VLD 421: Veterinary Clinical Biochemistry and Laboratory Diagnosis-II (0+1)

Indirect ELISA

INDIRECT ELISA FOR ANTIBODY DETECTION

Principle

• In indirect ELISA antigen is directly attached to solid phase and targeted by added antibodies (detecting antibodies).
• These added antibodies are not labeled with enzyme but are themselves targeted by antispecies antibodies linked to enzyme termed as conjugates.
• The bound enzyme is developed by the addition of substrate /chromogen, then stopped and finally read using a spectrophotometer

Materials

• ELISA plates
• Micro pipettes, both single and multiple channel
• Antigen
• Antibody
• Phosphate buffered saline (PBS) pH 7.4
• Carbonate bicarbonate buffer pH 9.6
• Substrate solution
• 1% Bovine serum albumin
• Antispecies IgG – HRP conjugate
• Stopping reagent – 1M H₂So₄
• ELISA reader

Procedure

• Calculate the amount of antigen to be used for coating the plates by checker board titration.
• Add 100 µl of antigen to all the wells and incubate the plates at humid chamber at 4^◦ C over night.
• Empty the plates and wash thrice (5min each) in PBST washing solution. Tap the plate against any soft paper or towel after each washing to get rid of any unabsorbed material in the well.
• For blocking the unsaturated sites of polystyrene plate well, add 100 µl of 1 % BSA to all the wells and incubate at 37^◦C for one hour.
• Wash the plates 3 times with washing solution and tape as mentioned above.
• Add 100 µl of appropriate diluted serum to the first well and serially dilute serum as required
• Use triplicates of l in 100 dilution of negative serum keep as negative control.
• Keep positive serum controls.
• After the addition of test positive and negative serum incubate the plate at 37^◦C for 1 hr .
• Wash and tap the plate as mentioned above.
• Add appropriate dilution of 100 µl of anti species IgG HRP conjugate to all the wells and incubate the plate at 37^◦ C for 1 hr
• Wash and tap the plate as mentioned above.
• Add 100 µl of substrate to all the wells. Keeping a substrate controls, the plates are incubated at room temperature for 30 minutes for development of colour.
• At the end of incubation period stop the reaction by adding 100 µ of appropriate stopping reagent.
• Read the absorbance value on a ELISA reader using 492 nm wavelength filter.

Analysis

• The optical density (OD) value twice and above the negative value are taken up as cutoff value and considered as + ve reading.