Toxoplasma gondii antibody ELISA kit for bovine, sheep   

Catalog No. LSY-30038

1.Introduction

This kit is used to detect Toxoplasma gondii (TOX) antibody in bovine, sheep serum.

This kit use indirect ELISA method, TOX antigen is pre-coated on enzyme micro-well strips. When testing, add diluted serum sample, after incubation, if there is TOX specific antibody, it will combine with the pre-coated antigen, discard the uncombined antibody and other components with washing; then add enzyme labled anti-TOX virus antibody, combine with specific antigen-antibody complex; discard the uncombined enzyme conjugate with washing; Add TMB substrate in micro-wells, there will be blue product, use ELISA reader at 450nm wavelength to measure the absorbance A value in reaction wells after adding stop solution to stop the reaction.

2.Component

3.Materials required but not provided

1) Micropipettors and disposable tips: 0.5μL~10μL、10μL~100μL、100μL~1000μL

2) Disposable pipette tips

3) Measuring cylinder: 500 ml

4) 96 wells microplate reader

5) Distilled water or deionied water

6) Bottle or microplate washing machine 

4. Sample preparation

Take animal whole blood, make serum according to regular methods, the serum should be clear, have no hemolysis.

5. Preparation of washing buffer

Return 10XConcentrated washing buffer to room temperature before use, if there is salty crystals, shake to make the crystals dissolve, then use distilled water or deionied water to dilute it at 10 times. The diluted washing solution can store for 1 week at 4 ℃.

6. Sample dilution

At serum dilution plate, dilute serum at 1:100 with sample diluent. 

Notice: Negative control and Positive control do not need dilute. Exchange tip after taking sample every time, record the situation of the sample on plate accurately. Shake the sample evenly before adding it.

7. Notes

1) All reagents should return to the room temperature and shake evenly before using, store back to 2-8 ℃after using

2) Do not exchange the reagents from the kits of different lot numbers to use. Avoid reagent pollution when using.

3) Substrate and stop solution may have excitant to skin and eyes, pay attention when using.

4) Do not expose Substrate to strong light and avoid it contact with antioxidants.

5) The wells should avoid damp or touching water after unsealing (Put the un-using microplate back to bag with dehydrator in 2~8 ℃soon )

6) Deal all waste reasonable before dumping to avoid pollution.

7) Strictly adhere to instruction to get best result. All procedure including pipetting, timing and washing etc. must be accurate.

8) Serum dilution plate is disposable, do not use for second time; the MAX volume of it is 300μL/well.

8. ELISA procedure

1) Take pre-coated microplate (Can unseal for several time use as per sample quantity), add 100μL diluted serum to sample well, meanwhile set 2 wells for Negative control, 1 well for Positive control wells separately. Add 100 μL Negative/Positive control to its wells. Shake softly (do not spill),

2) Cover andincubate at 37℃ for 30 min.

3) Pour the liquid out of the wells, add 250 μL diluted washing solution to each well, pour out. Repeat 4-6 times, then pat to dry on absorbent paper.

4) Add 50 μL Enzyme conjugate to each well, andincubate at 37℃ for 30 min.

5) Repeat the step 3(washing). Remember pat to dry on absorbent paper at last.

6) Add 100 μL substrate to each well, mix properly,react for 10 min at 37℃ in dark.

7) Add 50 μL stop solution in each well, and measure the result within 10 min.

9. Results

Read OD value with ELISA Reader at 450nm (630nm as a reference) .

For the test to be valid:

OD value of Negative Control (N) <0.20, meanwhile OD value of Positive control (P)> 0.5

Result judgment

If the sample’s A450 value is greater than 0.25+ absorbance of negative control mean, it is judged to be positive; and if less than 0.25 + absorbance of negative control mean, negative. If absorbance of negative control mean is less than 0.05, calculate as 0.05

Expiry date:12 months.

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