Brucellosis antibody diagnostics for bovine, sheep. pig and dog etc. brucella abortus elisa kit

Brucellosis antibody ELISA kit for bovine, goat, sheep, camels, pig and dog

Catalog No. LSY-30036

1. Introduction

The Brucellosis antibody ELISA kit is used to test Brucellosis antibody in serum of bovine, goat, sheep, camels, pig and dog etc.

This kit use competitive ELISA method, the purified Brucella abortus antigen is pre-coated on enzyme micro-well strips. When testing, add diluted serum sample and Monoclonal antibody working solution, after incubation, if there is Brucellosis specific antibody in the sample, it will compete with antibody in Monoclonal antibody working solution to combine with the pre-coated antigen, discard the uncombined antibody and other components by washing; then add Enzyme Conjugate, the enzyme reacts with the monoclonal antibody that binds on the plate, discard the uncombined enzyme conjugate by 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 wavelength to measure the absorbance OD value in reaction wells after adding stop solution to stop the reaction.

2. Reagents and contents

3. Material required not provided

1) Micropipette: 0.5ul-10ul, 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. Preparation of Monoclonal antibody working solution

Monoclonal antibody working solution: dissolve the Lyophilized monoclonal antibody with 6ml diluted washing buffer for each bottle when use.

7. Notes

1) Return all reagents into room temperature before use, put all reagents at room temperature for at least 1 hour. 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.

8. Test procedure

1. Set one well for positive control and two wells for negative controls for each testing;
2. Add Sample diluent to each reaction well, 45ul/well; then add negative control, positive control, serum sample into it’s well accordingly, 5ul/well; then add Monoclonal antibody working solution to each well, 50ul/well, gentle shake and mix it evenly(do not mix using tips);
3. Cover the plate with Adhesive Foil, incubate at (25±3) ℃ for 45 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 Enzyme Conjugate, 100ul/well, cover the plate with Adhesive Foil, incubate at (25±3) ℃ for 30 minutes;
6. 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;
7. Add substrate, 100ul/well, mix it evenly then cover it with Adhesive Foil, incubate at (25±3) ℃ in dark for 10 minutes;
8. Add stop solution, 50ul/well to stop the reaction, measure the result in 10 minutes.

9. Results judgement

Read the OD value by ELISA Reader at 450nm (630nm as reference).

For the assay to be valid:

OD value of negative control(N) > 0.6, OD value of positive value (P)/ average OD value of negative control(N) ＜0.3.

Calculate method:

PI(blocking rate)= {1- (Sample OD value/ Negative control OD average value)}x 100%

Results interpretation

PI(blocking rate)> 70%: Positive

PI(blocking rate)≤ 70%: Negative

10. Storage and expire date

Store at 2~8℃ in dark, no frozen, expiry date: 12 months.

Shenzhen Lvshiyuan Biotechnology Co., Ltd

D Building, National Biological Industrial Park of Marinelife, No.2 Binhai Road, Dapeng, Shenzhen, 518120 China

Tel. 86-755-28438788

Fax 86-755-28938800

Email: info@lsybt.com

www.lsybt.com