2. Dr. Jeevan Shetty MBBS MD Associate Professor Reference Book:  Text book of Biochemistry By DM Vasudevan 5th edition

3. At the end of the topic student shall be able to  Define and classify ELISA  Principle of ELISA technique  Instrumentation in ELISA  Procedure of measurement  Interpretation of results  Advantages of ELISA over RIA  Application of ELISA in medicine and pharmacy

4. Definition - Is a biochemical technique used mainly in immunology to detect the presence of an antibody or an antigen in a sample. The technique is divided into 1- Competitive ELISA - antigen detection or antibody detection 2- Sandwich ELISA (also called direct ELISA) 3- Indirect ELISA

5. Principle - Single antibody (competitive ELISA)  The labelled antigen competes for primary antibody binding sites with the sample antigen (unlabelled).  The more antigen in the sample, the less labelled antigen is retained in the well and the weaker is the signal.

6. Single antibody (competitive ELISA)  Antibodies are coated on to the micotitre plate  Serum (unlabeled antigen) and labeled antigen is added to the plate.  Both antigen in the serum and labeled antigen binds to coated antibody and excess antigen is washed.  Substrate for the enzyme is added and observe the color development.  Intensity of the color developed is inversely proportional to the amount of antigen in the serum.

8.  The ELISA plate is coated with Antibody(T4) to detect specific antigen.  Patient serum is added in the well and incubated for 30 minutes at 37 0 C.  Antigen(T4) present in the serum is fixed on the antibody. Excess antigen and other unwanted proteins are washed out.  Then add specific antibody against T4 tagged with enzyme (horse radish peroxidase) is added

9.  If the antigen is already fixed, antibody-HRP- conjugate will be fixed in the well,  Then a color reagent, containing hydrogen peroxide (H 2 O 2 ) and diamino benzidine (DAB) are added.  The reaction is as follows H 2 O 2 H 2 O + Nascent oxygen Diamino benzidine Oxidized DAB (Colorless) (Brown color)  This is called sandwich ELISA.  Development of brown color indicates the presence of antigen in the serum  Color developed is proportional to the antigen in the serum.

11.  The antigen for antibody of interest (testing antibody) is added to each well of ELISA plate,  It will adhere to the plastic through charge interactions.  A solution of non-reacting protein is added to block any plastic surface in the well that remains uncoated by the protein antigen.

12.  Then the serum is added, which contains a mixture of the serum antibodies, of unknown concentration, some of which may bind specifically to the test antigen that is coating the well.  Afterwards, a secondary antibody is added, which will bind to the antibody bound to the test antigen in the well.  This secondary antibody often has an enzyme attached to it

13.  A substrate for this enzyme is then added.  Often, this substrate changes colour upon reaction with the enzyme.  The colour change shows that secondary antibody has bound to primary antibody, which strongly implies that the donor has had an immune reaction to the test antigen.  The higher the concentration of the primary antibody that was present in the serum, the stronger the colour change.  Often a spectrometer is used to give quantitative values for colour strength

14.  Solid Phase : Plastic tubes / Micro titre plates  Enzymes : Peroxidase (Hydrogen Peroxide) Alkaline Phosphatase (PNPP – para nitrophenyl phosphate)

16.  Micropipettes  Incubator ( 37 0 C ± 1 0 C )  ELISA Reader / Spectrophotometer  Micro well Washer ( Automatic / Manual )

17.  Before starting the work read kit instruction carefully  1- The 96 well plate is labeled carefully and the first wells are used to draw the standard curve

18.  The sample is added to plate in duplicate or triplicate and then the mean result is calculated  The quality control sample which is provided with the kit is treated as the test samples

19.  After reading the results the standard curve is drawn were the concentration is blotted on the X-axis and the absorbance on the Y-axis Concentration ng/ml Absorption nm

20.  The standards concentrations is specified on the x-axis and the reading of each standard is specified on the y-axis and the standard curve is drawn

21.  This standard curve is used to determine the unknown concentration of each sample by finding the opposite concentration to the absorbance Concentration ng/ml Absorption nm

22.  The quality control sample concentration is determined from the standard curve and if the result is in the range given by the kit manufacturer the results could be accepted.

23.  Screening donated blood for evidence of viral contamination by ◦ HIV-1 and HIV-2 (presence of anti-HIV antibodies) ◦ hepatitis C (presence of antibodies) ◦ hepatitis B (testing for both antibodies and a viral antigen)  Measuring hormone levels ◦ β-HCG (as a test for pregnancy) ◦ LH (determining the time of ovulation) ◦ TSH, T3 and T4 (for thyroid function) ◦ Hormones (e.g., anabolic steroids, HGH) that may have been used illicitly by athletes.

24.  Detecting infections ◦ sexually-transmitted agents like HIV, syphilis, and chlamydia ◦ hepatitis B and C ◦ Toxoplasma gondii  Detecting allergens in food and house dust  Measuring "rheumatoid factors" and other autoantibodies in autoimmune diseases like systemic lupus erythematous (SLE).  Measuring toxins in contaminated food  Detecting illicit drugs, e.g., ◦ cocaine ◦ opiates ◦ Δ-9-tetrahydrocannabinol, the active ingredient in marijuana

25. Serum Hormone estimation T 4, TSH, FSH, LH, Insulin etc Tumor Marker estimation AFP, PSA, HCG,CEA, CA-125 etc AFP, PSA, HCG,CEA, CA-125 etc Detection of Bacterial toxins, Viruses etc to Rubella virus Antiviral antibodies eg to Rubella virus to Salmonella Antibacterial antibodies eg to Salmonella Auto antibodies ANA, anti-DNA, anti Thyroglobulin

26.  It is cheaper than RIA  Lacks radiological hazards of RIA  Reagents have more shelf-life compared to RIA  It is equally sensitive and specific as RIA  Suitable for use even in small laboratories