6 reviews with the highest overall score scored 5 to 8 points, indicating moderate quality.
The authors concluded that systematic reviews are useful to evaluate the efficacy of SLIT in children. Although more reviews have become available, the methodological quality could be improved. They stated that SLIT for children could be promising, but methodological flaws in the reviews and individual studies are too serious to draw definite conclusions. In a Cochrane review, Calderon et al (2011) evaluated the effectiveness of SLIT compared with placebo for reductions in ocular symptoms, topical ocular medication requirements and conjunctival immediate allergen sensitivity.
These investigators searched CENTRAL (which contains the Cochrane Eyes and Vision Group Trials Register) (The Cochrane Library 2011, Issue 1), MEDLINE (January 1950 to January 2011), EMBASE (January 1980 to January 2011), Latin American and Caribbean Literature on Health Sciences (LILACS) (January 1982 to January 2011), Web of Science (January 1970 to January 2011), Biosis Previews, (January 1979 to January 2011), the metaRegister of Controlled Trials (mRCT) (www.
controlled-trials.comuyftcvyuffwtzvdutrywwxtx) (January 2011), ClinicalTrials.gov (www.clinicaltrials.gov) (January 2011), the Australian New Zealand Clinical Trials Registry (ANZCTR) (www.actr.org.au) (July 2010), SCOPUS (November 2008) and the UK Clinical Trials Gateway (January 2010). There were no language or date restrictions in the search for trials. All electronic databases except for SCOPUS, the UK Clinical Trials Gateway and ANZCTR were last searched on 19 January 2011.
Randomized controlled trials (RCTs), double-masked and placebo controlled, which evaluated the efficacy of SLIT in patients with symptoms of allergic rhino-conjunctivitis (ARC) or allergic conjunctivitis (AC) were included in this analysis. The primary outcome was the total ocular symptom scores. Secondary endpoints included individual ocular symptom scores (such as itchy eyes, red eyes, watery eyes, swollen eyes), ocular medication scores (eye drops) and conjunctival immediate allergen sensitivity (CIAS).
Data were analyzed and reported as standardized mean differences (SMDs) using Review Manager software. A total of 42 trials (n = 3,958 total participants; n = 2,011 SLIT and n = 1,947 placebo) had available data to evaluate the efficacy of SLIT on AC and were included in the meta-analyses. Heterogeneity among studies (I(2) statistic) was around 50 % or below for all endpoints. Sublingual immunotherapy induced a significant reduction in both total ocular symptom scores (SMD -0.
41; 95 % confidence interval [CI]: -0.53 to -0.28; p < 0.00001; I(2) = 59%) and individual ocular symptom scores for red eyes (SMD -0.33; 95 % CI: -0.45 to -0.22; p < 0.00001; I(2) = 27 %), itchy eyes (SMD -0.31; 95 % CI: -0.42 to -0.20; p < 0.00001; I(2) = 46 %) and watery eyes (SMD -0.23; 95 % CI: -0.34 to -0.11; p < 0.0001; I(2) = 42 %) compared to placebo. Those participants having active treatment showed an increase in the threshold dose for the conjunctival allergen provocation test (SMD 0.
35; 95 % CI: 0.00 to 0.69; p = 0.05; I(2) = 43 %). No significant reduction was observed in ocular eye drops use (SMD -0.10; 95 % CI: -0.22 to 0.03; p = 0.13; I(2) = 34 %). The authors concluded that overall, SLIT is moderately effective in reducing total and individual ocular symptom scores in participants with ARC and AC. There were however some concerns about the overall quality of the evidence-base, this relating to inadequate descriptions of allocation concealment in some studies, statistical heterogeneity and the possibility of publication bias.
They stated that there is a need for further large rigorously designed studies that examine long-term effectiveness after discontinuation of treatment and establish the cost-effectiveness of SLIT.Allergoids Allergoids are formalin treated allergens which have been shown to be as effective as conventional aqueous extracts and superior to placebo in terms of reduction of symptom medication scores, production of an increase in ragweed IgG levels, and a decrease in seasonal rise in ragweed IgE levels.
Allergoids are licensed and manufactured for general distribution in Europe, but not yet in the United States.Enzyme Potentiated Desensitization (EPD)Enzyme potentiated desensitization is patented in Europe under the brand name of Epidyme. This immunotherapy consists of a mixture of allergens to molds, grass, weeds, trees, dust mites, dog and cat dander, and house dust. These allergens are administered in the doctor's office.
