Rabies virus antibody ELISA kit for Dogs and Cats
1. Introduction
The Rabies Virus (RBV) antibody ELISA kit is used to test rabies virus antibody in serum of dogs, cats etc., used to assess the status of rabies vaccination.
This kit use block ELISA method, rabies antigen is pre-coated on enzyme micro-well strips. When testing, add diluted serum sample, after incubation, if there is rabies virus specific antibody, it will combine with the pre-coated antigen, discard the uncombined antibody and other components with washing; then add enzyme labled anti-rabies 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 | RBV-AgCoatedplates | 1/2 plate of 96 wells | 6 | Stop solution | 11 ml |
| 2 | EnzymeConjugate | 11/22 ml | 7 | Negative control | 1/2ml |
| 3 | 10X Concentrated washing buffer | 100 ml | 8 | Positive control | 0.5/1 ml |
| 4 | Substrate | 11/22 ml | 9 | Adhesive Foil | 2/4 pieces |
| 5 | Sample diluent | 100 ml | 10 | Instruction sheet | 1 piece |
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 A 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) RBV-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 pullution 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) RBV-Ag coated plates is disposable, do not repeat use.
7.Test procedure
1) For every test, set 1 well for positive control and 2 wells for negative control, negative control and positive control do not need to dilute, directly add to its corresponding wells, 100ul/well;
2) Add sample diluent into reaction wells, 80ul/well, then add serum, 20ul/well, blow and beat it to even. (Do not mix use the tips);
3) Cover it with Adhesive Foil,incubateat 37 ℃ for 30 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 5 times, at last flap to dry with the absorbent paper;
5) Adding Enzyme Conjugate,100ul/well, cover it with Adhesive Foil,incubateat 37 ℃ for30 minutes;
6) Open the adhesive foil, discard the liquid of the well, washing for 5 times as step 4), remember at last flap to dry with the absorbent paper;
7) Add substrate, 100ul/well, mix it evenly then cover it with Adhesive Foil,incubateat 37 ℃in darkfor15 minutes;
8) Add stop solution, 50ul/well to stop the reaction, measure the result in 10 minutes.
8. Results judgment
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 > 60%
Calculate method:
PI(blocking rate)= 1- (Sample OD value/ Negative control OD average value)
Resultsinterpretation
PI(blocking rate)> 60%: Positive
PI(blocking rate)≤60%: Negative
(Note: when PI=60%, it means antibody titer 0.5 IU/ml )
9. Storage and expire date
Store at 2~8℃ in dark, no frozen, expiry date: 12 months.