TOXOCARA CANIS

Enzyme immunoassay for the diagnosis of human toxocariasis

96 assays on individual wells for in vitro diagnostic use and for professional laboratory use

Instructions for use for article N° 9200

EC reg. N°: H-CH/CA01/IVD/01755

Intended use:
The Bordier Toxocara canis ELISA kit is intended for the quantitative detection of IgG antibodies against
Toxocara canis in human serum. Serology is an aid for diagnosis and cannot be used as the sole
method of diagnosis.

Background:
Toxocariasis is a worldwide zoonosis caused by Toxocara canis, a parasitic nematode of dogs, or T.
mystax of cats. Humans can be infected by accidentally ingesting Toxocara embryonated eggs.
Intestinal hatching of Toxocara larvae from eggs allows them to migrate to a wide variety of tissues,
including liver, lungs, muscles, brain or eyes. Most infected people do not show any symptoms.
However, in some cases, migrating larvae can induce Visceral Larva Migrans (VLM) or Ocular Larva
Migrans (OLM) syndromes. Diagnosis is based on the presence of signs of VLM (eosinophilia, fever,
cough, abdominal pain, hepatomegaly and rash), or OLM (ocular problems) plus a history of exposure
and a positive result by serological testing.

Principle and presentation:

The kit provides all the material needed to perform 96 enzyme-linked immunosorbent assays (ELISA) on
breakable microtitration wells sensitized with Toxocara canis excreted/secreted (E/S) larval antigens.
Specific antibodies in the sample will bind to these antigens and washing will remove unspecific
antibodies. The presence of parasite specific antibodies is detected with a Protein A - alkaline
phosphatase conjugate. A second washing step will remove unbound conjugate. Revealing bound
antibodies is made by the addition of pNPP substrate which turns yellow in the presence of alkaline
phosphatase. Color intensity is proportional to the amount of Toxocara canis specific antibodies in the
sample. Potassium phosphate is added to stop the reaction. Absorbance at 405 nm is read using an
ELISA microplate reader.
The test can be performed with automatic systems, but this must be validated by the user.

Material contained in the kit (96 assays):

WELL 9200-01 Breakable ELISA strips sensitized with 96 wells

Toxocara canis E/S antigens
DILB 9200-02 Dilution buffer (10 x) concentrate, coloured purple 50 ml
WASH 9200-03 Washing solution (10 x) concentrate 50 ml
ENZB 9200-04 Enzyme buffer 50 ml
STOP 9200-05 Stopping solution (0.5M K3PO4) 25 ml
CONTROL - 9200-06 Negative control serum (20 x), green cap 200 l
CONTROL -/+ 9200-07 Weak positive control serum (cut off, 20 x), yellow cap 200 l
CONTROL + 9200-08 Positive control serum (20 x), red cap 200 l
CONJ 9200-09 Protein A - alkaline phosphatase conjugate (50 x), purple cap 300 l
SUBS 9200-10 Phosphatase substrate (para-nitrophenylphosphate) 20 tablets
Multipipette reservoir, 25 ml 1 piece
Frame for ELISA 8-well holder 1 piece

50204_05 9200 Eng 01.2018

Shelf life and storage:
Store the kit at 2° to 8°C (transport at ambient temperature), avoid long term exposure of the
components to direct light. The expiry date and the lot number of the kit are printed on the side of the
box. After initial opening, all reagents are stable until the expiry date when stored at 2-8°C.

Equipment needed but not provided with the kit:

Pipettes (ml and µl). Flasks. Dilution tubes. Adhesive tape to cover wells during incubations. Distilled
water. Incubator set at 37°C. ELISA reader set at 405 nm. Manual or automatic equipment for rinsing
wells. Vortex mixer. Timer.

Preparation of reagents before use:

Bring all reagents to room temperature and mix before use.

ELISA wells: open side of aluminum bag 9200-01 and remove number of wells needed (one for blank,
three for controls plus the number of samples). Place sensitized wells in 8-well holder(s). If needed,
complete the empty positions in the holder with used wells. Insert holder(s) in the frame in the correct
orientation. Reseal open package with desiccant pad.

Dilution buffer: dilute dilution buffer (10 x) concentrate 9200-02, 1/10 in distilled water. This is used for
the dilution of controls, samples and conjugate. The diluted buffer is stable for 2 months at 2-8°C.

