presentation for activity antigen assays.pptx

presentation for activity work

1. Antigen-Based Assays Medical Research Institute Prepared by : Ahmed Abdelwahab Ragab Diagnostic Immunological Application II (1208705) Under supervision : DR. Hossam Ghoneim Professor of immunology and Allergy 1

2. OUTLINE Introduction Enzyme-linked immunosorbent assay (ELISA ) The principle of ELISA test The types of ELISA latex immunoassay Types of immunoassay Factor VIII Binding Assay [VWF:FVIII] References 2

3. Introduction Antigen-based assays can be used to distinguish quantitative (reduced protein expression or secretion) from qualitative (functional) deficiencies. Two main types of antigen-based assays are commonly used: the enzyme-linked immunosorbent assay ( ELISA ) and the latex immunoassay. The ELISA uses a specific antibody to capture an antigen and a second enzyme-linked antibody to measure the amount of antigen, whereas latex immunoassays are single- antibody systems. 3

4. . The von Willebrand factor/factor VIII-binding (VWF-FVIII-binding) assay is used to distinguish the 2N subtype from other VWD type 2 subtypes, and von Willebrand factor multimer analysis is typically performed after initial  VWD  assays to help define the VWD variant subtype. Clotting factor antigen assays may be used to differentiate functional from quantitative defects. 4

5. Enzyme-linked immunosorbent assay (ELISA) ELISA is a common laboratory testing technique that detects and counts certain antibodies, antigens, proteins and hormones in bodily fluid samples. This includes blood, plasma, pee, saliva (spit) and cerebrospinal fluid (CSF). “ELISA” stands for “enzyme-linked immunosorbent assay.” Another name for it is an EIA test. Advantages is quick results , often low coast and portable. 5

6. The principle of ELISA test In ELISA, various antigen-antibody combinations are used, always including an enzyme-labeled antigen or antibody, and enzyme activity is measured colorimetrically . The enzyme activity is measured using a substrate that changes color when modified by the enzyme. Light absorption of the product formed after substrate addition is measured and converted to numeric values 6

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8. the types of ELISA There are four major types of ELISA: Direct ELISA (antigen-coated plate; screening antibody) Indirect ELISA (antigen-coated plate; screening antigen/antibody) Sandwich ELISA (antibody-coated plate; screening antigen) competitive ELISA. 8

9. Direct ELISA (antigen-coated plate; screening antibody) A target protein (or a target antibody) is immobilized on the surface of microplate wells and incubated with an enzyme-labeled antibody to the target protein (or a specific antigen to the target antibody). After washing, the activity of the microplate well-bound enzyme is measured. 9

10. Indirect ELISA (antigen-coated plate; screening antigen/antibody) A target protein is immobilized on the surface of microplate wells and incubated with an antibody to the target protein (the primary antibody), followed by a secondary antibody against the primary antibody. After washing, the activity of the microplate well-bound enzyme is measured. Although indirect ELISA requires more steps than direct ELISA, labeled secondary antibodies are commercially available, eliminating the need to label the primary antibody. 10

11. Sandwich ELISA (antibody-coated plate; screening antigen) An antibody to a target protein is immobilized on the surface of microplate wells and incubated first with the target protein and then with another target protein-specific antibody, which is labeled with an enzyme. After washing, the activity of the microplate well-bound enzyme is measured. The immobilized antibody (orange) and the enzyme-labeled antibody (green) must recognize different epitopes of the target protein. 11

12. competitive ELISA. An antibody specific for a target protein is immobilized on the surface of microplate wells and incubated with samples containing the target protein and a known amount of enzyme-labeled target protein. After the reaction, the activity of the microplate well-bound enzyme is measured. 12

13. When the antigen level in the sample is high, the level of antibody-bound enzyme-labeled antigen is lower and the color is lighter. Conversely, when it is low, the level of antibody-bound enzyme-labeled antigen is higher and the color, darker. The graph above and to the right illustrates the correlation between absorption and antigen levels in samples. 13

14. latex immunoassay A general method for the determination of antibodies, antigens, and haptens . Latex particles coated with antibody specific to an antigen agglutinate in the presence of that antigen. The degree of agglutination is directly proportional to the concentration of the antigen in the sample and is determined by measurement of transmitted light or light scattering. 14

15. types of immunoassay According to the difference of label and signal detection strategy, immunoassay can be classified as the following types: Western Blot (WB) Radioimmunoassay (RIA)   Fluoroimmunoassay (FIA)  Chemiluminescence Immunoassay (CLIA)  Immunochromatograohic Assay (ICA)  Immunohistochemistry (IHC) 15

16. WESTERN BLOT (WB) The key principles of western blotting are equal loading of proteins, separation of proteins by molecular weight, electrophoretic transfer to a suitable membrane, and antibody probing. The five most common types of western blotting equipment used by researchers in labs include gels, imaging systems, filter papers, tank blotting transfer systems, and semi-dry blotting systems. SDS-PAGE is typically used for western blotting, where proteins are denatured and reduced to obtain their primary structure. Coating with SDS molecules (Sodium dodecyl sulfate) imparts a relative negative charge proportional to their molecular weight, allowing separation of the protein by size only. 16

