Principle of the Test
The BRU antibody ELISA test kit use to detection of Brucellosis antibodies in the serum of pigs, cattle, sheep and goat .
This kit use competitive ELISA method to pre-coated BRU antigens on microplate wells. When testing, add diluted serum sample and enzyme labled anti-BRU monoclonal antibody, after incubation, if there have BRU antibody, it will combine with the pre-coated antigen, antibody in sample block the combination of monoclonal antibody and pre-coated antigen; discard the uncombined enzyme conjugate with washing; Add TMB substrate in micro-wells, the blue signal by Enzyme catalysis is in inverse proportion of antibody content in sample.
Notes
1) Return all reagents into room temperature before use, shake it evenly before use, and store back to 2-8℃ after usage.
2) Do not mix use reagents from different kits and different lot no., prevent the reagents been polluted when using.
3) Substrate and stop solution may have irritation to skin and eyes, be careful to use.
4) Do not expose Substrate to strong light and avoid contact with the oxidant.
5) pre-coated plates should be sealed and moisture-proof. Put bac unused Microwell plate into dry foil bag and sealed at 2~8 ℃.
6) All wastes should be treated well to avoid pollution before discarding.
7) Strict compliance with the operating instructions can get the best results. Pipetting operation, timing, and washing of the whole process must be precise.
8) pre-coated plates is disposable, do not repeat use.
7. Test procedure
1) Dilute the sample to be tested and negative/positive controls (195ul sample dilution + 5ul sample serum) by 1:40 on the dilution plate and mix ;
2)Take the antigen coated microplate(the plate can be open and used for several times according to sample quantity each time), add the diluted serum to reaction wells, 20ul/well .At the same time set the negative control 2 wells, positive control 1 well, negative and positive control dilution of 20ul were added to the reaction well.
3) Adding Enzyme Conjugate, 80ul/well, shake gentle to mix it evenly, cover it with Adhesive plate sealer, incubate at 37 ℃ for 60 minutes;
4) Open the adhesive plate sealer, discard the liquid of the well, add diluted washing buffer to each well, 250ul/well, discard the liquid, repeat the above step for 4-6 times, at last flap to dry with the absorbent paper;
6) Add substrate, 100ul/well, mix it evenly then cover it with Adhesive plate sealer, incubate at 37 ℃ in dark for 15 minutes;
7) Add stop solution, 50ul/well to stop the reaction, measure the result in 10 minutes.
8. Results judgement
Set zero at blank control well, read the OD value at 450nm (630nm as reference).
For the assay to be valid:
OD value of negative control(N) > 0.5, meanwhile positive value blocking rate > 80%
Calculate method:
PI(blocking rate)=
(1-)× 100%
Results interpretation：
PI(blocking rate)> 70%: Positive
PI(blocking rate)≤ 70%: Negative
9. Storage and expire date
Store at 2~8℃ in dark, no frozen, expiry date: 12 months.