While this is common practice in Europe, it is not on the United States market or regulated/approved by the U.S. FDA. The FDA has banned importation of EPD. There is a lack of clinical trials supporting the efficacy of this product.A variant of enzyme potentiated desensitization is ultra low dose enzyme activated immunotherapy (also known as low dose allergens or LDA), which has been described as a method of immunotherapy enhanced by a minute dose of the enzyme, beta glucuronidase.
According to proponents, the beta glucuronidase activates extremely miniscule doses of various allergens and stimulates the production of T-suppressor cells. The T-suppressor cells, in turn, down regulate the T-helper cells that are causing allergic symptoms by misidentifying normal substances in the body as allergens. LDA uses the same active components as EPD, but utilizes more more pollens, foods and other allergens.
Photo-InactivationPhoto-inactivation of an antigen with ultraviolet may allow larger doses of antigen to be administered with fewer adverse effects. Currently, these preparations are used for research purposes only and not in clinical practice.Polymerized Ragweed ExtractPolymerized ragweed extract has been employed for treatment of ragweed hay fever in placebo-controlled trials and has been shown to produce a significant decrease in symptoms and medication scores.
However, polymerized ragweed extracts have not yet been licensed or manufactured for general distribution in the United States.RhinophototherapyPhototherapy has a profound immunosuppressive effect and is able to inhibit hypersensitive reactions in the skin. Leimgruber (2006) stated that phototherapy applied inside the nose (rhinophototherapy) is among new therapeutic options being developed for allergic rhinitis to counteract its impact on quality of life and health costs.
The author noted that the immunosuppressive effect of phototherapy has been tested in nasal mucosa. This application has shown anti-inflammatory results in nasal cleaning fluid and, consequently, may reduce allergic rhinitis. The author noted that long-term studies involving large cohorts of patients are needed if rhinophototherapy is going to be prescribed without restrictions.In a randomized, double-blind study (n = 49), Koreck et al (2005) examined if phototherapy using a combination of UVB (5 %), UVA (25 %), and visible light (70 %), referred to as mUV/VIS (rhinophototherapy), is effective in treating allergic rhinitis.
The study was carried out during the ragweed season. Each intra-nasal cavity was illuminated 3 times a week for 3 weeks with mUV/VIS or with low-intensity visible light (control group). Symptom scores, inflammatory cells, and their mediators were assessed in nasal lavages. In vitro effects of mUV/VIS irradiation on T-cell and eosinophil apoptosis, and its inhibitory effect on mediator release from basophils were examined.
Rhinophototherapy was well-tolerated, and resulted in a significant improvement of clinical symptoms for sneezing (p < 0.016), rhinorrhea (p < 0.007), nasal itching (p < 0.014), and total nasal score (p < 0.004). None of the scores improved significantly in the control group. The investigators reported that scores for nasal obstruction slightly improved after rhinophototherapy and significantly increased in the control group (p < 0.
017). In the nasal lavage, rhinophototherapy significantly reduced the number of eosinophils and the level of eosinophil cationic protein and IL-5. In vitro irradiation of T-cells and eosinophils with rhinophototherapy dose-dependently induced apoptosis. In addition, rhinophototherapy inhibited the mediator release from RBL-2H3 basophils. These promising results would need to be replicated in a larger clinical trial with longer-term follow-up.
The Helminth Trichuris Suis TherapyIn a double-blind, placebo-controlled, parallel group study, Bager et al (2010) ascertained the effectiveness of helminth Trichuris suis therapy for the treatment of allergic rhinitis. A total of 100 subjects aged 18 to 65 years with grass pollen-induced allergic rhinitis were randomly assigned to ingest a total of 8 doses with 2,500 live Trichuris suis ova or placebo with an interval of 21 days.
The primary outcome was a change in mean daily total symptom score for runny, itchy, sneezing nose (maximum change, 9.0) or in percentage of well days during the grass pollen season. Treatment with Trichuris suis ova (n = 49) compared with placebo (n = 47) caused transient diarrhea peaking at day 41 in 33 % of participants (placebo, 2 %), and increased eosinophil counts (p < 0.001) and Trichuris suis-specific IgE (p < 0.
05), IgG (p < 0.001), IgG(4) (p < 0.003), and IgA (p < 0.001), whereas there was no significant change in symptom scores (0.0; 95 % confidence interval [CI]: -0.5 to 0.4; p = 0.87), well days (3 %; 95 % CI: -9 % to 14 %; p = 0.63), total histamine (p = 0.44), grass-specific IgE (p = 0.76), or diameter of wheal reaction on skin prick testing with grass (p = 0.85) or 9 other allergens. The authors concluded that repeated treatment with the helminth Trichuris suis induced a substantial clinical and immunologic response as evidence of infection, but had no therapeutic effect on allergic rhinitis.
Title: Elisa Act Allergy Test