Brucellosis antibody ELISA kit for bovine, goat,sheep, pig and dog
1. Introduction
The Brucellosis antibody ELISA kit is used to test Brucellosis antibody in serum of bovine, goat, sheep, pig and dog etc.
This kit use competitive ELISA method, Brucellosis antigen is pre-coated on enzyme micro-well strips. When testing, add diluted serum sample, after incubation, if there is Brucellosis specific antibody, it will combine with the pre-coated antigen, discard the uncombined antibody and other components with washing; then add enzyme labled anti-Brucellosis virus monoclonal antibody, 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, use ELISA reader at 450nm wavelenth to measure the absorbance A value in reaction wells after adding stop solution to stop the reaction.
2. Reagents and contents
| Code | Item | Spec. | Code | Item | Spec. |
| 1 | Brucellosis-AgCoatedplates 96wells | 1/2 plates | 7 | Stop solution | 15 ml |
| 2 | EnzymeConjugate | 10/20 ml | 8 | Negative control | 200 ul |
| 3 | 10X Concentrated washing buffer | 100 ml | 9 | Positive control | 60 ul |
| 4 | Substrate | 11/22 ml | 10 | Adhesive Foil | 2/4 pieces |
| 5 | Sample diluent | 100 ml | 11 | Instruction sheet | 1 piece |
| 6 | Serum dilution plate | 1/2 pieces |
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3. Materialrequired notprovided
1) Micropipette: 10ul-100ul, 100ul-1000ul.
2) Disposable pipette tips.
3) Graduate: 500ml.
4) Microplate Reader: 96 wells.
5) Distilled water or deionized water.
6) Microplate Washer
4. Sample preparation
Take animal whole blood, get serum by using regular method, the serum should bright and no hemolysis
5. Washing buffer preparation
Return 10X Concentrated washing buffer into room temperature before use, if there is salt crystals, shake to make it dissolved, then dilute it at 10 times with distilled water or deionized water. The diluted washing buffer can store at 4℃for about 1 week.
6. 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) Brucellosis-Ag coated plates should be sealed and moisture-proof. Put back 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) Brucellosis-Ag coated plates is disposable, do not repeat use.
7. Test procedure
1) On serum dilution plate, dilute serum, negative control, positive control at 1:40 (for example: 195ul sample diluent + 5ul serum sample), mix it evenly;
2) Take Brucellosis-Ag Coated plates (open and take the quantity required for use according to sample quantity), add diluted serum into test wells, 20ul/well; meanwhile set 1 well for positive control and 2 wells for negative control, add diluted negative control and diluted positive control into its wells accordingly, 20ul/well;
3) Adding Enzyme Conjugate,80ul/well, shake gentle to mix it evenly, cover it with Adhesive Foil, incubate at 37℃for 60 minutes;
4) Open the adhesive foil, 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;
5) Add substrate, 100ul/well, mix it evenly then cover it with Adhesive Foil,incubateat 37 ℃in darkfor15 minutes;
6) 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 (P) blocking rate > 80%
Calculate method:
PI(blocking rate)= {1- (Sample OD value/ Negative control OD average value)}x 100%
Resultsinterpretation
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.