Elisa - a Rapid Immunochemical Assay

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The diluted sera were discarded and washed four times using PBS-Tween. Consequently, 100 µL of 1:5000 of anti rabbit-HRP in PBS-Tween was added to the wells and another round of incubation at 37oC for 30 minutes was carried out. The antibody-enzyme conjugate was discarded and the plate was washed four times with PBS-Tween. Hundred microliters of ABTS substrate solution were added to all wells and the plate was incubated at room temperature. The plate was monitored for colour change and approximately 5 minutes after, 50 µL of 0.5M oxalic acid were added to stop the reaction in all wells and the optical density was measured at 405 nm using ELISA plate Reader.

Part II: Titration of antibody serum by ELISA

Materials and Equipment

- 96-well microtiter plate

- Purified antigen

- PBS-Tween (0.15M Phosphate buffer saline containing 0.5% Tween)

- Anti rabbit- HRP conjugate

- ABTS solution

- Carbonate-bicarbonate buffer pH 9.6

- 0.5M oxalic acid

- Water bath

- ELISA plate Reader/Filter (405nm)

- Rabbit anti-BSA serum

- Normal rabbit serum

- Affinity purified rabbit Ig anti-BSA samples (bound and unbound)

Method

The 96-well microtiter plate was previously coated with the BSA antigen except in row H and washed five times with PBS-Tween. The last wash was discarded and 100 µL of test serum, diluted bound fraction, unbound fraction and normal serum were added to column 1 of rows A, B, C and D respectively. E1 was loaded with 100 µL PBS while F1, G1 and H1 were loaded with 100 µL of positive control serum. Fifty microliters of PBS-Tween were loaded to all the other wells. In order to perform serial dilutions, 50 µL sample was transferred by using a multichannel auto pipette from well 1 to well 2 of all eight rows, and mixed by pipetting up and down. The process is repeated all the way up to column 12 in which 50 µL of the well sample was removed and discarded. The microtiter plate was then incubated at 37oC for 30 minutes.

Consequently, the diluted sera were discarded and washed four times using PBS-Tween. Then, 100 µL of 1:5000 of anti rabbit-HRP in PBS-Tween was added to all wells except those of row G into which PBS was added instead. Another round of incubation of the plate at 37oC for 30 minutes was carried out. The antibody-enzyme conjugate was discarded and the plate was washed four times with PBS-Tween. Hundred microliters of ABTS substrate solution were added to all wells and the plate was incubated at room temperature. The plate was monitored for colour change and approximately 5 minutes after, 50 µL of 0.5M oxalic acid were added to stop the reaction in all wells and the optical density was measured at 405 nm using ELISA plate Reader.

Results

Part I: Screening test for detection of Antibody in culture media by ELISA

The 96-well microtitre plate was coated with BSA and undergone several rounds of PBS-Tween wash. Different types of sera were added to column 1 in respective rows and incubated. The plate was washed again several times and the antibody-HRP conjugate was added and incubated again. Finally, the plate was washed four times and the ABTS substrate was added for the production of a coloured precipitate upon incubation at room temperature. After generation of the coloured product, oxalic acid was added to stop the enzymatic reactions in the wells and absorbance of the wells was measured at 405nm using ELISA plate Reader and noted in Table 1.

Table 1. The absorbance values of serial diluted positive, negative, A, B and C samples and calculated average absorbance values of A, B and C serum samples.

Absorbance at 405nm

Dilution factor

+ve control

-ve control

A1

A2

Average

A

B1

B2

Average

B

C1

C2

Average

C

0

2.274

1.904

1.915

1.965

1.940

2.249

2.273

2.261

0.133

0.139

0.136

1:2

1.995

0.756

0.801

0.801

0.801

1.713

1.795

1.754

0.124

0.124

0.124

1:4

1.400

0.393

0.408

0.437

0.378

1.138

1.312

1.225

0.116

0.117

0.117

1:8

1.308