Patent ID: 12222353

DETAILED DESCRIPTION OF THE INVENTION

It is a novel invention for brucellosis diagnosis, because due to the expression of antibodies against Native Hapten only occurs during a field infection, this test allows to discriminate between truly positive animals from the false positives, ensuring that the animals sent to slaughterhouse correspond only to the infected animals. It avoids the presence of false negative animals within the healthy population, preventing the spread of the disease. Besides, to being used in blood serum, this test can also be used for milk analysis, a novel feature since there are no reports on diagnostic tests that use NH in milk. This test can be used using individual cow milk or bulk milk, with a detection capacity of 969.162 liters of positive milk in a tank of 30,000 liters, proving to be a highly sensitive and specific test. For all the mentioned before, this test can be taken as a tool that contributes to the eradication of the disease thus preventing the spread to humans.

1.—Antigen Production

Brucella melitensis16M cultivation

The production of the antigen is carried out in an isolated area, using biosecurity measures typical of a microbiology laboratory (biosecurity level 2). The equipment used in the area consists of a CO2 incubator, centrifuge, autoclave, refrigerator, Fischer burners and analytical scale. The first culture is done from theBrucella melitensis16M strain preserved in liquid nitrogen using the streak plate technique in a Petri dish with trypticase soy agar (TSA). The Petri dish is incubated at 37° C. in a 5% CO2 incubator for 72 to 120 hours. As the colonies grow on the dish they should be checked against light, a bluish color should be observed, characteristic ofB. melitensisstrain. These colonies are regrown (re-cultivated) on new Petri dishes with TSA medium to start a new production batch. Subsequently, they are incubated under the same incubation conditions mentioned before. Of those ten dishes, those that do not present contamination are selected to be regrown in approximately 80 new Petri dishes and are incubated under the same conditions.

Cell Harvest

After the incubation time the cells within the 80 Petri dishes are harvested using a cell scraper or a pasteur pipette bent into an L shape and placing the cells into a Falcon tube with 10 ml of sterile saline solution, the volume of saline may vary according to the amount of cells harvested. The cells are washed; centrifuging at 6000 rpm for 30 minutes. The supernatant obtained is discarded and the pellet precipitate is resuspended, adding the same volume of saline as previously added. The washings continued until the supernatant obtained is clear. At the end of the washings, the strain is inactivated by heat in the autoclave, sterilizing at 120° C. and 15 lb. for 25 minutes.

Extraction

Wait for it to cool down and perform a centrifugation at 6000 rpm for 30 minutes. The supernatant is taken with a syringe to control the volume obtained. This should be taken on the opposite side of the pellet to avoid contamination. The supernatant is poured into a 100-500 ml beaker, depending on the volume obtained. Three volumes of cold ethanol are added to the supernatant (example: if 10 ml of supernatant are contained in the beaker, 3 volumes of 10 ml of ethanol must be added). It is placed in magnetic stirring, maintaining it at 4° C. for 18 hours to precipitate the antigens. It is then centrifuged at 6000 rpm for 30 minutes, take the pellet and resuspend in saline, adding 0.5 ml, mix and observe the turbidity, if it is observed too saturated you can add 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. 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. When finished, centrifuge at 6000 rpm for 30 minutes. The formed pellet is taken and resuspended in 0.5 ml of saline, observe the turbidity, add more saline solution if necessary. This suspension contains the NH antigen.

Lyophilization of Raw Native Hapten Antigen

The lyophilization (or freeze drying) of the antigen and controls is carried out in an exclusive area for this procedure. The area in general has a negative pressure preventing possible contamination to adjacent areas. The equipment consists of a freeze dryer and a freezer. After the antigen extraction is completed, aliquots of the NH suspension are made in glass vials of the same size, placing 1 ml in each previously labeled vial. The vials are frozen at −80° C. for 30 minutes to 1 hour, placing the stoppers of the vials half-closed to facilitate the extraction of the vacuum. After freezing the vials are placed in the trays of the freeze dryer balancing the amount of vials on each side. The pressure and temperature of the freeze dryer is monitored during the process, the temperature should be about −80° C. The lyophilization process should be carried out for at least 6 hours. At the end of lyophilization, the vials should be capped and sealed. To open the vials, gently puncture the cap until you see that the antigen stops releasing pressure to prevent the antigen from being lost due to the release of the vacuum. 2 mg of the antigen are weighed and passed to microtubes properly identified. The vials with antigen can be stored in refrigeration at 4° C. until use. The processes for antigen production are shown inFIG.1.

