Antibody Reactions Precipitation Reactions

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Antibody Reactions Precipitation Reactions

Immunological testing Perspective 17.1 Monoclonal Antibodies • Monoclonal antibodies • Serology • Quantifying antigen – antibody reactions 17-1 17-2 Serology Quantifying antigen – antibody reactions • Antibodies • Seroconversion or rise in titer • Antibodies detect and identify antigens • Serial dilutions 17-3 17-4 Precipitation reactions • Immunodiffusion • Immunoelectrophoresis Figure 17.2 - Quantitation of immunologic tests 17-5 17-6 1 Figure 17.4 Immunodiffusion Figure 17.3 Precipitation reaction 17-7 17-8 Agglutination reactions • Direct agglutination • Indirect agglutination • Hemagglutination Figure 17.6 Immunoelectrophoresis 17-9 17-10 Direct agglutination Indirect agglutination • Cross – linking and lattice formation • Soluble antigen is coated onto particles (red bood cells, • Antibodies react with particulate antigens (red blood latex beads) cells, bacteria, fungi) • Allow for visible clumps (agglutination) • Visible clumps • Estimate amount of antibody 17-11 17-12 2 Immunofluorescence tests • Direct fluorescent antibody test • Indirect fluorescent antibody test Figure 17.7 - Agglutination reaction 17-13 17-14 Figure 17.8 - Direct and indirect fluorescent antibody test Antigen – antibody assays • Radioimmunoassay (RIA) • Enzyme – linked immunosorbant assay (ELISA) • Western blot 17-15 17-16 Enzyme – linked immunosorbant assay Radioimmunoassay (RIA) (ELISA) • Competitive inhibition assay • Color reaction assay • Measure antigen or antibody • Indirect ELISA • Ex. Measure small amounts of hormones or drugs in a • Direct ELISA clinical sample • Ex. Measure small amounts of IgE antibody (radioallergosorbent test) 17-17 17-18 3 Figure 17.9 - Indirect ELISA Figure 17.1 - Direct ELISA 17-19 17-20 Figure 17.1 Complement fixation test Western blot • Measures the binding of complement by an antigen – antibody interaction • Indicator system determine positive or negative reactions 17-21 17-22 Figure 17.1 Complement Neutralization test fixation test • Antibody bind to specific antigen (virus, toxin) • Antibody – antigen complex prevents antigen from binding (neutralization) 17-23 17-24 4 Cellular immunology test • Identification of subsets of lymphocytes • Lymphocyte response to mitogens • Cytoxic T – cell function • Cell – mediated immunity to infectious agents 17-25 5.

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Food Allergy Outline
Allergy Evaluation-What it all Means & Role of Allergist Sai R. Nimmagadda, M.D.. Associated Allergists and Asthma Specialists Ltd. Clinical Assistant Professor Of Pediatrics Northwestern University Chicago, Illinois Objectives of Presentation • Discuss the different options for allergy evaluation. – Skin tests – Immunocap Testing • Understand the results of Allergy testing in various allergic diseases. • Briefly Understand what an Allergist Does Common Allergic Diseases Seen in the Primary Care Office • Atopic Dermatitis/Eczema • Food Allergy • Allergic Rhinitis • Allergic Asthma • Allergic GI Diseases Factors that Influence Allergies Development and Expression Host Factors Environmental Factors . Genetic Indoor allergens - Atopy Outdoor allergens - Airway hyper Occupational sensitizers responsiveness Tobacco smoke . Gender Air Pollution . Obesity Respiratory Infections Diet © Global Initiative for Asthma Why Perform Allergy Testing? – Confirm Allergens and answer specific questions. • Am I allergic to my dog? • Do I have a milk allergy? • Have I outgrown my allergy? • Do I need medications? • Am I penicillin allergic? • Do I have a bee sting allergy Tests Performed in the Diagnostic Allergy Laboratory • Allergen-specific IgE (over 200 allergen extracts) – Pollen (weeds, grasses, trees), – Epidermal, dust mites, molds, – Foods, – Venoms, – Drugs, – Occupational allergens (e.g., natural rubber latex) • Total Serum IgE (anti-IgE; ABPA) • Multi-allergen screen for IgE antibody Diagnostic Allergy Testing Serological Confirmation of Sensitization History of RAST Testing • RAST (radioallergosorbent test) invented and marketed in 1974 • The suspected allergen is bound to an insoluble material and the patient's serum is added • If the serum contains antibodies to the allergen, those antibodies will bind to the allergen • Radiolabeled anti-human IgE antibody is added where it binds to those IgE antibodies already bound to the insoluble material • The unbound anti-human IgE antibodies are washed away.
