IMMUNOLOGICAL ASSAYS.pptx

IMMUNOLOGICAL ASSAYS- M.PHARMA 1ST YEAR
TOPICS COVERED-
1) RADIO IMMUNO ASSAY
2) ENZYME LINKED IMMUNOSORBANT ASSAY

1)VARIOUS TYPES OF ELISA TECHNIQUE
METHODOLOGY AND ADVANTAGES AND DISADVANTAGES OF ELISA
2) METHODOLOGY OF RADIO IMMUNOASSAY ITS ADVANTAGES AND DISADVANTAGES
3) APPLICATION OF BOTH RIA AND ELISA
4) QUALITATIVE AND QUANTITATIVE DETERMINATION TECHNIQUES OF RESULT.
5) NEED OF IMMUNOLOGICAL ASSAYS.
DIAGRAMATIC REPRESENTATIONS WITH FLOW CHART FOR METHODS EXPLAATION.

1. IMMUNOLOGICAL ASSAYS RADIOIMMUNO ASSAY AND ENZYME LINKED IMMUNOSORBANT ASSAY SUBMITTED BY: PANDEY AJAY SANJAY M.PHARMA 1st Year PHARMACEUTICS SUBMITTED TO: Dr. KAISAR RAZA ASSISTANT PROFESSOR DEPARTMENT OF PHARMACY CENTRAL UNIVERSITY OF RAJASTHAN

2. CONTENTS Introduction RadioImmunological Assay Advantages and Disadvantages Of RIA Enzyme linked immunosorbant Assay Types Of ELISA Advantages and Disadvantages of ELISA Detection Of Results Aplication of ELISA and RIA Referenes

3. INTRODUCTION IMMUNOLOGICAL ASSAY: It is a Highly selective Bio-analytical method that measures the Presence or Concentration of analyte ranging from small molecule to macromolecule in a solution through an Antigen or Antibody as biorecognition agenet. Play a critical role in Clinical Diagnostic, Bio-Pharmaceutical techniques, Food Testing etc The investigation on the antigenicity of small molecules by Landsteiner in 1917 has provided the framework for the development of immunological assays for detection of low molecular weight compounds. In typical immunoassay the antibody act as a reagent to detect analyte (antigen) of intrest.

4. REQUIREMENT OF IMMUNOLOGICAL ASSAY

5. RADIOIMMUNO ASSAY RIA ( 1959 ) by Solomon,Belson,and yalow for estimation of Insulin in Human Serum. This technique is used for the biological fluids that are found in very low concentration(nanogram or picogram). PRINCIPLE: It involves a combination of three principles. An immune reaction i.e. antigen, antibody binding. A competitive binding or competitive displacement reaction. (It gives specificity) Measurement of radio emission.

6. PROCEDURE OF RADIOIMMUNOASSAY

7. Advantages of RIA (Radioimmunoassay) It is an extremely sensitive assay as it can measure antigen up to picogram quantities. It is a highly specific test as the antibody-antigen reaction is highly specific. A large number can be processed. It is an indirect method of analysis. Disadvantages of RIA (Radioimmunoassay) Radiation hazardous . Require special arrangements for storage of radioactive material. The high cost of waste disposal. Lengthy counting time There are some difficulties in the automation of this assay. The reaction time is long due to the use of highly diluted reagent.

8. ENZYME LINKED IMMUNOSORBANT ASSAY ELISA works on the principle that specific antibodies bind the target antigen and detect the presence and quantity of antigens binding. The enzyme-linked immunosorbent assay (ELISA) is an immunological assay commonly used to measure antibodies, antigens, proteins and glycoproteins in biological samples. Some examples include: diagnosis of HIV infection, pregnancy tests, and measurement of cytokines in cell supernatant or serum. ELISA assays are generally carried out in 96 well plates , allowing multiple samples to be measured in a single experiment. These plates need to be special absorbant plates (e.g. NUNC Immuno plates ) to ensure the antibody or antigen sticks to the surface.