2.—Microplate Coating

The coating of the plates is carried out in an isolated area inside a type II biosafety cabinet, incubator and refrigerator. The area in general should have a slightly positive pressure. There are ten (10) coated microplates of 96 wells contained in the kit, the wells are distributed in 12 strips of 8 wells each, the material of the microplates is polystyrene with a specially treated surface (by the manufacturer) for achieving a high capacity of adhesion of the antigen, with a maximum capacity of 360 microliters per well, flat and clear bottom. The coating procedure is as follows: A microtube with 2 mg of the freeze dried Native Hapten antigen is reconstituted with 1 ml of sterile distilled water, making sure to dissolve all the freeze dried content. Once dissolved, the milliliter is added in 99 ml of bicarbonate carbonate buffer solution (CABI) to obtain a total of 100 ml (20 μg/ml), and it is mixed perfectly. To each well of the microplates is added 50 μl (1 μg of antigen) of this solution and sealed with parafilm. The microplates are incubated at 4° C. for 18 hours (Overnight).

The microplates are washed with a 0.05% PBS-Tween 20 washing solution by adding 250 μl of this solution to each well and discarding it immediately. The washings are done 4 times. The excess of the washing solution is removed by shaking the plate twice and gently tapping it on a flat surface covered with a disposable towel. Subsequently, 50 μl of blocking solution (3% skim milk) is added to each well and the microplates are sealed. They are incubated at 37° C. for 1 hour and at the end another series of four washes are carried out as mentioned above. The excess solution is removed from the microplates and covered with the plastic microplate adhesive cover. They are sealed in a plastic bag by removing the vacuum, placing 10 plates in each bag labeled with the product name, batch number, date of manufacture and expiration date. The microplates are stored in refrigeration at 2-8° C. The process mentioned above is described inFIG.2.

3.—Procedure of the Indirect ELISA Anti-NH

Preparation of Samples

Before being analyzed, the samples should be diluted to a concentration of 1:20 with the Sample Diluent solution (CABI buffer), using a pre-dilution Microplate.

Blood Serum:

Blood samples are left to coagulate, and are centrifuged at 2500 rpm for 10 minutes. In samples taken from 12-24 hours prior to the test, it is not necessary to centrifuge. Samples with fibrin debris should be centrifuged before the test. Highly hemolyzed or lipemic samples should not be processed. The samples are stable for 2 days stored at 2-8° C. or 3 months at −20° C.

Milk:

Milk samples can be processed as whole milk, by shaking the sample before adding it in the pre-dilution plate, or it can be added as whey. To obtain whey, the samples are centrifuged at 2500 rpm for 15 minutes and then the top layer containing fat is removed using an applicator. The sample is stable 3 days at 2-8° C. or 3 months at −20° C.

Preparation of ELISA Reagents

The preparation of washing and diluting solutions is carried out in an isolated area within a laminar flow hood. The weighing of the reagents is carried out using an analytical scale and the pH adjustment is made out with a potentiometer. The area in general should have a slightly negative pressure.

Sample Diluent Solution. Bicarbonate Carbonate Buffer (CABI):

Dilute the 10× Sample Diluent solution in a 1:10 rate in distilled water. Example, to prepare 30 mL, dilute 3 mL of solution in 27 mL of distilled water.

Washing Solution. PBS-TWEEN 20 (0.05%):

Dilute the 10× Wash Solution in a 1:10 rate. Example, to prepare 250 mL, dilute 25 mL in 225 mL of distilled water.

Concentrated Conjugate:

The conjugate is an anti-bovine IgG produced in goat conjugated with horseradish peroxidase. Dilute the Concentrated Conjugate to a 1:50 rate using the Conjugate Diluent Solution. The conjugate should be diluted 15 minutes before being used. Once diluted, the concentration of the conjugate is 1:2000 and cannot be stored again. Example, to prepare 5 mL, dilute 100 μl of Concentrated Conjugate in 4.9 mL of Conjugate Diluent Solution.

Stop Solution. SDS 4%:

It is recommended to keep the solution at room temperature to avoid the formation of crystals before its use. In case of crystallization, temper the solution at 37° C. and homogenize correctly, 30 minutes before its use. Do not shake the solution immediately before using, to avoid the formation of bubbles.

Positive and Negative Controls of Blood Serum and Milk Whey:

The production of controls is carried out in the process area, which consists of a spectrophotometer, an automated plate washer, an incubator at 37° C., a centrifuge, micropipettes of different volumes of capacity, and refrigerator. In this area, individual sera are selected to later elaborate pools. The following tests are performed for blood serum: RBT, Rivanol, RID, BruScreen anti-LPS ELISA and BruPlus anti-Native Hapten ELISA; and for milk serum: MRT, BruScreen anti LPS ELISA and BruPlus anti-Native Hapten ELISA are performed. The controls are freeze dried as a conservation method. The controls are freeze dried so they must be resuspended by adding 1 ml of sterile tridestilated or distilled water, and shaking gently until completely homogenize. Once reconstituted store the controls in refrigeration at 2 to 8° C. It is recommended to make aliquots of the controls and store in freezing those that are not in use, to avoid contamination.

