Rabies virus antibody ELISA kit

Catalog No. LSY-30012

1.     Introduction

The Rabies Virus (RBV) antibody ELISA kit is used to test rabies virus antibody in serum of dogs, cats, pigs, bovine and goat etc. It is used to evaluate the immune status of rabies virus vaccine or the infection of non immunized animals. This product is only used for veterinary diagnosis reference, not for human clinical diagnosis.

This kit use indirect 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 labeled anti-rabies virus antibody, antibodies in sample combine with the enzyme labeled anti-rabies virus antibodies; 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 wavelength to measure the OD value in reaction wells after adding stop solution to stop the reaction.

2.     Reagents and contents

Code	Components	Specifications & Quantity
		96T	192T	480T
1	Rabies-Ag Coated plates 96wells	1 plate	2 plates	5 plates
2	Enzyme Conjugate	11ml *1	11ml *2	11ml *5
3	10X Concentrated washing buffer	100ml *1	100ml *1	100ml *3
4	Substrate	11ml *1	22ml *1	22ml *2, 11ml *1
5	Sample diluent	15ml *1	15ml *2	15ml *5
6	Stop solution	15ml *1	15ml *1	15ml *3
7	Negative control	2ml*1	2ml*1	2ml*3
8	Positive control	1ml*1	1.6ml*1	1.6ml*3
9	Serum dilution plate	1 piece	2 pieces	5 pieces
10	Adhesive Foil	2 pieces	4 pieces	10 pieces
11	Instruction sheet	1 piece	1 piece	1 piece

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. Serum sample dilution

Dilute serum at 1:25 on Serum dilution plate (for example: 144uL Sample diluent+ 6uL serum)

Notice: Negative control and Positive control do not need dilute. Change tips after adding each sample, mark and record the sample position on plate correctly. Each sample should be gently mixed to evenly before adding to micro-well of Rabies-Ag Coated plate.

7. Notes

1) Return all reagents into room temperature before use, put all reagents in the kit at room temperature for at least 1hour. Shake it evenly before use, and store back to 2-8℃ after usage.

2) When add samples by using pipette, change tips for each sample.  

3) Do not mix use reagents from different kits and different lot no., prevent the reagents been polluted when using.

4) Stop solution may have irritation to skin and eyes, be careful to use.

5) Do not expose Substrate to strong light and avoid contact with the oxidant.

6) RBV-Ag coated plates should be sealed and moisture-proof. Put back unused MicroWell plate into dry foil bag and sealed at 2~8 ℃.

7) RBV-Ag coated plates is disposable, do not repeat use. All wastes should be treated well to avoid  pollution before discarding.

8) Strict compliance with the operating instructions can get the best results. Pipetting operation, timing, and washing of the whole process must be precise.

8. Test procedure

1) Take out the Rabies-Ag Coated plates (According to the number of samples, they can be disassembled and used in batches), add the diluted serum sample into micro-wells, 100uL/well; Meanwhile, for every test, set 2 wells for positive control and 1 well for negative control, add positive control 100Ul/well and negative control 100uL/well into their wells accordingly; gently shake to mix sample evenly(do not spill).

2) Cover it with Adhesive Foil, incubate at 37 ℃ for 30 minutes;

3) 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;

4) Adding Enzyme Conjugate,100ul/well, cover it with Adhesive Foil, incubate at 37 ℃ for 30 minutes;

5) Open the adhesive foil, discard the liquid of the well, washing for 4-6 times as step 3), remember at last flap to dry with the absorbent paper;

6) Add substrate, 100ul/well, mix it evenly then cover it with Adhesive Foil, incubate at 37 ℃ in dark for 15 minutes;

7) Add stop solution, 50ul/well to stop the reaction, measure the OD value in 10 minutes with reader at 450nm wavelenth.

9. Results judgment

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

For the assay to be valid:

OD value of negative control(N) ＜ 0.2, meanwhile OD value of positive value (P) > 0.5

Calculate method:

S/P value = OD value of Sample / average OD value of Positive control

Results interpretation

S/P value≥ 35%: Positive

S/P value＜ 35%: Negative

(Note: when S/P =35%, it means antibody titer 0.5 IU/ml )

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