CRUDE NATIVE HAPTEN-BASED INDIRECT ELISA ASSAY KIT AND LYOPHILISED CONTROLS FOR THE CONFIRMATORY DIAGNOSIS OF BOVINE BRUCELLOSIS IN BLOOD SERUM AND MILK BY ANIMAL AND TANK

Abstract

A diagnostic kit for a confirmatory assay using the indirect ELISA method that measures the levels of anti-Native Hapten antibodies produced during a real infection, thereby preventing large financial losses to livestock farm, by discerning false positives that present anti-LPS antibodies due to doss-reactions with enterobacteria and post-vaccinal antibodies for the diagnosis of bovine brucellosis in blood serum and individual milk (per animal) and bulk milk (tank), characterised by using the crude Native Hapten antigen, extracted from B. melitensis 16M strain, with no purification treatment and an effective adherence capacity, which is used to antigenize plates at a known concentration (1 g per well), where using as reference positive and negative controls subjected to the lyophilisation (freeze drying) method to ensure their preservation, thereby avoiding the contamination and degradation of the antibodies present and ensuring the stability of the optical densities in said controls for a correct results interpretation of an indirect ELISA, are taken as reference. The lyophilisation method for controls that may be used in other diagnostic methods is also presented.

Claims

1. KIT for indirect ELISA test based on Raw Native Hapten for confirmatory diagnosis of bovine brucellosis in blood serum and individual milk (per animal) and bulk milk (tank), characterized by ten (10) microplates coated with crude Native Hapten, each plate containing 96 wells (distributed in 12 strips of 8 wells each strip) of flat and clear bottom, with a surface treated specially for a high capacity of adhesion of the antigen, with a maximum capacity of 360 microliters per well. It includes five (5) microplates of 96 wells, without treatment, to perform the predilution of samples; four (4) bottles of sixty (60) milliliters each with PBS-Tween 20 Washing Solution, 0.05%, at (10) concentration; one bottle (1) of forty (40) milliliters of Sample Diluent Solution, based on CABI (carbonate bicarbonate) Buffer at (10) concentration; one (1) bottle of fifty (50) milliliters of Conjugate Diluent, based on CABI (carbonate bicarbonate) buffer at concentration (1); one (1) 50 milliliter bottle of Substrate, which is ABTS (commercial product); one (1) vial of one (1) milliliter consisting of 25 microliters of Concentrated Conjugate, which is an Immunoglobulin G-anti Bovine, conjugated with horseradish peroxidase produced in goat (commercial product) which is prediluted in a preservative HRP Protector, which is a peroxidases stabilizer; one (1) bottle of fifty (50) milliliters of Stop Solution, which is sodium Duodecyl Sulfate (SDS) at 4% concentration. One (1) freeze dried positive blood serum vial, one (1) freeze dried negative blood serum vial, and one (1) freeze dried positive milk serum vial and one (1) freeze dried milk serum vial. All control vials contain one (1) milliliter of freezed dried serum for reconstitution in one (1) milliliter of distilled water.

2. Indirect ELISA Test Procedure based on Raw Native Hapten for confirmatory diagnosis of bovine brucellosis in blood serum and individual milk (per animal) and bulk milk (tank), using this crude antigen obtained from B. melitensis 16M strain, without applying any purification treatment to it, and with an effective adhesion capacity to perform an indirect ELISA for brucellosis diagnosis in blood serum and milk.

3. The indirect ELISA test procedure based on a crude Native Hapten for confirmatory diagnosis of bovine brucellosis in blood serum and individual milk (per animal) and bulk milk (tank) according to claim 1, the coating of ELISA plates with Native Hapten antigen at a 1 microgram per well concentration for the diagnosis of brucellosis in blood serum and milk.

4. The indirect ELISA Test Procedure based on the use of freeze dried controls for confirmatory diagnosis of bovine brucellosis in blood serum and individual milk (per animal) and bulk milk (tank) according to claim 1, with the use of blood serum and milk controls freeze dried as a conservation method to avoid contamination and degradation of the antibodies present, guaranteeing the stability of the optical densities in positive and negative controls for a correct results interpretation in an indirect ELISA.

5. Procedure for the Selection and Freeze-drying of Positive and Negative Controls for use in other diagnostic tests.

Description

BRIEF FIGURES DESCRIPTION

[0126] FIG. 1 shows the flow diagram of the Native Hapten antigen obtaining process. From the B. melitensis 16M strain cultivation, the cell harvest and the antigen extraction until the lyophilization process of the antigen obtained.

[0127] FIG. 2 shows the flow diagram of the antigen coating process of the ELISA plates. This diagram describes the steps that are carried out to coat the plates with the Native Hapten antigen.

[0128] FIG. 3 shows the flow diagram of the indirect ELISA process, which describes the steps to be performed during the assay.

[0129] FIG. 4 shows the flow diagram of the blood serum controls and milk controls obtaining process. Showing the conditions by which the controls are selected and their validation before being freeze dried.

[0130] FIG. 5 shows the result on the use of this KIT in a herd with 2533 dairy cows in production, and their results to conventional tests, as well as the False Positive animals and those really infected.