General Information Kit Components Precautions For sera and plasma – dilution at 1/10 :

This kit is used to detect anti-Toxoplasma gondii 1. Do not pipette by mouth. 1. Add :
Reagents*
antibodies in ruminant, dog, cat or pig sera, plasma or - 90 µl of Dilution Buffer 2 to each microwell.
Microplates coated with p30 antigen 2. The substrate solution can be irritating to the skin.
meat juice. Please contact IDvet for use in other species. - 10 µl of the Negative Control to wells A1 and B1.
This indirect ELISA uses the P30 antigen of Toxoplasma 3. The stop solution (0,5 M) may be harmful if - 10 µl of the Positive Control to wells C1 and D1.
Concentrated Conjugate (10X)
gondii. swallowed. It may cause sensitisation by skin contact - 10 µl of each sample to be tested to the remaining
Positive Control (R22-43). Avoid contact with skin (S24-37). wells.
Description and principle
Negative Control For meat juice – dilution at 1/2:
4. Do not expose the substrate solution to bright light
Microwells are coated with the P30 antigen of Dilution Buffer 2 nor to oxidating agents.
1. Add:
Toxoplasma gondii.
Dilution Buffer 3 5. All single-use material used for the assays should be
- 90 µl of Dilution Buffer 2 and 10 µl of the Negative
Samples to be tested and controls are added to the decontaminated by immersion in freshly prepared 5% Control to wells A1 and B1.
Concentrated Wash Solution (20X)
wells. Anti-Toxoplasma antibodies, if present, form an sodium hypochlorite for minimum 1 hour before - 90 µl of Dilution Buffer 2 and 10 µl of the Positive
antigen-antibody complex. Substrate Solution elimination, or by autoclaving at 120°C. Control to wells C1 and D1.
- 50 µl of Dilution Buffer 2 and 50 µl of each sample
After washing, a multi-species peroxidase (HRP) Stop Solution (0,5 M) Sample Preparation to be tested to the remaining wells.
conjugate is added to the wells. It fixes to the antibodies,
* Quantities supplied are indicated on the kit label. For all applications:
forming an antigen-antibody-conjugate-HRP complex. In order to avoid differences in incubation times between
After elimination of the excess conjugate by washing, the
1. The conjugate, the controls and the substrate specimens, it is possible to prepare a 96-well plate 2. Incubate 45 min ± 4 min at 21°C (± 5°C).
solution must be stored at 5°C (± 3°C). containing the test and control specimens, before
substrate solution (TMB) is added. 3. Empty the wells. Wash each well 3 times with
transferring them into an ELISA microplate using a
2. Other reagents can be stored between +2°C and approximately 300 µl of the Wash Solution. Avoid
The resulting coloration depends on the quantity of multichannel pipette.
+26°C. drying of the wells between washings.
specific antibodies present in the specimen to be tested:
3. Components bearing the same name (wash Wash Solution Preparation 4. Prepare the Conjugate 1X by diluting the Concentrated
- in the presence of antibodies, a blue solution
solution, dilution buffers) can be used for the entire Conjugate 10X to 1/10 in Dilution Buffer 3.
appears which becomes yellow after addition of
IDvet product range. If necessary, bring the Wash Concentrate (20X) to room
the stop solution. 5. Add 100 µl of the Conjugate 1X to each well.
temperature and mix thoroughly to ensure that the Wash
- in the absence of antibodies, no coloration Materials required but not provided Concentrate (20X) is completely solubilized.
6. Incubate 30 min ± 3 min at 21°C (± 5°C).
appears.
Prepare the Wash Solution (1X) by diluting the Wash
1. Mono or multi-channel micropipettors capable of 7. Empty the wells. Wash each well 3 times with
The microplate is read at 450 nm. Concentrate (20X) to 1:20 in distilled/deionized water.
delivering volumes of 10 µl, 100 µl, and 200 µl. approximately 300 µl of the Wash Solution. Avoid
Testing Procedure drying of the wells between washings.
2. Disposable tips.
8. Add 100 µl of the Substrate Solution to each well.
3. 96-well microplate reader. Allow all reagents to come to room temperature (21°C ±
5°C) before use. Homogenize all reagents by inversion or 9. Incubate 15 min ± 2 min at 21°C (± 5°C) in the dark.
4. Distilled or deionized water.
Vortex.
10. Add 100 µl of the Stop Solution to each well in order to
5. Manual or automatic wash system.
stop the reaction.

11. Read and record the O.D. at 450 nm.

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TOXOS-MS ver 1014 GB TOXOS-MS ver 1014 GB
 Certified
management
Validation Interpretation system
The test is validated if: For each sample, calculate the S/P percentage (S/P%):

the mean value of the Positive Control O.D.

ODsample – ODNC
(ODPC) is greater than 0.350.
S/P% = x 100

ODPC > 0.350

ODPC – ODNC

Samples (sera, plasma or meat juice) presenting a

ID Screen®
the ratio of the mean O.D. values of the Positive S/P%:
and Negative Controls (ODPC and ODNC) is greater
than 3. - less than or equal to 40% are considered Toxoplasmosis Indirect
negative.

ODPC / ODNC > 3 - between 40% and 50% are considered doubtful. Multi-species
- greater than or equal to 50% are considered
positive.

Result Status

S/P % ≤ 40% NEGATIVE

40% < S/P % < 50% DOUBTFUL

S/P % ≥ 50% POSITIVE Kit for the detection of antibodies directed against Toxoplasma gondii by
indirect ELISA in sera, plasma and meat juice.
Nota: For canine sera, IDvet validation studies (report
available upon request) showed that ELISA results
between 40% and 70% were difficult to interpret by
IFAT. IDvet therefore recommends a wider doubtful For in vitro use
zone (40-70%) for canine sera.

October 2013: Note about canine sera interpretation

TOXOS-MS ver 1014 GB

IDvet, 310, rue Louis Pasteur – Grabels - FRANCE

Tel:+ 33 (0)4 67 41 49 33 - Fax: + 33 (0)4 67 45 36 95
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TOXOS-MS ver 1014 GB