SwineTrichinella spiralis (T.s) AntibodyELISATest Kit
1. Principle
This kit use indirect enzyme-linked immunoassay (ELISA) to detect Trichinella spiralis (T.s) antibody in Swine serum or plasma qualitatively, assist the detection of swineTrichinella spiralis.
If there is Trichinella spiralis (T.s) antibody in the sample, it will combine with coated Trichinella spiralis (T.s) antigen on plate and enzyme conjugate on the second step. The color reaction will reflect whether there is Trichinella spiralis (T.s) antibody in the sample.
2. Reagents and contents
| Code | Item | Spec. | Code | Item | Spec. |
| 1 | T.s-AgCoatedplate | 2*96 wells | 7 | Stop solution | 15 ml |
| 2 | EnzymeConjugate | 12 ml | 8 | Positive control | 1ml |
| 3 | 10X Washing solution | 100 ml | 9 | Negative control | 1ml |
| 4 | Substrate A | 15 ml | 10 | Adhesive Foil | 6 pieces |
| 5 | SubstrateB | 15 ml | 11 | Instruction sheet | 1 piece |
| 6 | Sample diluent | 100 ml | 12 | Serum diluent plate | 2 piece |
3.Materialrequired notprovided
1) Microplate Reader with 450nm and 630nm
2) 37 ℃ thermostatic device
3) Micropipettes, adjustable.
4. Samplerequirement
1) This kit only used to testing swine serum or plasma.
2) To get the best test result, avoid using sample with severe hemolysis, precipitate, contaminated by bacteria or protein suspension.
3) The serum sample can store at 2-8 ℃for short term (in 3 days), if for long term, it should be kept at -20℃or lower, avoid repeated freezing and thawing.
5.Test procedure
1) Washing Solution preparation: Dilute 10X Washing solution with distilled water or deionized water at 1:10 (for example: take 100ml 10X Washing solution, add 900ml distilled water or deionized water, mix),mix it evenly to get washing solution.
2) Sample dilute: Dilute serum sample with sample diluent at 1:100 (for example: 5ul serum + 495ul sample diluent), mix it evenly, add the diluted serum sample to sample wells, 100ul/well.
If use Serum diluent plate to dilute sample, the method is as following:
Dilute at 1:100: firstly, add sample diluent into sample wells on T.s-Ag Coated plates, 50ul/well. Add 245ul sample diluent into Serum diluent plate, then add 5ul serum, use pipettes blow and beat evenly, then take 50ul into T.s-Ag Coated plates for react.
(Note: Serum diluent plate is disposable, do not repeat use)
3) Adding controls:Positive control and Negative control do not need to dilute, add it directly; Set 2 wells for negative control, only add 100ul negative control; 2 wells for Positive control, only add 100ul Positive control. Cover plate with adhesive foil,incubate in dark at 37℃for 30 minutes.
4) Washing plate: discard the liquid of the well, fill full with washing solution to each well, washing for 3 times, be static for 1 min for each time; at last time, flap to dry with the absorbent paper.
5) Adding Enzyme Conjugate: add Enzyme Conjugate to each well, 50ul/well, Cover plate with adhesive foil,incubate in dark at 37℃for 30 minutes. Discard liquid of the wells and wash 3 times as described in step 4).
6). Adding substrates: Measure as required, take equal volume of Substrate A and Substrate B mix evenly, then add the mixture 100ul/well. Cover plate with adhesive foil,Incubate at 37℃in dark for 10 min.
7). Stop reaction: Add 50ul Stop solution into all wells to stop reaction. Read the OD value at ELISA reader 450nm (630nm as a reference).
6. Reference
1) Cut-off Value (COV) = Average OD Value of Negative control X 2.1 (calculate as 0.08 when the OD Value of Negative control < 0.08).
2)Validation judge
Negative control OD value≤0.20 (If more than 0.20, the test is invalid)
Positive control OD value ≥ 0.50 (If less than 0.50, the test is invalid)
7. Resultsinterpretation
Sample OD value ≥ COV: Trichinella spiralis (T.s) antibody positive;
Sample OD value < COV: Trichinella spiralis (T.s)antibody negative.
Limitation: This kit is for research use only, not for clinical diagnosis.
8. Notes
1) Return to room temperature before use. Seal the unused micro-well strip and store at 2~8℃.
2) Mix reagent evenly before adding sample.
3) Before incubation, seal the plate with adhesive film, do not repeat use adhesive film.
4) Tighten all caps after use, don’t mix caps. Never mix up reagents and manual from test kits in different batch number.
5) The wasted kits and sample should be treated as infection agent. The stop reaction solution is corrosive, be careful to use.
9. Storage and expire date
Store at 2~8℃ in dark,no frozen, expiry date: 12 months.