Foot-and-mouth disease virus (FMDV) Type Asia-ⅠAntibody ELISA Test Kit

Catalog No. LSY-30033

1. Principle

This kit is used to detectspecific antibody againstfoot and mouth disease virus (FMDV)TypeAsia-ⅠAntibody in serum of pig, cattle and sheepqualitatively, formonitoring antibody after FMDV Type Asia-Ⅰimmune.

This kit use indirect ELISA method, the purified FMD virus antigen ispre-coated on enzyme micro-well strips. In the test,add the diluted serum, and after incubation, if the sample contains FMD virus-specific antibodies, it will bind to the antigen on the coated plate, and after washing to remove unbound antibodies and other components; Add the enzymeconjugate to specifically bind to the antigen-antibody complex on the detection plate; then wash to remove the unbound enzyme conjugate; add the substrate to the microwell, and the enzyme catalyzes the formation of a blue product, andadd stop solution to terminate the reaction, the absorbance A value in the reaction well was measured with a microplate reader at a wavelength of 450 nm.

2.Reagents and contents

3.Materialrequired notprovided

1)Micropipette: 0.5-10ul, 10ul-100ul, 100ul-1000ul.

2) Disposable pipette tips.

3) Graduate: 500ml.

4) Microplate Reader: 96 wells with 450 wavelength.

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 dissovled, 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. Sample dilution

Dilute serum at 1:100 on Serum dilute plate.

Note: both positive control and negative control do not need dilute, change tips after taking every sample, mark the sample location on plate accurately, mix every sample evenly before adding to the pre-coated micro-wells.

7. 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 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) FMD-Ag coated platesshould be sealed andmoisture-proof. Put backunused Micro-Well plateintodry foil bagandsealedat 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) Serum dilute plate is disposable, do not repeat use; the max volume of the plate is 300ul/well.

8.Test procedure

1) Take the antigen coated plate(the plate can be open and used for several times according to sample quantity each time), add the diluted serum to reaction wells, 100ul/well; meanwhile, set 2 wells for positive control and 2 wells for negative control, take 100ul directly and add into its well, mix gently(do not overflow);

2) Cover it with Adhesive Foil,incubateat 37 ℃ for30 minutes;

3) Open the adhesive foil,discard the liquid of the well, add diluted washing buffer to each well, 250ul/well, thendiscard the liquid, repeat the above step for 4-6 times, at lastflap to dry with the absorbent paper;

4) Adding EnzymeConjugate 100ul/well, Cover it with Adhesive Foil,incubateat 37 ℃ for30 minutes;

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

6) Add substrate: add substrate, 100ul/well, mix it evenly then cover it with Adhesive Foil,incubateat 37 ℃in darkfor10 minutes;

7) Add stop solution 50ul/well to stop the reaction, measure the result in 10 minutes.

9. Results judgment

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

For the assay to be valid:

Negative control (N) OD value< 0.2, meanwhile positive control (P) OD value > 0.3;

Calculate method:

(Sample OD value - Negative control OD average value)/(Positive control OD value - Negative control OD average value) = IRPC value

Resultsinterpretation

IRPC<0.35: Negative

0.35≤IRPC<0.4: Skeptical

IRPC≥0.4: Positive

9. Storage and expire date

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