Washing solution: dilute washing solution (10 x) concentrate 9200-03, 1/10 in distilled water. You may
also use your own washing solution. Avoid buffers containing phosphate which could inhibit the
enzymatic activity of the alkaline phosphatase. The diluted washing solution is stable for 2 months at 2-
8°C.

Control sera: dilute 10 µl control sera 9200-06 to -08 in 190 µl dilution buffer solution (final dilution
1/20). The diluted control sera are stable for 2 months at 2-8°C.

Conjugate: dilute conjugate 9200-09 in dilution buffer solution (final dilution 1/50). Dilute conjugate on
the day of the assay. Do not store diluted conjugate.

Substrate solution: dissolve tablet(s) of phosphatase substrate 9200-10 in undiluted enzyme buffer
9200-04 (1 tablet in 2.5 ml buffer). Vortex until complete dissolution of the tablet(s). Dilute substrate on
the day of the assay and protect the tube from direct light. Tablets and substrate solutions should be
colourless or should have only a slight yellow tinge. If a tablet or a substrate solution turns yellow, it may
have been partially hydrolysed and should be discarded. Do not store the substrate solution.

Stopping solution: use reagent 9200-05 undiluted.

Specimen collection and preparation:

Use human serum. Serum should be stored at 2-8°C if analysed within a few days, otherwise store at
–20°C or lower. Avoid repeated freezing and thawing.
Vortex samples and dilute 1/201 in dilution buffer solution (for instance 5 µl sample in 1.0 ml).

Warnings and precautions:

Toxic compounds are found in following concentration:

Component Reference Sodium azide (NaN3) Merthiolate

Dilution buffer (10 x) 9200-02 0.1 % 0.02 %
Washing solution (10 x) 9200-03 0.05 % /
Enzyme buffer 9200-04 0.01 % /
Control sera (20 x) 9200-06 to –08 0.1 % 0.02 %
Conjugate (50 x) 9200-09 0.1 % /

At the used concentrations, sodium azide and merthiolate do not have any toxicological risk on contact
with skin and mucous membranes.

50204_05 9200 Eng 01.2018

- The stopping solution 9200-05 (0.5 M K3PO4) is irritant.
- The negative, weak positive, and positive control sera (9200-06 to -08) are from rabbits.
- Treat all reagents and samples as potentially infectious material.
- Do not interchange reagents of different lots or Bordier ELISA kits.
- Do not use reagents from other manufacturers with reagents of this kit.
- Do not use reagents after their expiry date.
- Close reagent vials tightly immediately after use and do not interchange screw caps to avoid
contamination.
- Use separate and clean pipettes tips for each sample.
- Do not re-use microwells.

Disposal consideration:
All materials used for this test are generally considered as hazardous waste. Refer to national and
regional laws and regulations for the disposal of hazardous waste.

Procedure:
When running the assay, avoid the formation of bubbles in the wells.

Step 1: Blocking:
Fill completely wells with dilution buffer solution.
Incubate for 5 to 15 minutes at ambient temperature (blocking).
Remove dilution buffer by aspiration or by shaking the wells over the sink.

Step 2: Incubation with samples:

Fill the first well of the first strip with 100 µl dilution buffer only (no-serum blank).
Fill the subsequent three wells with respectively 100 µl diluted negative, weak positive (cut off) and
positive control serum. For assays of more than 25 samples, we recommend to fill the three last wells
with control sera as a duplicate.
Fill remaining wells with the diluted samples (100 µl each).
Cover wells with adhesive tape and incubate for 30 minutes at 37°C.
Remove sera and wash 4 x with ~ 250 µl washing solution.

Step 3: Incubation with conjugate:

Distribute 100 µl diluted conjugate in each well (including no-serum blank).
Cover wells with adhesive tape and incubate for 30 minutes at 37°C.
Remove conjugate and wash 4 x with ~ 250 µl washing solution.

Step 4: Incubation with substrate:

Distribute 100 µl substrate solution per well.
Cover wells with adhesive tape and incubate for 30 minutes at 37°C.
Stop the reaction by the addition of 100 µl stopping solution to each well.

