Swine Foot and Mouth Disease IgG Distinguishing Test Kit
Catalog No. LSY-30003
1. Usage
The GreenSpring® Swine Foot and Mouth Disease Distinguishing ELISA Test kit, or called FMD-3ABC ELISA kit is used for detection the non-structural 3ABC-protein of swine foot and mouth disease (FMD-3ABC) antibodies in the serum, and differentiation naturally infected from vaccined swine.
2. Brief Introduction
The GreenSpring® FMD-3ABC test kit is made of the antigen coated microplate, enzyme conjugation (HRP goat-anti-pig IgG) and other reagents, which applies the indirect ELISA principle to test the antibodies against FMD-3ABC in the serum.
3. Reagents
1 3ABC Antigen coated microplate....................................... 2 pieces (96 wells)
2 Negative (green lid)/positive (red lid) serum............. 1 tube each (1.5 mL/tube)
3 3 ABC Enzyme conjugate (yellow lid)........................... 1 bottle (22 mL/bottle)
4 20× concentrated washing solution (white lid)............... 1 bottle (50 mL/bottle)
5 Substrate A (purple lid)/B (yellow lid).................... 1 bottle each (12 mL/bottle)
6 Stop solution (blue lid)................................................ 1 bottle (12 mL/bottle)
7 Sample diluent solution (transparent lid)....................... 1 bottle (25 mL/bottle)
8 Adhesive Foil................................................................................. 6 pieces
9 Instruction....................................................................................... 1 piece
(Note: Never mix up reagents from test kits in different batch number)
4. Material Required But not Provided
- Microplate Reader(single-wave length: 630 nm).
- Microplate Washer.
- Micropipettes, adjustable.
- Constant temperature box or incubator.
- Oscillator.
5. Sample requirement
1. The test sample is porcine serum, collecting sample without bacteria, store at 2℃～8℃ for less than a week, store at lower than -20℃ for long-term storage.
2. Avoid using sample of severe hemolysis, sediments, containing suspended long fibrin and pollution bacteria.
3. Samples with conventional dosage of EDTA, sodium citrate or sodium heparin anticoagulant do not affect the experiment.
6. Test Procedure
- Adding sample: Take out the required coated plates according to sample quantity (Can be detached) and record the sample position on a worksheet. Set one blank control well (Add nothing); Set 2 wells for negative control serum and 2 wells for positive control serum, add undiluted negative and positive control serum to its well accordingly, 100 μL/well. Others are sample wells, use sample diluent to dilute sample at 1: 200, add the diluted sample to each well, 100 μL each well.
- Incubation: mix and shake for 10s after adding sample, incubate at 37℃ for 30 min.
- Wash each well with 300 μL washing buffer (dilute 20×concentrated washing buffer with deionized water at 1:20) for 5 times, and each static for 1 min. The last time, pat to dry on absorbent paper.
- Add 100 μL enzyme comjugate into each well, and incubate at 37℃ for 30 min.
- Repeat step 3.
- Add 50 μL substrate A and 50 μL substrate B into each well, mix properly, Color for 10 min at room temperature (25℃) in the dark.
- Add one drop stop solution (50 μL) into each well, and determine the result within 10 min.
- Read A value of each well with ELISA Reader at single-wave length: 630nm, set zero at blank control well. Note: if haven’t set zero at blank control well before reading, then the result should minus A value of blank control well.
7. Results
- Generally speaking, the 3ABC-Positive control OD630nm value should be equal or greater than 0.7, the 3ABC-Negative control OD630nm value should be less than 0.1.
- Results:
If OD630nm value ≥0.55, the sample is classified as POSITIVE for 3ABC antibodies;
If OD630nm value＜0.5, the sample is classified as NEGATIVE for 3ABC antibodies.
Between 0.5-0.55, it is skeptical.
9. Interpretation of the result
1. Severe hemolysis, fiber protein in the serum separation is not sufficient, containing erythrocytes, a precipitate, a sample with bacteria may lead to false positive.
2. Negative results may occur on individual pigs after vaccines due to individual differences or immune duration.
3. Positive results for serological diagnosis and epidemiological investigation of swine to be combined with other methods and clinical data.
10. Product performance
- Specificity: to test 30 negative control serums, no false positive.
- Sensitivity: to test 30 positive control serums, no negative.
- Precision: CV（%）no bigger than 15%（n=10）
- Stability: Store at 2℃～8℃ for 6 months or store at 37℃ for 3 days, the result can reach the above 3 standard.
11. Precautions and warnings for users
1. This test kit is suitable for in vitro diagnostics.
2. Wear glove and working clothes when operate, treat the test kit as containing infectious material.
3. Experiment rubbish should be dealt with high pressure steam sterilization at 121 ℃ for 30 minutes, or treated with 5.0g/L sodium hypochlorite disinfectant for 30 minutes, then discard.
4. MicroWell plate removed from the refrigerated environment should be balanced moisture to dry at room temperature, then can be opened. Put back unused MicroWell plate into dry foil bag and sealed at 4 ℃. Unused liquid reagent should cover caps, store at 2-8 ℃ in dark with other group components.
5. If the 20×concentrated washing buffer appears crystal, it is normal, put at 37℃ until been dissolved.
6. Should use Micropipettor to add sample and reagents, and often proof its accuracy.
7. When adding washing buffer, should be full but no overflow, avoid appearing free enzyme at mouth of well or cross pollution between wells.
8. Stop solution is corrosive, use large amount of water to wash immediately when touch the skin or clothes.
Specifications: 96 wells × 2.
Expiry date: 6 months.
Storage: at 2-8 ℃, don’t expose in strong light.
Production Date: On outer-packing of the test kit.
Shenzhen Lvshiyuan Biotechnology Co., Ltd
D Building, National Biological Industrial Park of Marinelife, No.2 Binhai Road, Dapeng, Shenzhen, 518120 China
Tel. 86-755-28438788
Fax 86-755-28938800
www.lsybt.com
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