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ELISA kit for serodiagnosis of Neospora caninum and BVDV


BIO K 367

Código: BIO K 367. Categorías: , , .
   
Descripción

I – INTRODUCTION

Serotesting calves before they ingest any colostrum is an excellent way to detect the congenital transmission of various infections such as neosporosis and BVD. In the case of BVD, serotesting detects foetal infections between the 180th and 300th day of gestation in gestating cows as well as infections in heifers and the results are not upset by interference from vaccine antibodies. That makes the method very useful for all BVD control strategies, especially before or after the detection and culling of immunotolerant animals on the farm. Precolostral calves that are seropositive for BVD are not a risk for the rest of the herd but show that the virus is circulating in the livestock. In such a situation it is important to set up a strategy for detecting and culling immunotolerant animals that involves strict verification of new introductions to the herd. The decision to vaccinate or not to vaccinate animals after removing all immunotolerant animals from the herd will depend on the farm’s veterinary surgeon. In the case of neosporosis, seropositive female calves will have to be taken out of the breeding programme for such individuals will perpetuate the infection on the farm and have a heightened risk of first-gestation abortion. Their dams will also have to be checked to determine if they were infected during their period of gestation (horizontal contamination) or are themselves the offspring of Neospora-positive cows (vertical contamination).

 

II – PRINCIPLE OF THE TEST

The kit contains a multi-parametric ELISA test (combined test) that can be utilised to test blood serum and plasma collected before any colostrum is ingested, ideally just post-partum. It is also possible to use umbilical cord blood collected on blotter paper that is designed for this and is available from Bio-X Diagnostics. The kit’s 96-well microtitration plates have been sensitised with monoclonal antibodies specific for the two pathogens listed above. The antibodies capture and purify these pathogens from lysates of the pathogen’s host cells. The reagents are distributed across each microplate as follows:

Columns 1, 5,and 9: Neospora caninum (protein SRS2)

Columns 2, 6, and 10: BVDV (protein NS3)

Columns 3, 7, and 11: precolostral control

Columns 4, 8, and 12: negative control

Columns 3, 7, and 11 have been sensitised with a control antigen in order to ensure that the serum was indeed taken before all colostrum intake and the serological profile that results is definitely that of the calf and not its mother’s. Columns 4, 8, and 12 are the kit’s negative controls. They contain no antigens. The inclusion of such negative controls makes it possible to reduce the number of false positive serum reactions considerably. The serum and the plasma samples are diluted 100-fold in the dilution buffer and incubated at 21 +/- 3 °C for one hour. If blotter paper is used, two 6 mm in diameter circles are punched out and placed in 600 µl of dilution buffer. After incubation and washing of the preparation, the conjugate – a peroxidase-linked monoclonal antibody specific for bovine IgG1 – is added. After a second incubation at 21 +/- 3 °C for one hour and a second washing, the chromogen solution (single-component TMB) is added. This chromogen has the advantageof being more sensitive than the other peroxidase chromogens and not being carcinogenic. If the specific immunoglobulins are present in the samples, the conjugate remains bound to the corresponding well and the enzyme catalyses the transformation of the colourless chromogen into a blue compound. The intensity of the colour is proportionate to the titre of the specific antibody present in the sample. The signals recorded for the negative control wells (Columns 4, 8, and 12) are subtracted from the signals of the corresponding positive wells. An interpretation grid with cut-off values for Neosporaand BVDV is provided to evaluate the results.

 

III – COMPOSITION OF THE KIT

– Microplate: Two 96-well microtitration plates (24 strips of 8 wells). The distribution of the various valences is indicated on the metal envelope.

– Washing solution: One 100-ml bottle of 20-fold concentrated washing solution. The solution crystallises spontaneously when cold. If only part of the solution is to be used, bring the bottle to 21 +/- 3 °C until all crystals have disappeared completely, mix the solution, and remove the necessary volume. Dilute the solution twentyfold with distilled or demineralised water.

– Dilution buffer: One 30-ml bottle of coloured, 5-fold concentrated dilution buffer. The contents of the bottle must be diluted in distilled or demineralised water.This solution is used to dilute the blood serum,the plasma samples, the blotter paper and the conjugate. If a deposit forms at the bottom of the container, filter the solution on Whatman filter paper.

– Conjugate: One vial of peroxidase-linked anti-bovine immunoglobulin conjugate (anti-bovine IgG1 antibody linked to horseradish peroxidase).

– Positive serum: One vial containing the positive serum. Store this reagent between +2 and +8 °C.

– Negative serum: One vial containing the negative serum. Store this reagent between +2 and +8 °C.

– Single-component TMB:One 25-ml vial of the chromogen tetramethylbenzidine (TMB). Store between +2°C and +8°C protected from the light.This reagent is ready to use.

– Stop solution: One 15-ml vial of stop solution containing 1 M phosphoric acid.

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