This kit is for research use only.

96T

Purpose of use:

This kit is used to determine bluetongue virus antibodies in sheep serum, plasma, and related liquid samples（BLU Ab）Expressing.

Experimental Principle

This reagent kit is designed for dual useantigenSandwich method determinationspecimenmiddleSheep Blue Tongue Virus Antibody（BLU Ab）ExpressingUsing purifiedantigenCoated microporous plate, made into solid phaseantigen，Can interact with bluetongue virus antibodies in the sample（BLU Ab）Combined, washed to remove unbound antibodies and other components, and then combined withHRPBinding of labeled antigens to form antigens-antibody-Enzyme linked antigen complexAfter washingplussubstrateTMBColor rendering.TMBinHRPUnder the catalysis of enzymes, it is converted into blue, and under the action of acid, it is converted into the final yellow. Using an enzyme-linked immunosorbent assay (ELISA) reader450nmMeasure absorbance at wavelength（ODValue), withCUTOFFCompare the values to determine the presence of antibodies against sheep bluetongue virus in the specimen（BLU Ab）Whether it exists or not.

Kit components

1	30Concentrated washing solution	20ml×1bottle	7	Stop Solution	6ml×1bottle
2	Enzyme linked immunosorbent assay (ELISA) reagents	6ml×1bottle	8	positive control	0.5ml×1bottle
3	enzymeThe package is labeled asboard	12Kong×8item	9	negative control	0.5ml×1bottle
4	samplediluent	6ml×1bottle	10	Instruction manual	1copy
5	Chromogenic agentAliquid	6ml×1bottle	11	Sealing film	2Zhang
6	Chromogenic agentBliquid	6ml×1/bottle	12	sealed bag	1a

Specimen requirements

1Extract the specimen as soon as possible after collection, according to relevant literature, and conduct experiments as soon as possible after extraction. If the experiment cannot be conducted immediately, the specimen can be placed in-20Stored at ℃, but shouldAvoid repeated freeze-thaw cycles

2. Samples containing NaN3 cannot be detected becauseNaN3Inhibition of horseradish peroxidase（HRP）Activity.

operating steps

Number: Number the corresponding micropores of the sample in sequence, and each plate should have a negative control2Kong, positive control2Hole and blank control1Hole (blank control hole without sample or enzyme-linked immunosorbent assay, all other steps are the same)

Sample addition: Add negative control and positive control to the negative and positive control wells respectively50μlThenSample hole to be testedAdd sample diluent first40μlThen add the sample to be tested10μlAdd the sample to the bottom of the enzyme-linked immunosorbent assay plate well, avoiding contact with the well wall as much as possible,gentlyshakeMix well,

Incubation: Seal the plate with a sealing film and place it on the back37Incubate at 3 ℃0minute.

Liquid preparation: will30Concentrated washing solution with distilled water30Dilute twice before use

Washing: Carefully remove the sealing film and discard the liquid,spin-dryFill each hole with detergent and let it stand30Abandon in seconds, repeat like this5Next time, shoot dry.

Enzyme addition: Add enzyme labeled reagent to each well50MlExcept for blank holes.

Incubation: same operation3.

Washing: same operation5.

Color development: Add color developer to each hole firstA50MlAdd color developer againB50MlGently shake and mix well,37Color development in the dark for 10 minutes at ℃

Termination: Add termination for each holeliquid50μlTerminate the reaction(At this moment, blue turns to yellow).

Measurement: Zero with blank hole,450nmMeasure the wavelengths of each hole in sequenceabsorb lightDegree（ODValue). The measurement should be conducted after adding the termination solution15Conducted within minutes.

Calculation and result determination:

Experimental effectiveness: average value of positive control wells≥1.00;Mean value of negative control≤0.20

Critical value（CUT OFF）Calculation: critical value=Mean value of negative control wells+0.15

Negative judgment: sampleODvalue<Critical value（CUT OFF）It is an antibody against bluetongue virus（BLU Ab）Negative

Positive judgment: sampleODvalue≥Critical value（CUT OFF）It is an antibody against bluetongue virus（BLU Ab）Positive.

Precautions

1Strictly follow the instructions for operation, and components from different batch numbers of this reagent must not be mixed.

2The reagent kit should be taken out of the refrigerated environment and equilibrated at room temperature15-30Minutes later, the enzyme label coated plate can be used. If it is not used up after opening, the Flat noodles should be stored in a sealed bag.

3．Concentrated detergent solutionpossiblethere will becrystallizationPrecipitation can be dissolved by heating in a water bath during dilutionWashing does not affect the results.

4.The sealing film is only for one-time use to avoid cross contamination.

5Please store the substrate away from light.

6The determination of experimental results must be based on the reading of the enzyme-linked immunosorbent assay (ELISA) reader. When using dual wavelength detection, the reference wavelength is630nm

7All samples, detergents, and various waste materials should be treated as infectious agents. Termination liquid is2MWhen using sulfuric acid, safety must be taken into account.

Storage conditions and expiration date

1．Storage of reagent kit:；2-8℃.

2．Validity period:6month