Transcript ELISA Lab
ELISA ( Enzyme-Linked Immunosorbent Assay) What is an ELISA? • Enzyme-linked immunosorbent assay • Name suggests three components – Antibody • Allows for specific detection of Ag – Solid phase (sorbent) • wash away all the material that is not specifically captured – Enzymatic amplification • Convert a little capture into a visible color change that can be quantified using an absorbance plate reader ELISA PRINCIPLE The principle of the ELISA is that the target analyte (the antigen) is recognized with high specificity by antibodies, which are proteins produced by the immune system of animals . These antibodies labeling with enzyme and after reaction the labeling Ab with Ag the substrate add and read the color by spectrophotometer . What is it used for? • • • • • • • • • • Measure antibody levels (allergies, vaccines) Detect viruses (hepatitis, HIV, venereal diseases) Detect hormonal changes (Exa :pregnancy) Detect circulatory inflammatory markers (cytokines) •Infections ( bacterial infection ,parasitic infection ) •Specific disease factors •Drugs •Allergens of food •Residues in food •Toxins Advantages • •Fast—90 samples tested in 2-3 hr • •Sensitivity (up to 10 pg/mL) • •Specificity ( even sample with high concentration contaminants) • •Many samples can be processed at once • •Small sample size required (10 μL~ 100μL ) • •Colorimetric results –easily observed and measured (spectrophotometer) • •Test for presence of Ag or Ab • •Flexible usage for research design • •Easy to learn, simple procedure The materials for your kit • • • • • • • • • • • 1. ELISA plate 2. Positive controls 3. Negative controls 4. Dilution Buffer (already in dilution tubes) 5. Conjugate (secondary antibody labeled with enzyme ) 6. Substrate (ex.TMB) 7. Stop solution 8. wash solution 9-procedure leaflet 10- blank solution 11- Standards( may be 2 or more ) Types of ELISA assay • • • • 1.Direct-ELISA 2.Indirect-ELISA 3.Sandwich-ELISA 4.Competition-ELISA Direct-ELISA • –Enzyme conjugated Ab is directly bound to the Ag • Detect antigen in the sample (e.g., HIV viral proteins) Indirect method Detect antibodies in the sample (e.g., antibodies against HIV proteins Sandwich ELISA Sandwich standard curve Competitive ELISA Enzymes used in ELISA • 1- Horse radish peroxidase . • 2-Alkaline phosphatase . • 3- Beta-D-galactosidase Substrate used in ELISA • 1- TMB (tetra methyl benzidine) : soluble substrate yield blue color when react with HRP. Very sensitive, quick oxidized resulting in faster color development . • OPO (o-phenylenediamine dihydrochloride yield yelloworenge color when react with HRP. • PNPP (p-nitrophenyl phosphate ,Disodium salt ) Is a widely used substrate for detecting alkaline phosphatese .produce yellow color . • ABTS ( Azinobis [3-ethelbenzothiazoline-6-sulfonic acide]-diammonium salt .give green reaction when react with HRP. ELISA resultes • 1-Quantitative : • The results of patient sample compared to standard curve (a serial dilution of known ,purified Ag [standards ] ) • 2-Qualitative : • Give yes or no compared with blank (well not containing Ag . • 3- Semi-quantitative • The result compare with relative levels of Ag or AD