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Medicare Local Coverage Determination Policy Allergy Testing RAST Type Tests CPT: 86003 Medically Supportive CMS Policy for Kentucky and Ohio ICD Codes are listed Local policies are determined by the performing test location. This is determined by the state on subsequent page(s) in which your performing laboratory resides and where your testing is commonly performed. of this document. Coverage Indications, Limitations, and/or Medical Necessity Radioallergosorbent test (RAST), fluoroallergosorbent test (FAST), and multiple antigen simultaneous tests are in vitro techniques for Add full policy information determining whether a patient's serum contains IgE antibodies against specific allergens of clinical importance. As with any allergy testing, the need for such tests is based on the findings during a complete history and physical examination of the patient. The multiple antigen simultaneous testing technique is similar to the RAST/FAST techniques in that it depends upon the existence of Template structure: allergic antibodies in the blood of the patient being tested. With the multiple antigen simultaneous test system, several antigens may be used to test for specific IgE simultaneously. First level is for headers such as limitations, ELISA (enzyme-linked immunosorbent assay) is another in vitro method of allergy testing for specific IgE antibodies against allergens. indications and usage guidelines This method is also a variation of RAST. Second level is for main body copy Limitations • Third level is for bullet (if needed) The following tests are considered to be not medically necessary and will be denied. • ELISA/Act qualitative antibody testing. This testing is used to determine in vitro reaction to various foods and relies on lymphocyte blastogenesis in response to certain food antigens.
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Radioallergosorbent Test (RAST)—Reliable Tool Or Poor Substitute?
Radioallergosorbent test (RAST) VII Ö1 1 reliable tool or poor substitute? Edward W. Hein, M.D. An in vitro method, the radioallergosorbent test (RAST) has been developed for the detection of allergen-specific antibodies of the IgE class. Review of the literature shows that in comparison to skin testing, the RAST has a high degree of correlation (60% to 90% depending on the antigen); however, this method is not as sensitive as other tests (50% false-negative). The RAST is affected by blocking antibodies (IgG), resulting in false-negative values and high levels of IgE that bind on the allergen discs, giving false- positive findings. Because of these problems, RAST is somewhat limited for use in the clinical setting. Index terms: Allergy and immunology • Radioallergosor- bent test (RAST) Cleve Clin Q 50:361-366, Fall 1983 Skin testing has been the traditional method for diagnosis of IgE-mediated allergic disorders. This bioassay is highly sensitive, cost-effective, and safe when used by experienced personnel. In 1967, Wide et al,1 in Sweden, reported a new technique capable of detecting the minute quantities of allergen-specific IgE antibodies that circulate in the serum of allergic patients. This laboratory procedure, called RAST (radioallergosorbent test), utilized a solid- phase radioimmunoassay method. During the last decade this in vitro test has been refined and is now a commercially 1 Departments of Pediatric and Adolescent Medi- available laboratory test for clinical laboratories and, in kit cine, and Allergy and Immunology, The Cleveland form, for physicians' offices. Proponents of this new Clinic Foundation. (E.W.H., Head, Section Pediatric method claim that its results are more objective, safer for Allergy and Clinical Immunology.) Submitted for pub- lication May 1983; accepted June 1983.
