Indirect ELISA test procedure to detect antibodies anti Native Hapten for confirmatory diagnosis of bovine brucellosis in blood serum and individual milk (per animal) and bulk milk (tank) with a detection capacity of 969,162 liters of positive milk in a 30,000-liter tank, wherein the antigen Native Hapten is crude Native Hapten, which is obtained by the next procedure of extraction with ethanol from B. melitensis 16M strain, with no further purification treatment:
- a. strain harvest is inactivated by heat in the autoclave, sterilizing at 120° C. and 15 lb. for 25 minutes;
- b. waiting for it to cool down and perform a centrifugation at 6000 rpm for 30 minutes;
- c. three volumes of cold ethanol are added to the supernatant;
- d. placing the supernatant in magnetic stirring, maintaining it at 4° C. for 18 hours to precipitate the antigens;
- centrifuging the supernatant at 6000 rpm for 30 minutes, taking the pellet and resuspend in saline, adding 0.5 ml, mixing and observing the turbidity, if it is observed too saturated, adding 0.5 ml more, avoiding to reach transparency as this could dilute the antigen so that a low concentration of it will be obtained, this is labeled as LPS antigen (lipopolysaccharide);
- e. two more volumes of cold ethanol are added to the supernatant, and it is kept in freezing (−20° C.) for 18 hours without agitation to precipitate the NH antigen;
- f. when finished, centrifuge at 6000 rpm for 30 minutes;
- g. the formed pellet is taken and resuspended in 0.5 ml of saline, observing the turbidity, and adding more saline solution if necessary;
- h. This suspension contains the Crude Native Hapten antigen with no further purification treatment;
- and
- wherein the procedure further comprises the following steps:
- i) coating of ELISA plates with the crude Native Hapten antigen, obtained in steps a through h, wherein the coating of ELISA microplates is performed with crude Native Hapten antigen at a 1 microgram per well concentration for confirmatory diagnosis of bovine brucellosis in blood serum and individual milk (per animal) and bulk milk (tank);
- ii) producing lyophilized controls of blood serum and milk serum (whey) used as the reference for determining the cut-off point of the test, wherein positive controls have an absorbance of ≥1.0, and negative controls of 0.20-0.28;
- both at a wavelength of 405 nm;
- iii) conducting Indirect ELISA process, which comprises:
- Distributing 285 μL of a Sample Diluent Solution to each well in a pre-dilution Microplate, wherein the Sample Diluent Solution is a Bicarbonate Carbonate Buffer (CABI);
- Adding 15 μL of Negative Control in wells A1 and B1;
- and 15 μL of Positive Control in wells C1 and D1, continuing adding 15 μL of the sample (blood serum or milk) in the remaining wells;
- Taking 50 μL of the diluted samples and controls in the pre-dilution microplate and transferring them to the microplate coated with Native Hapten of step i);
- Incubating the coated microplate for 1 hour at 37° C.;
- Washing each well with 250 μL of the previously diluted Washing Solution, and making a total of 4 washes;
- wherein the washing solution is PBS-TWEEN 20;
- Adding 50 μL of the previously diluted Conjugate (dilution 1:2000) to each well, wherein the conjugate is an anti-bovine IgG produced in goat conjugated with horseradish peroxidase;
- Incubating the Microplate for 1 hour at 37° C.;
- Performing 4 more washes;
- Adding 50 μL of Substrate (ABTS) to each well;
- Incubating the Microplate for 15 minutes at room temperature (20° C.-25° C.) in darkness;
- Distributing 50 μL of a Stop Solution, wherein the stop solution is a solution of sodium dodecyl sulfate (SDS) at 4%;
- and
- Reading at 405 nm of optical, wherein the reading is stable for 30 minutes once the Stop Solution has been added.