LSY-30039 Avian leukosis virus(ALV) antigen ELISA kit 96*2 wells/kit

Avian leukosis virus(ALV) antigen ELISA kit    

Catalog No. LSY-30039

1. Introduction

This kit is based on double antibody sandwich enzymatic immunoassay (DAS-ELISA) to detect Avian leukosis virus(ALV) P27 antigen.When testing, add positive control, negative control and samples into its wells, after incubation, if there is Avian leukosis virus(ALV) P27 antigen in sample, it combines with coated antibody on micro-plate, form antigen-antibody complex, otherwise can not. After washing, add enzyme conjugate, combine with antigen-antibody complex. Washing again, add substrate, with reaction there will be blue color,

The darkness of color has positive correlation with ALV P27 antigen in sample, reach the goal of quick test.

2. Reagents

3. Materials required but not provided

ELISA Reader(650nm), deionized water, centrifuge tube, Micropipettors, timer, centrifuge(4000r/min), cotton swab, NaCl, KCl, Na2HPO4, KH2PO4.

4. Notes

1) This kit is for research use only.

2) Do not exchange the reagents from the kits of different lot numbers.

3) Do not use kit out of date.

4) Must use water (like distilled water, deionized water or pure water) according to Pharmacopoeia rule to dilute Concentrated washing buffer.

5) All reagents should return to the room temperature(25±2℃) before use.

6) Put the unused microplate back to foil bag and sealed, store at 2~8 ℃ soon.

7) Avoid to use metal material to load and stir reagents.

8) Do not expose substrate to strong light.

9) Stop solution may have excitant to skin and eyes, pay attention when using.

10) Ensure the Pipetting system is clear and accurate, to ensure solution volume is accurate and do not avoid cross pollution.

5. Procedure

5.1 Reagent preparation

1) 0.01mol/L pH=7.4 PBS buffer: weigh 8.0g NaCl, 0.2g KCl, 1.14gNa2HPO4 and 0.24g KH2PO4, add deionized water to dissolve to 1L.  

2) Washing buffer: return the 25x Concentrated washing buffer to room temperature(20-25℃) before use, shake to dissolve precipitation( best to heat at 37℃incubator for 5-10min), then dilute with deionized water at 25 times (for example: 30ml 25x Concentrated washing buffer + 720ml distilled water). Mix evenly, store diluted washing buffer at 2-8℃ for 7 days.

5.2 Sample preparation

1) Egg white: take whole egg, crush it and directly take 100ul egg white.

2) Cloacal swab: collect sample by cotton swab from chicken cloacal, then put in 1ml PBS buffer (0.01mol/L, pH=7.4), then thaw at blow -20℃ refrigerator for one time, after return to room temperature, take 100ul to test.

3) Vaccine: dissolve vaccine with vaccine diluent, then dilute with 0.01mol/L pH=7.4 PBS buffer for 10-20 times, take 100ul test.

4) Meconium: do not need dilute, take it directly to test, if there is Precipitation or impurities, centrifuge at 2000-3000 r/min for 10min, take 100ul up-layer clear liquid to test.

5.3 ELISA procedure

1) Take pre-coated microplate (Can unseal for several time use as per sample quantity), add 100μL sample to a well, meanwhile set 2 wells for Negative control, 2 wells for Positive control separately. Add 100 μL Negative/Positive control to its wells. Cover and Shake softly for 10s evenly, incubate at room temperature (25±2℃) in dark for 60 min.

2) Pour the liquid out of the wells, add 300 μL diluted washing solution to each well, shake and wash for 4 times, each time for 30s, pour out. At last, pat to dry on absorbent paper.

3) Add 100 μL Enzyme conjugate to each well, cover and incubate at room temperature (25±2℃) in dark for 60 min.

4) Repeat the step 2(washing). Remember pat to dry on absorbent paper at last.

5) Mix substrate A and substrate B at 1:1 evenly, then add mix solution to each well, 100ul/well, cover and incubate at room temperature (25±2℃) for 15 min. Note: use the substrate mix solution in 5 minutes.

6) Add 100 μL stop solution in each well, and measure the result within 5 min. Note: if there is flocculent crystalline in stop solution, heat at 37℃ to dissolve then use.

6. Results

6.1 Use ELISA Reader to read OD650nm value, calculate NC (Average OD value of negative control) and PC(Average OD value of positive control).

Note: NV< 0.200 and 0.500< PC≤2.000, the test is valid, otherwise, re-test again.

6.2 Calculate S/P: S/P=(SC-NC)/(PC-NC), SC is OD value of sample.

6.3 If S/P< 0.17, there is no ALV P27 protein in sample; if S/P≥0.17, there is ALV P27 protein in sample

Specifications: 96*2 wells/kit.

Expiry date: 12 months.

Storage: Storing at 2-8℃, in the dark.