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ELISA Enzyme Linked Immune Sorbent Assay in basic science and clinical practice ELISA plate well Different plastic coatings for different materials enzyme linked immune sorbent enzyme Antibody conjugated with enzyme Antigen/antibody adsorbed to solid surface ENZYME ACTIVITY IN ELISA IS DIRECTLY PROPORTIONAL TO THE AMOUNT OF ANTIGEN PRESENT Enzyme activity is measured by the color reaction due to conversion of substrate Similar principle applies to many other antibody-based detection methods BASIC SETUPS IN ELISA / IMMUNOHISTOCHEMISTRY / FLOW CYTOMETRY Direct method Indirect method Label Label Secondary antibodies Primary antibodies Antigen BASIC SETUPS IN ELISA / IMMUNOHISTOCHEMISTRY / FLOW CYTOMETRY Enzyme/anti-enzyme system PAP – peroxidase / anti-peroxidase APAAP – alkaline phosphatase / anti- alkaline phosphatase Enzyme Enzyme-specific antibody, same isotype as the primary antibody Primary antibody Antigen Secondary antibody BASIC SETUPS IN ELISA / IMMUNOHISTOCHEMISTRY / FLOW CYTOMETRY Indirect systems combined with biotin-avidin signal amplification (Avidin binds biotin with very high affinity ) Basic Avidin-enzyme complexes ABC Avidin-biotin enzyme complexes Biotin-enzyme complex Avidin Biotinylated antibody Antigen STEPS OF BASIC INDIRECT ELISA Detection of antigen specific antibody Adsorption of antigen (coating) Removal of excess antibody Removal of excess antigen Saturation of uncovered surface area with proteins Addition of Ag-specific antibodies Addition of Secondary Ab conjugated with enzyme Addition of chromogenic substrate Removal of excess protein EXAMPLES FOR DIRECT AND INDIRECT ELISAs TESTING VIRAL INFECTION Viral antigens cannot be detected efficiently in lot of case (latency), but you can efficiently detect the antibodies that were produced in response to the viral infection. These antibodies can serve as diagnostic markers. Human Ig specific labeled antibodies indicate the presence of the virus specific antibodies. The serum of the infected person with virus specific antibodies. The antibodies could bind the virus antigens. viral antigen precoated test plate EXAMPLES: • Epstein-Barr Virus (EBV) test kit → ELISA method for the qualitative detection of IgG antibody to Epstein-Barr Virus nuclear antigen-1 (EBNA-1) in human serum • HIV assay kit → enzyme-linked immunosorbent assay for the detection of antibody to HIV-1 in serum, plasma or dried blood spots •Toxoplasmosis (Toxoplazma gondii – parasitic protozoa) → chromatographic immunoassay for the qualitative detection of human IgM antibodies against Toxoplasma in serum or plasma AUTOIMMUNITY Autoantibodies from different autoimmune diseases can be detected similarly. In 60% of SLE patients autoantibodies can be detected against double strand DNA. Anti-ENA antibodies can be seen in many systemic AIDs. Type I (autoimmune) diabetes can be detected by the presence of antibodies against islet-cell specific antigens. Glutamic acid decarboxylase (GAD65), specific antibodies have been found in 70-90% of prediabetic and Type 1 diabetic patients (including approximately 7-10% of adult onset diabetics with Type 1 diabetes) IA-2 (a tyrosine phosphatase-like protein) specific Ab. are found in 50-75% of Type 1 diabetic patients at and prior to disease onset, are generally more prevalent in younger patients, and are associated with rapid progression to overt disease. These autoantibodies have been detected in some ICA positive/GAD Ab negative patients, and therefore can be considered independent markers of disease. Insulin: anti-insulin ABs are found predominantly, though not exclusively, in young children (<5 years) developing Type 1 diabetes. In insulin-naive (untreated) patients, the prevalence of autoantibodies to insulin is almost 100% in very young individuals and almost absent in patients with adult onset of Type 1 diabetes. (It should be noted that insulin autoantibodies are indistinguishable from insulin antibodies that commonly develop with insulin therapy). DETERMINATION OF ANTIBODY ISOTYPE Sometimes the antigen specific antibodies indicate the presence of the antigen in the body (see the ”viral infection testing” part ) The isotypes of these antibodies additionally could reveal whether aan ongoing immune response is a primary (IgM dominance) or a or a secondary/memory response (IgG dominance). To determine the isotype of reacting antibodies, one should use isotype specific secondary antibodies. α-IgM α-IgG Memory response IgM IgG Antigen Increased IgE level can be seen in atopic allergy cases, and some autoimmune process, and in the case of some parasite infection Abnormal levels of serum immunoglobulin isotypes can be seen in the case of some immunodeficiencyies (e.g. hyper IgM syndrome, multiplex myeloma (case study!)) isotype specific antibody antibody from the serum antibody capture