Goat ORFV-Ab ELISA Kit

INTENDED USE

This ORFV-Ab ELISA kit is intended Laboratory for Research use only

and is not for use in diagnostic or therapeutic procedures.The Stop Solution

changes the color from blue to yellow and the intensity of the color is measured

at 450 nm using a spectrophotometer. In order to measure the concentration of

ORFV-Ab in the sample, this ORFV-Ab ELISA Kit includes a set of calibration

standards. The calibration standards are assayed at the same time as the samples

and allow the operator to produce a standard curve of Optical Density versus

ORFV-Ab concentration. The concentration of ORFV-Ab in the samples is then

determined by comparing the O.D. of the samples to the standard curve.

PRINCIPLE OF THE ASSAY

The kit assay ORFV-Ab level in the sample，use Purified antigen to coat

microtiter plate wells, make solid-phase antigen, then add ORFV-Ab to wells,

Combined With ORFV-Ab, after washing and removing non-combinative

antibody and other components ,then Combined antigen which with HRP

labeled become antigen - antibody - enzyme- antigen complex, after washing

Completely, Add TMB substrate solution,, TMB Chromogen Solution Becomesblue color At HRP enzyme-catalyzed, reaction is terminated by the addition of a

sulphuric acid solution and the color change is measured spectrophotometrically

at a wavelength of 450 nm. Compared with the CUTOFF value, according to

this to judge ORFV-Ab exist in the sample or not.

REAGENTS PROVIDED

All reagents provided are stored at 2-8° C. Refer to the expiration date on

the label.

1. MICROTITER PLATE 96 wells

2. Sample diluent

6.0 mL 1 tube

3. Negative control

0.5ml 1 tube

4. Positive control

0.5ml 1 tube

5. STREPTAVIDIN-HRP 6.0 mL 1 tube

6. Chromogen Solution A 6.0 mL 1 vial

7. Chromogen Solution B 6.0 mL 1 vial

8. STOP SOLUTION 6.0 mL 1 vial

9. WASH SOLUTION x30 20 mL 1 vial

10. Instruction 1

SAMPLE COLLECTION AND STORAGE

Serum- Use a serum separator tube(SST) and allow samples to clot for

30minutes before centrifugation for 15minutes at approximately 1000 x

g.Remove serum and assay immediately or aliquot and store samples at -20 °C

or -80°C.

Plasma - Collect plasma using EDTA or heparin as an

anticoagulant.Centrifuge samples for 15 minutes at 1000 x g at 2-8°C within

30minutes of collection.Store samples at -20°C or -80°C.Avoid repeated

freeze-thaw cycles.

Cell culture fluid and other biological fluids - Remove particulates by

centrifugation and assay immediately or aliquot and store samples at -20°C or

-80°C.Avoid repeated freeze-thaw cycles.

NOTE: Serum, plasma, and cell culture fluid samples to be used within 7

days may be stored at 2-8°C, otherwise samples must stored at -20°C(≤2months)

or -80°C(≤6months) to avoid loss of bioactivity and contamination. Avoid

freeze-thaw cycles .When performing the assay slowly bring samples to room

temperature.

DO NOT USE HEAT-TREATED SPECIMENS.

MATERIALS REQUIRED BUT NOT SUPPLIED

1. Microplate reader capable of measuring absorbance at 450 nm.

2. Precision pipettes to deliver 2 ml to 1 ml volumes. .

3. Adjustable 10ml -100ml pipettes for reagent preparation.

4. 100 ml and 1 liter graduated cylinders.

5. Calibrated adjustable precision pipettes, preferably with disposable plastic

tips. (A manifold multi-channel pipette is desirable for large assays.)

6. Absorbent paper.

7. 37°C incubator.

8. Distilled or deionized water.

9. Data analysis and graphing software..

10. Tubes to prepare standard or sample dilutions.

PRECAUTIONS

1. Do not substitute reagents from one kit lot to another. Standard, conjugate

and microtiter plates are matched for optimal performance. Use only the

reagents supplied by manufacturer.

