CS277024B6 - Indication for detection of antibodies against Fasciola hepatica in cattle - Google Patents

Abstract

The solution relates to a device consisting of an indicator material in the form of a nitrocellulose membrane-based strip applied to a plastic substrate, with three separate drops of an antigen solution with a volume of 2 pg/ml of diluent in three concentrations of 150, 100 and 50 ug antigen/ml of diluent applied to the indicator strip and the remaining surface of the membrane is covered with a blocking layer of a non-reactive protein, e.g. gelatin. The device is used in a test in which the antigen reacts with an antibody in the sample and the resulting immunocomplex is then visualized by reaction with an anti-species-specific immunoglobulin labeled with peroxidase, with a positive reaction demonstrating the presence of antibodies in the sample being indicated by a change in the color of the substrate material from white to blue.

Description

The invention relates to an indicator composition for the detection of antibodies against Fasciola hepatica in cattle.

Serological detection of most parasitic diseases is irreplaceable where diagnosis by direct evidence is impossible or unlikely, ie in tissue helminthiasis and those where the prepatent period is long, or there is a risk of severe host damage during this period.

In veterinary and human medicine, fasciolosis can be counted among such helminth diseases, because, as experience has shown, coproscopy is not always reliable and the period until the discovery of the first eggs in the host faeces is relatively long about 42 to 56 days / Sinclair, K. B. Helmint. Abstr. 36: 115-134, 1967; de Leon, D. et al. J. Parasitol. 67: 734-735, 1981. In most cases, eggs can be detected regularly after 3 to 4 months of invasion, as opposed to antibodies, which can be detected in the experiment after only 3 to 5 weeks. Therapeutic intervention is conducted already in the period of developed pathogenesis and often the host is no longer able to regenerate the damage.

Bovine and ovine fasciolosis is a persistent potential source of danger to cattle and sheep farming, as confirmed by experience in many developed and developing countries. / Malone: Vet. Clin. N. Amer., 2: 262-275, 1986; Bundy et al Paho Bull. 17: 243-258, 1983. In the Czechoslovak Socialist Republic, about 0.7 to 1.0% of the state and 8.5 to 12.1% of farms are still infected in some cattle farms; it is necessary to treat 60,500 cattle per year, which represents a loss in the cost of anthelmintics of 750 to 1,300,000 CZK per year. Losses on gains, milk, confiscations, etc. are estimated at approximately CZK 1,300 per infected animal. The state of distribution in sheep is similar to that in cattle to the expensity and main localities. .........

Due to the zoonotic nature of the disease in humans, diagnostics is also of great practical importance in human medicine / Hilleyer, G. V .: Fasciolosis, In .: Immunology of Parasitic Diseases, vol. 1. Walls, Schantz eds., Pp. 39-68, 1986 /. Classic serological tests are difficult to prepare and perform, difficult to standard! easy to read. Recently, an almost exclusively immunoenzymatic ELISA has been developed. / Boulard et al. Ann. Rech. Vet., 16: 363-368, 1985 /. A major obstacle to the application of this highly sensitive and effective serological test is the occurrence of frequent non-specific or cross-reactions, which in many cases make it completely impossible to interpret the test results for clinical practice.

In the work on experimental fasciolosis in sheep, nitrocellulose was used as a transport medium for introducing antigen into the wells of a plate for an otherwise classical ELISA procedure. / Zimmerman et al .: J. Vet. Res. 46: 1513-1515, 1985. The same technique was used, for example, in toxocariosis in dogs / Matsumura et al .: Zbl. Bakt. Hyg. A., 266: 580-585, 1987 /. Another disadvantage of these tests is the need to use well plates dependent on washers, multichannel pipettes and evaluation equipment.

The subject of the invention is an indicator device for the detection of Fasciola hepatica antibodies in cattle, the essence of which is that it consists of an indicator tape formed of a nitrocellulose membrane fixed on a plastic substrate, on which three drops of 2 μΐ antigen solution are applied in three concentrations 150, 100 and 50 μg antigen / ml diluent, the remaining membrane area being covered by a blocking layer of a non-reactive protein selected from the group consisting of gelatin, horse serum and bovine serum albumin.

The method of using this composition is based on the principle that the antigen-loaded composition is contacted with a test sample, which is then allowed to incubate, after which the resulting immunocomplex is visualized by reaction with peroxidase-labeled antibovine immunoglobulin, with positive reactions showing antibodies in the sample. is indicated by a change in the color of the support material by the products of the peroxidase reaction with the substrate.

The change in color at two lower antigen concentrations is considered to be a specifically positive reaction, and the color of the highest antigen concentration is considered non-specifically positive.

The advantage of the composition according to the invention is in particular:

1 / The test is performed with one dilution of serum on one strip

2 / The limit of detection is given by the use of three different concentrations of antigen, of which a reaction of two or more is evaluated as a positive reaction, one as a negative non-specific result (internal control of the reactivity of the system).

3 / The test does not require any special equipment.

4 / The indicator tape is long-term storable and permanently ready for use.

Example

Preparation of a composition for the detection of antibodies against Fasciola hepatica in the form of strips

A 2 μΐ spot of antigen in three concentrations, 150, 100 and 50 μg of antigen in 1 ml of diluent / buffered saline „H 7.2 with the addition of 0, 05% wt. Tween 20, hereinafter referred to as PBS-T20 /. ^F

The strip was left for 30 minutes at room temperature and then washed in PBS-T20 for 10 minutes and blocked in PBS-T20 with 10% by weight of normal horse serum (hereinafter referred to as PBS-720-NKS).

It was then rinsed in PBS-T20 and allowed to dry on filter paper. For later use, the tape is stored sealed in a PVC bag at room or refrigerator temperature for at least 1 week, preferably 1 month. Using the composition of the invention, the strip was rehydrated in PBS-T20 for 10 minutes, further incubated in a 1: 1000 serum sample in PBS-T20-NKS for one hour on a shaker and then washed 3 times in 5 minutes in PBS-T20. After incubation in antibacterial conjugate 1: 1000 / or other suitable concentration according to its quality / one hour, the strip was washed 3x 5 minutes in PBS-T20, incubated for about 30 minutes in a substrate solution of 4-chloro-1-naphthol in TRIS- / hydroxymethyl / -aminomethane-HCL buffer _n H 7.6 and the reaction was stopped after staining. CS 277024 B6 first stain with 0.5% by weight, sodium azide in water and washed well with water. The blue reaction of two spots with a lower antigen concentration is evaluated as a positive result.

Efficacy was verified on a set of 30 sciences of cattle autopsy on fasciolosis and 30 sera of cattle autopsy negative on this disease.

Of the 30 sera from sick animals, 25, ie 83.3%, responded positively. Of the sera from healthy animals, 4 sera reacted positively, representing a specificity of 86%. Overall, 85% of the correct results were obtained, which is sufficient accuracy in the animals.

Claims (1)

PATENT CLAIMS Indicating agent for the detection of antibodies against Fasciola hepatica in cattle, characterized in that it consists of an indicating strip made of a nitrocellulose membrane fixed on a plastic support, on which three separate drops of antigen solution with a volume of 2 μΐ are applied in three concentrations 150, 100 and 50 μg antigen / ml diluent, the remaining membrane area being covered by a blocking layer of a non-reactive protein selected from the group consisting of gelatin, horse serum and bovine serum albumin.