antibody STEPS OF COMBINED SANDWICH ELISA For antigens present at low concentration in complex biological samples Coating with Ag-specific „capture” antibody Blocking free plastic surface with inert protein Addition of antigencontaining solution Addition of biotinylated antibody specific to a different epitope on target protein Addition of avidinconjugated enzyme Addition of substrate Removal of excess enzyme Removal of unbound material Removal of unbound protein Removal of unbound material PRACTICAL USE OF IMMUNOASSAYS Detection of tumor antigens, cytokines, hormones doping/drug assay: EPO (erythropoietin), steroids hCG (human chorionic gonadotropin) – pregnancy test sandwich assays TUMORDIAGNOSTICS Tumor specific (TSA) and tumor associated (TAA) antigen recognizing antibodies can be used in diagnostics The antigens can be detected by sandwich techniques Tumor Ag specific detecting/reporter antibody (suplemented) Tumor antigen (patient serum) PROSTATE CANCER: Tumor Ag specific capture antibody precoated plate (as a part of the kit) Prostate-Specific Antigen (PSA) Human prostatic acid phosphatase (PAP) in human serum can be used similarly in quantitative measurement. Bladder cancer: NMP22 is a Nuclear Matrix Protein found in human epithelial cells. The majority of patients with bladder cancer release large quantities of NMP22 into their urine,. Thyroid Cancer : Increased Serum Thyroglobulin (sTG) can be detected by immunoassay Hepatocarcinoma Alpha Fetoprotein (AFP) is a 68 kDa protein, Its normal concentration in serum is below 9 ng/mL; higher concentrations are associated with hepatomas and ovarian cancer. Carcinoembryonic Antigen (CEA) is a glycoprotein involved in cell adhesion. It is normally produced during fetal development. serum from individuals with colorectal and other carcinomas had higher levels of CEA than healthy individuals and can be used to monitor the response to colon cancer treatment. ELISA methodical errors Aspecific adhesion of the materials The antibodies (as proteins generally) could also bind to the surface aspecifically - directly - indirectly the color is the same Aspecific adhesion of the proteins can be reduced by: •blocking of the surface •applying detergent Hook effect - It could happen in ”one step”, ”without washing” sandwich tests. Large antigen concentration could give invalid small value. Unwashed soluble antigens compete with the captured surface bound ones. It can be avoided by efficient washing steps, or with the dilution of the sample. ELISA PLATES - RESULTS (Chromogenic substrates can have different colors) Today’s Practical: Pregnancy test based on the principle of capture ELISA You may have met such diagnostic tools or you certainly are are going to meet them during your career Test is based on detection of human chorionic gonadotropin in serum or urine (pregnancy test) The principles of these tools are similar to the capture ELISA assay we discussed earlier. hCG Rapid One-Step Immunochromatographic Assay strip front view side view absorbtion pad (cellulose) control antibody lane (detection antibody capture) hCG capture antibody lane nitrocellulose membrane (signal detection pad) glass fiber membrane with visually labeled detection antibodies sample application pad urine detection antibody capture antibodies control antibody lane hCG capture antibody lane hCG positive hCG negative control lane (C) test lane (T) detection antibodies similar to sandwich ELISA hCG Competitive system hCG positive control lane detection antibody capture antibody ( hCG lane bound hCG) hCG negative control lane test lane similar to competitive ELISA You don’t need to use additional enzymatic incubation steps, because the labels on the detection antibodies can be observed by naked eye: • colloidal gold („surface plasmon” resonance) • colored latex beads The assay can be used in semi-quantitative manner Other uses of immunechromatographic test strips: detection of toxins in food e.g.: aflatoxins (mycotoxins of Aspergillus spp.) ITT ÉRDEMES BEFEJEZNI, DE HA GONDOLJÁTOK A KÖVETKEZŐ KÉT DIA AZ EIA TESZT KVANTITATÍV KIERTÉKELÉSÉRŐL SZÓL. AZ IDŐBE VALÓSZÍNŰLEG BELEFÉR Tamás, mennyire tanítottátok meg nekik a számításokat? DETERMINATION OF THE CONCENTRATION A quantitative property of an indicator refers to the concentration: color (absorbance, optical density) fluorescence Quantified concentration can be obtained by comparison with signals obtained with a serial dilution of a known standard sample The principle of comparison: equal absorbances equal concentrations PARTIAL TRUTH !!! The serial dilution of the standard OD The sample with unknown concentration You should also dilute the unknown sample Only this region indicates valid concentrations All these concentrations could be choosen? concentration 0.004 0.007 0.015 0.030 0.061 0.12 0.24 0.49 0.97 1.9 3.9 7.8 16 31 62 125 250 500 1000 ? 0