2. Allow kit reagents and materials to reach room temperature (20-25°C) before

use. Do not use water baths to thaw samples or reagents.

3. Do not use kit components beyond their expiration date.

4. Use only deionized or distilled water to dilute reagents.

5. Do not remove microtiter plate from the storage bag until needed. Unused

strips should be stored at 2-8°C in their pouch with the desiccant provided.

6. Use fresh disposable pipette tips for each transfer to avoid contamination.

7. Do not mix acid and sodium hypochlorite solutions.

8. Serum and plasma should be handled as potentially hazardous and capable of

transmitting disease. Disposable gloves must be worn during the assay

procedure, since no known test method can offer complete assurance that

products derived from human blood will not transmit infectious agents.

Therefore, all blood derivatives should be considered potentially infectious

and good laboratory practices should be followed.

9. All samples should be disposed of in a manner that will inactivate viruses.

10. Solid Waste: Autoclave 60 min. at 121°C.

11. Liquid Waste: Add sodium hypochlorite to a final concentration of 1.0%.

The waste should be allowed to stand for a minimum of 30 minutes toinactivate the viruses before disposal.

12. Substrate Solution is easily contaminated. If bluish prior to use, do not use.

13. Chromogen Solution B contains 20% acetone, keep this reagent away from

sources of heat or flame.

14. Remove all kit reagents from refrigerator and allow them to reach room

temperature ( 20-25°C).

REAGENT PREPARATION

Wash Buffer - If crystals have formed in the concentrate, warm to room

temperature and mix gently until the crystals have completely dissolved. To

prepare enough Wash Buffer for one plate, add 20mL Wash Buffer Concentrate

into deionized or distilled water to prepare 600mL of Wash Buffer.

ASSAY PROCEDURE

Prepare all Standards before starting assay procedure (see Preparation

Reagents). It is recommended that all Standards and Samples be added in

duplicate to the Microtiter Plate.

1. Number: to sample correspond microtitration well and Number Sequence,

each plate should be set feminine comparison 2 wells, masculine comparison 2

wells, blank comparison 1 well(don’t add sample and HRP-Conjugate reagent

to blank comparison well, other each step the operation are same).

2. Add sample：separately add Positive control and Negative control 50μL to the

Positive and Negative well . add Sample dilution 40μl to testing sample well,

then add testing sample 10μl. add sample to the bottom of ELISA plates

coated well , don’t touch the well wall as far as possible, and Gently mix.

3.Incubate: After closing plate with Closure plate membrane ,incubate for 30

min at 37℃.

4. Configurate liquid: 30-fold (or 20-fold)wash solution diluted 30-fold (or

20-fold) with distilled water until 600ml,and reserve.

5. Washing：Uncover Closure plate membrane, discard Liquid, dry by swing,

add washing buffer to every well, still for 30s then drain, repeat 5 times, dry by

pat.

6. Add enzyme：Add HRP-Conjugate reagent 50μL to each well, except the

blank well.

7. Incubate：Operation with 3.

8. Washing：Operation with 5.9. Color：Add Chromogen Solution A 50μL first，then add Chromogen Solution

B 50μL to each well, evade the light preservation for 15 min at 37℃

10. Stop the reaction：Add Stop Solution50μl to each well, Stop the

reaction(the blue color change to yellow color).

11. Assay：take blank well as zero , Read absorbance at 450nm after Adding

Stop Solution and within 15min.

TYPICAL DATA

Determine the result

Test validity: the average of Positive control well≥1.00; the average of

Negative control well ≤0.20.

Calculate Critical(CUT OFF) : Critical= the average of Negative control well +

0.15.

Negative control: sample OD< Calculate Critical(CUT OFF) is ORFV-Ab

Negative control.

Positive control: ample OD≥ Calculate Critical(CUT OFF) is ORFV-Ab

Positive control.