Antibody potency assay
Summary
Antibody potency assay is used to test the potency of immune serum. Currently there are three main methods used to determine antibody potency: tube agglutination reaction, agar diffusion test and enzyme-linked immunosorbent assay (ELISA).
Principle
The basic principle of the ELISA method for determining antibody potency is a highly sensitive test technique based on an immunologic reaction that combines the specific reaction of antigen and antibody with the efficient catalytic effect of enzymes on the substrate (Figure 5-1).
Operation method
Determination of antibody potency by ELISA
Principle
The basic principle of ELISA is based on immunological reaction, combining the specific reaction of antigen and antibody with the efficient catalytic effect of enzyme on the substrate, which is a highly sensitive test technique.
Materials and Instruments
Equipment: enzyme labeling apparatus
Reagents:
① 1x PBST (1 x PBS ＋ 0.1% Tween 20)
② Enzyme secondary antibody
① Enzyme secondary antibody ③ Coating buffer (0.1 M Na
0.1 M Na
① Enzyme secondary antibody ③ Coating buffer (0.1 M Na
① 1x PBST (1 x PBS + 0.1% Tween 20)
pH 9.6)
Move
The basic procedure for antibody potency determination by ELISA can be divided into the following steps:
(1) Dilute the antigen in coating buffer (0.1 M Na2CO3, pH 9.6) at a final concentration of 5 ng/μL, add 100 μL to each well, and set up a blank control (only add coating buffer), and then coat overnight at 4 ℃.
(2) Wash with 1 x PBST (1 x PBS + 0.1% Tween 20) three times at room temperature for 5 min each time, and then add 200 μL of blocking solution (10% calf serum dissolved in 1 x PBS) to each well and block at 37 ℃ for 1 h. (3) 1 x PBST (1 x PBS + 0.1% Tween 20) was used as the final concentration of 5 ng/μL, and 100 μL of blocking solution was added to each well.
(3) Wash 3 times with 1x PBST. Add serum to be tested, serum samples were diluted in antibody diluent (5% BSA dissolved in 1x PBS) at 1:1000, 1:2000, 1:4000 ...... times, 100 μL per well, 100 μL of 1:2000 diluted negative serum was added to negative wells, 100 μL of 1:1000 sample serum was added to blank wells. Incubate for 1 h at 37 ℃.
(4) Wash 3 times with 1x PBST and add the secondary antibody. The enzyme secondary antibody was diluted 1:2500 in antibody diluent according to the instruction, 100 μL per well, and ABTS color development was performed at 37 ℃ for 1 h. 100 μL per well was incubated at 37 ℃ for 15 min, and the result was read at 405 nm on the enzyme counter when the negative wells showed color.
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