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18. Radioimmunoassay (RIA)   Radio immuno assay is a highly sensitive method to determine an antigen’s concentration in sample. RIA can be used to measure hormones in a variety of body fluids, including blood, urine, and saliva. A vast number of samples can be processed, It is an indirect analysis method. The main advantage of RIA is its high specificity and sensitivity. It can detect very low levels of antibodies, making it a valuable tool for diagnosing autoimmune diseases and other conditions that involve antibody abnormalities.   18

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20. Fluoroimmunoassay (FIA)  Fluorescence immunoassay (FIA) is a simple, rapid, and sensitive technique that is used to measure many compounds including drugs, hormones, and proteins. This method has been widely applied in the  in vitro  diagnostics (IVD) industry 20

21. the development of fluorescent labeling technologies and instrumental technologies, a variety of FIA-related technologies have been developed, including : 1) Fluorescent excitation transfer immunoassay 2) Fluorescence polarization immunoassay (FPIA) 3) Time-Resolved Fluorescence Immunoassays (TRFIA) 4) Fluorescence Energy Transfer Immunoassays 5) Phase-Modulation Fluorescence Spectroscopy 6) Phase-Resolved Fluoroimmunoassays 7) Phase Fluorescence and Fluorescence Lifetime Immunoassays 8) Liposome Fluoroimmunoassays 21

22. Chemiluminescence Immunoassay (CLIA)  The basis of the  CLIA method  is similar to that of  ELISA , except that CLIA substrates can generate light emission in the presence of an enzyme, providing a more sensitive process compared to ELISA. Substrates such as isoluminol or acridinium ester produce the luminescence signal in the presence of hydrogen peroxide and enzyme. The electro- chemiluminescence   immunoassay  (ECLIA), another type of CLIA, uses  electrical current  for oxidizing substrate. CLIA and ECLIA methods have higher sensitivity compared to ELISA ,and have a shorter analysis time 22

23. Sandwich ELISA Based CLIA 23

24. Immunochromatograohic Assay (ICA) A chromatographic immunoassay is a technique in which an antibody or antibody-related agent is used as part of a chromatographic system for the isolation or measurement of a specific target. The concept of immune-chromatography is a combination of chromatography (separation of components of a sample based on differences in their movement through a sorbent) and immunochemical reactions . Is immunochromatography accurate ? The sensitivity and specificity for the assay on serum samples are 100% and 99.4%, respectively . 24

25. Immunochromatography assay (ICA), namely lateral flow test, is a simple device intended to detect the presence or absence of the target analyte . 25

26. Immunohistochemistry (IHC) Immunohistochemistry (IHC) uses antibodies to detect antigens in a tissue sample. It's one lab technique a pathologist may use to check for . signs of disease following a biopsy Immunohistochemistry (IHC) is an important application of monoclonal as well as polyclonal antibodies to determine the tissue distribution of an antigen of interest in health and disease . IHC is widely used for diagnosis of cancers; specific tumor antigens are expressed de novo or up-regulated in certain cancers . 26

27. Immunohistochemistry (IHC) Staining 27 Immunostaining uses antibodies to detect an antigen in cells or tissue. The major benefit of immunostaining in immunohistochemistry (IHC ) is the ability to see the desired target in a tissue sample while maintaining the spatial context and tissue architecture .

28. Factor VIII Binding Assay [ VWF:FVIII]  is a recessively inherited disorder due to amutation in von Willebrand Factor that impairs its ability to bind to and transport Factor VIII [FVIII] in the plasma . As a result the half-life [T½] of FVIII is significantly reduced as it is no longer protected from proteolytic degradation within the plasma. Individuals with Type 2N VWD were and occasionally still are, misdiagnosed as having mild Haemophilia A . There are a number of methods available to measure the binding of FVIII to VWF. The ELISA method is commonly used . 28

29. References 1. Nichols WL, Hultin MB, James AH, et al. von Willebrand disease (VWD): evidence-based diagnosis and management guidelines, the National Heart, Lung, and Blood Institute (NHLBI) Expert Panel report (USA). Haemophilia 2008;14:171-232. 2. James AH, Eikenboom J, Federici AB. State of the art: von Willebrand disease. Haemophilia 2016;22 Suppl 5:54-9. 3.https:// ruo.mbl.co.jp /bio/e/support/method/ elisa.html 4. Wang, Wei, et al. "Rapid quantification of chlorpromazine residues in pork using nanosphere ‐based time‐resolved fluorescence immunoassay analyzer."  International Journal of Analytical Chemistry  2021.1 (2021): 6633016 . https://www.lsbio.com/products/elisakits/clia 29

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