9. TYPES OF ELISA DIRECT ELISA INDIRECT ELISA SANDWITCH ELISA COMPETITIVE ELISA (antigen -coated plate; screening antibody ) ( antigen -coated plate; screening antigen/antibody ) ( antibody -coated plate; screening antigen ) (screening antibody andAntigens) TYPES OF ELISA

10. STEPS INVOLVED IN ELISA DETECTION: Horseradish peroxidase (HRP)-B lue color Alkaline phosphatase (ALP)-Yellow color FOR WASHING : Phosphate-buffered saline (PBS) FOR SAMPLING Polystyrene plates, typically in 96-well plates.

11. Blood sample collected( SERUM In PHOSPHATE BUFFER SOLUTION) Added to Microtiter plates (Antigens adhered to plates) BLOCKING is done by adding BOVINE SERUM ALBUMIN ENZYME linked PRIMARY ANTIBODIES added(binds to antigens if present) SUBSTRATE is added which binds to ENZYME to give product as generation of COLOUR DIRECT ELISA INDIRECT ELISA ANTIGENS adhered plate is collected BLOCKING is done by adding BOVINE SERUM ALBUMIN Blood sample collected ( SERUM is placed In PHOSPHATE BUFFER SOLUTION) ( PRIMARY ANTIBODY adhered to ANTIGENS ALREADY PRESENT IN WELL) ENZYME linked antibodies added against Primary Antibodies ( Primary Antibodies acts as a Antigen for Enzyme lined Ab ) SUBSTRATE is added which binds to ENZYME to give product as generation of COLOUR

12. SANDWITCH ELISA COMPETITIVE ELISA CAPTURED ANTIBODY (specific to HIV) is added to Microtitere plate BLOCKING is done by adding BOVINE SERUM ALBUMIN Blood sample collected ( SERUM is placed In PHOSPHATE BUFFER SOLUTION) Added to wells( Antigens specific to HIV Antibodies binds with them Different detector protiens are present on antigens surface( EPITOPES ) ENZYME linked secondary antibodies( DETECTION ANTIBODIES ) added Binds to other epitope present on antigens surface SUBSTRATE is added which binds to ENZYME to give product as generation of COLOUR DONE WHEN IT IS CONFIRMED THAT PATIENT HAS SPECIFIC ANTIGENS(CONCENTRATION DETERMINATION) CAPTURED ANTIBODY (specific to HIV) is added to Microtitere plate BLOCKING is done by adding BOVINE SERUM ALBUMIN Blood sample Colleted ( SERUM is placed In PHOSPHATE BUFFER SOLUTION) ENZYME linked antigens specific to antibodies added Both antigens gets adhered to antibodies depending on their concentration SUBSTRATE is added which binds to ENZYME to give product as generation of COLOUR Depending on the colour produced , concentration of antigens were determined.

13. DETECTION OF RESULTS Data gathered from ELISA tests can be quantitative, qualitative, or semiquantitative. The quantitative concentration results are plotted and compared to a standard curve. The qualitative results confirm or deny the presence of a particular antigen/antibody in a sample. The semiquantitative results compare the intensity of the signals , which can compare relative antigen levels in a sample. Once color changes are measured from the assay, the results are graphed either on paper or software. Typically, the graph compares optical density to log concentration , which gives a sigmoidal curve.

14. APPLICATIONS RIA It used to check the plasma level of hormones . Also used in the analysis of vitamins Also used in the analysis of anti- DNA antibody in systemic lupus erythematosus. It is also used in the early detection of cancer. Also used in the research of brain chemicals called neurotransmitter. ELISA The presence of antibodies and antigens in a sample can be determined. It is used in the food industry to detect any food allergens present. To determine the concentration of serum antibody in a virus test. During a disease outbreak, to evaluate the spread of the disease, e.g. during recent COVID-19 outbreak, rapid testing kits are being used to determine presence of antibodies in the blood sample.

15. REFERENCES Aydin S. A short history, principles, and types of ELISA, and our laboratory experience with peptide/protein analyses using ELISA. Peptides. 2015 Oct;72:4-15. [PubMed] Weng X, Gaur G, Neethirajan S. Rapid Detection of Food Allergens by Microfluidics ELISA-Based Optical Sensor. Biosensors (Basel). 2016 Jun 07;6(2):24. [PMC free article] Journals of Immunoassaand Immunochemistry 25(3) 241-258 www.google.comimages www.slideshare.Net

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