Immunoassay

1. Immunoassay

2. Immunoassay • ● Highly specific in vitro tests that use antigen-antibody reaction to detect extremely low concentrations of a broad range of analytes in different types of samples (clinical, environmental, food, etc) • ● Antigen-antibody reaction - known since the end of the 19th ct: • -precipitation in gel • Agglutination • turbidimetry assays • gradually developed until their potential has fully been appreciated since 1960 when • higher sensitivitywas achieved bylabeling one of the components ● simple ●precise ●sensitive (nano- and picomolar range) ● high throughput measurement of more substances than any other analytical technique

3. Immunoassay All immunoassay require the same key reagents ● One or more antibodies raised against epitopes believed to be specific to the analyte in question ● A label (tracer) producing a measurable signal ●Calibrators in a fluid (matrix) similar to the sample Antibody The antibody used in immunoassay is usually of the IgG class

4. Immunoassay Natural Antigen Substance that naturally elicit immune response Usually a larger molecule (over 10 kD) with several epitopes (antigenic determinants) Conjugated haptens ● Smaller molecules (called haptens) are either weakly or not not at all immunogenic ● To produce the antibody it is necessary to couple them to an immunogenic carrier (e.g. BSA, KLH...) The optimal molar ratio (excess may range from 10 :1 to 80 :1) is important for production of good antisera

5. Immunoassay Natural Antigen Substance that naturally elicit immune response Usually a larger molecule (over 10 kD) with several epitopes (antigenic determinants) Conjugated haptens ● Smaller molecules (called haptens) are either weakly or not not at all immunogenic ● To produce the antibody it is necessary to couple them to an immunogenic carrier (e.g. BSA, KLH...) The optimal molar ratio (excess may range from 10 :1 to 80 :1) is important for production of good antisera

6. ELISA (Enzyme-Linked ImmunoSorbent Assay). Types and classification Analyte: antigen hapten (small molecule) one epitope multivalent antigen, many epitopes antibody Labelling: Direct Indirect Response: Competitive assay Non competitive Sandwich assay Antigen immobilized Antigen labelled

7. ELISA (Enzyme-Linked ImmunoSorbent Assay). Types and classification Direct competitive ELISA sulfonamida SFM-HRP anticuerpo anti-SFM

8. ELISA (Enzyme-Linked ImmunoSorbent Assay). Types and classification Indirect competitive ELISA anticuerpo anti-SFM sulfonamida SFM-BSA

9. ELISA (Enzyme-Linked ImmunoSorbent Assay). Types and classification Direct competitive ELISA (with antigen immobilized) anticuerpo anti-SFM-HRP sulfonamida SFM-BSA

10. S P S P ELISA (Enzyme-Linked ImmunoSorbent Assay). Types and classification - LOD - LOQ - IC50 - Linear range

12. ELISA (Enzyme-Linked ImmunoSorbent Assay). Types and classification Noncompetitive sandwich assay Salmonella anticuerpo anti-Salmonella-HRP

13. ELISA (Enzyme-Linked ImmunoSorbent Assay). Types and classification Noncompetitive sandwich assay Salmonella anticuerpo secundario-HRP

17. Elisa Competitivo

18. Signal amplification