Step 5: Measurement of absorbances:

If needed, wipe the bottom of the wells and eliminate bubbles. Measure absorbances at 405 nm within 1
hour after the addition of stopping solution.

Interpretation:
Subtract the value of the no-serum blank from all measured values. When applicable calculate the mean
absorbance values of duplicated serum controls. The test is valid if the following criteria are met:
- absorbance (A) of positive control > 1.200
- A of negative control < 8 % of A of positive control
- A of blank against air < 0.350

Quality controls of current lots are published on our website: www.bordier.ch.

50204_05 9200 Eng 01.2018

The antibody concentration of the weak positive (cut off) serum 9200-07 has been set to discriminate
optimally between sera of clinically documented cases of toxocariasis and healthy human sera.
The cut off index of a sample is defined, after subtraction of the no-serum blank, as:

Absorbance sample
Index =
Absorbance cut off serum

The result is negative when the index of the analyzed sample is lower than 1.0. In this case, the IgG
antibody concentration against Toxocara canis E/S antigens is clinically non-significant.
The result is positive when the index of the analyzed sample is higher than 1.0. In this case, the IgG
antibody concentration against Toxocara canis E/S antigens is considered as clinically significant. It
indicates that the patient has had a contact with the parasite.
A grey zone could be defined by each laboratory according to its patients population. In case of
borderline or doubtful results, we recommend repeating the test again 2-4 weeks later with a fresh
sample.

Sensitivity and specificity:

A sensitivity of 91% was found with 78 sera of patients with clinically suspected toxocariasis. A
specificity of 96% was found with 500 sera of blood donors (Swiss). A specificity of 98% was found with
500 sera of Swiss hospitalized children (not for toxocariasis).

Interferences:
Internal evaluation showed that hemorragic, lipemic or icteric sera do not interfere with the results of the
test.

Precision:
Repeatability were assessed by testing 2 human serum samples in 24 wells on 1 assay.
Reproducibility were assessed by testing the 2 human serum samples on 10 differents assays.

Repeatability Reproducibility
Sample 1 Sample 2 Sample 1 Sample 2
Average (absorbance) 1.067 2.383 0.960 2.152
Standard deviation (absorbance) 0.043 0.110 0.038 0.063
Variation coefficient (%) 4.0 4.6 4.0 2.9

Limitations:
A specificity of 86% was found with 199 sera of patients with other parasitic infections. Cross-reactivity
mainly occurs in patients with trichinellosis, fascioliasis, amebiasis and strongyloidiasis.
Diagnosis of an infectious disease should not be estabished on the basis of a single test results. A
precise diagnosis should take into consideration endemic situation, clinical history, symptomatology,
imaging as well as serological data.
In immunocompromised patients and newborns, serological data are of limited value.

References:
Jacquier, P., Gottstein, B., Singelin, Y. and Eckert, J. (1991) Immunodiagnosis of Toxocarosis in Humans: Evaluation of a
New Enzyme-Linked Immunosorbent Assay Kit. J. Clin. Microbiol. 29, 1831-1835.
Radman, N.E., Archelli, S.M., Fonrouge, R.D., Guardis, M.V. and Linzitto, O.R. (2000) Human Toxocariasis. Its
seroprevalence in the city of La Plata. Mem. Inst. Oswaldo Cruz. 95, 281-285.
Jin, Y., Shen, C., Huh, S., Sohn, W-M., Choi, M-H. and Hong, S-T. (2013) Serodiagnosis of Toxocariasis by ELISA Using
Crude Antigen of Toxocara canis Larvae. Korean J. Parasitol. 51, 433-439.
Rudzinska, M., Kowalewska, B. and Sikorska, K. (2016) Clinical usefulness of western blotting and ELISA avidity for the
diagnosis of human toxocariasis. Parasite Immunology 39.
Kim, H.B., Seo, J.W., Lee, J.H., Choi, B.S. and Park, S.G. (2017) Evaluation of the prevalence and clinical impact of
toxocariasis in patients with eosinophilia of unknown origin. Korean J. Intern. Med. 32, 523-529.

BORDIER AFFINITY PRODUCTS SA

Biokema building, Chatanerie 2, CH-1023 Crissier, Switzerland.
Phone: + 41 21 633 31 67, Fax : + 41 21 633 31 78, www.bordier.ch
50204_05 9200 Eng 01.2018