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Hypersensitivity Reactions (Types I, II, III, IV)
Hypersensitivity Reactions (Types I, II, III, IV) April 15, 2009 Inflammatory response - local, eliminates antigen without extensively damaging the host’s tissue. Hypersensitivity - immune & inflammatory responses that are harmful to the host (von Pirquet, 1906) - Type I Produce effector molecules Capable of ingesting foreign Particles Association with parasite infection Modified from Abbas, Lichtman & Pillai, Table 19-1 Type I hypersensitivity response IgE VH V L Cε1 CL Binds to mast cell Normal serum level = 0.0003 mg/ml Binds Fc region of IgE Link Intracellular signal trans. Initiation of degranulation Larche et al. Nat. Rev. Immunol 6:761-771, 2006 Abbas, Lichtman & Pillai,19-8 Factors in the development of allergic diseases • Geographical distribution • Environmental factors - climate, air pollution, socioeconomic status • Genetic risk factors • “Hygiene hypothesis” – Older siblings, day care – Exposure to certain foods, farm animals – Exposure to antibiotics during infancy • Cytokine milieu Adapted from Bach, JF. N Engl J Med 347:911, 2002. Upham & Holt. Curr Opin Allergy Clin Immunol 5:167, 2005 Also: Papadopoulos and Kalobatsou. Curr Op Allergy Clin Immunol 7:91-95, 2007 IgE-mediated diseases in humans • Systemic (anaphylactic shock) •Asthma – Classification by immunopathological phenotype can be used to determine management strategies • Hay fever (allergic rhinitis) • Allergic conjunctivitis • Skin reactions • Food allergies Diseases in Humans (I) • Systemic anaphylaxis - potentially fatal - due to food ingestion (eggs, shellfish,
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1 Bahrain Medical Bulletin, Vol. 30, No. 3, September 2008 Incidence of Allergen Specific IgE in Atopic Dermatitis Patients by Immunoblot Assay Abbas H Alsaeed M.Med, Sci, PhD, CLC* Objective: To determine the incidence of allergens specific IgE by immunoblot assay in atopic dermatitis patients. Design: Prospective study. Setting: South Riyadh, Dermatology clinic Alfalah International Hospital, Saudi Arabia. Method: Sixty-five adult patients and twenty volunteer controls sera were collected from atopic dermatitis patients. IgE antibodies specific to allergens of food, pollens, animal epithelia, house dust mites, and moulds were determined using an immunoblot assay. Result: The sensitization rate of all allergens was found to be 65% (42/65; 26 males, 39 females). The subjects were most commonly allergic to pollen (35.2%), food (31.5%) and animal epithelial dander (21.0%). House dust mite (11.4%) and mould (0.9%) were less frequent than the others were. Conclusion: The result indicates that pollen allergens, food allergens, animal epithelia and dander should be taken into account as possible exacerbating factors in adult patients with AD within the specific geographical area studied. Bahrain Med Bull 2008; 30(3): Atopic dermatitis (AD) is a rather common disorder as its name implies, it is often associated with the presence of IgE-mediated hypersensitivity and with other IgE–mediated disease states. Often diagnosed in infancy it can have its onset at any age in life. There is no definitive laboratory test which is able to define atopic dermatitis. Double-blind placebo controlled food challenges have documented that certain foods can exacerbate dermatitis in patients in whom there is immediate skin test reactivity to food extracts1.
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In Vitro Diagnostic Testing for Antibiotic Allergy Inmaculada Doña,1 Maria J
Review Allergy Asthma Immunol Res. 2017 July;9(4):288-298. https://doi.org/10.4168/aair.2017.9.4.288 pISSN 2092-7355 • eISSN 2092-7363 In Vitro Diagnostic Testing for Antibiotic Allergy Inmaculada Doña,1 Maria J. Torres,1,3 Maria I. Montañez,2,3* Tahía D. Fernández2* 1Allergy Unit, IBIMA-Regional University Hospital of Malaga-UMA, Málaga, Spain 2Research Laboratory, IBIMA-Regional University Hospital of Malaga-UMA, Málaga, Spain 3Andalusian Center for Nanomedicine and Biotechnology-BIONAND, Málaga, Spain This is an Open Access article distributed under the terms of the Creative Commons Attribution NonCommercial License (http://creativecommons.org/licenses/bync/4.0/) which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited. Allergy to antibiotics is an important worldwide problem, with an estimated prevalence of up to 10% of the population. Reaction patterns for differ- ent antibiotics have changed in accordance with consumption trends. Most of the allergic reactions to antibiotics have been reported for betalac- tams, followed by quinolones and macrolides and, to a lesser extent, to others, such as metronidazole clindamycin and sulfonamides. The diagnos- tic procedure includes a detailed clinical history, which is not always possible and can be unreliable. This is usually followed by in vivo, skin, and drug provocation tests. These are not recommended for severe, potentially lifethreaten reactions or for drugs that are known to produce a high rate of false positive results. Given the limitations of in vivo tests, in vitro test can be helpful for diagnosis, despite having suboptimal sensitivity. The most highly employed techniques for diagnosing immediate reactions to antibiotics are immunoassays and basophil activation tests, while lympho- cyte transformation tests are more commonly used to diagnose non-immediate reactions.
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