Indirect ELISA Process

It is necessary to keep all the included solutions at room temperature (21° C.±5° C.) and mixing before using. If the reconstituted controls were frozen they should be thawed completely and shaken perfectly before use. Distribute 285 μL of Sample Diluent Solution to each well in the pre-dilution Microplate. Add 15 μL of Negative Control in wells A1 and B1; and 15 μL of Positive Control in wells C1 and D1. Continue adding 15 μL of the sample (blood serum or milk) in the remaining wells. Take 50 μL of the diluted samples and controls in the pre-dilution Microplate (making sure to perfectly mix the samples and controls) and transfer them to the Microplate coated with Native Hapten, being careful to respect the order of the samples. Incubate the coated microplate for 1 hour at 37° C. Subsequently, wash each well with 250 μL of the previously diluted Washing Solution, avoiding the drying of the wells between each wash. Make a total of 4 washes. Remove the excess of Washing Solution by shaking the plate twice and gently tapping it on a flat surface covered with a disposable towel. Add 50 μL of the previously diluted Conjugate (dilution 1:2000) to each well. Incubate the Microplate for 1 hour at 37° C. Perform 4 more washes as mentioned above and remove the excess of washing solution. Add 50 μL of Substrate (ABTS) to each well, being careful not to expose the Substrate to the light, cover the Microplate with aluminum foil to avoid exposing the reaction to light. Incubate the Microplate for 15 minutes at room temperature (20° C.-25° C.) in darkness. Finally, distribute 50 μL of Stop Solution and read at 405 nm of optical. The reading is stable for 30 minutes once the Stop Solution has been added. The procedure is described inFIG.3.

4.—Selection and Lyophilization of Positive and Negative Controls of Blood Serum and Milk Serum (Whey).

Blood Serum and Milk Localization for Producing Controls

Negative: Are selected from samples of blood serum and milk processed with anti-NH ELISA, those that register an absorbance range of 0.20-0.28. Aliquots of the selected blood serum are made in a previously identified Eppendorf microtube. The aliquots are stored in freezing. In the case of milk, the samples must be skimmed first, centrifuging at 2500 rpm for 15 minutes and removing the top layer containing fat with an applicator. Once skimmed, the aliquots are made and stored in freezing.

Positive: Are selected from samples of blood serum and milk processed with anti-NH ELISA, those that register an absorbance of ≥1.0 nm. Aliquots of the selected blood serum are made and stored in freezing. In the case of milk, the procedure is followed before described.

Pre-Selection of Controls

Frozen Sera should be refrigerated at 2-8° C. before thawing to avoid a sudden change in temperature. Once defrosted, they are removed from the refrigerator and left to cool at room temperature. The samples of blood serum are processed to the official tests (RBT and Rivanol) and RID. And milk samples are tested with MRT. Once these pre-selected samples are approved, pools of positive and negative sera are formed.

Positive and Negative Control Pools Validation

The pools are validated once again with the official tests, RID and with the anti-LPS ELISA and anti-NH ELISA. Approved pools proceed to lyophilization process and with a previously assigned lot number.

Lyophilization of Positive and Negative Blood Serum and Milk Serum (Whey) Controls.

The control pools should be aliquoted in glass serum vials of the same size, placing 1 ml in each vial, previously labeled. Frozen at −80° C. for 30 min to 1 hour. Afterwards, they are placed in the freeze dryer trays, balancing the amount of vials in each side of the tray. Pressure and temperature are monitored during the process; the temperature should be about −80° C. The lyophilization process should be carried out for at least 6 hours. Freeze dried controls can be stored under refrigeration at 4° C. until use. Controls election and lyophilization procedures are shown inFIG.4.

PROCEDURES SIMILAR TO THE INVENTION

A search was made to corroborate the existence of procedures similar to the present invention, finding the following:

Summary of procedures found, similar to the invention.234511986198819931994Publication date1981Luis FernándezB. AlonsoEfren DiazEfren DiazAuthor (s)P. Diaz, etal.Lago, RamónUrmeneta, et alAparicio, et al.Aparicio, et al.Test methodRadialIndirect ELISACompetitiveRadialIndirect ELISAImunodiffusionELISAImunodiffusiontesttestSpeciesBovineHumanBovineBovine,Goatsheep-goatBacteria for antigenBrucellaBrucellaBrucellaBrucellaBrucellaproductionmelitensisM16melitensisM16melitensisM16melitensisM16;melitensisM16B. abortus2308;115, Rev 1;B. abortus19;Brucella abortusYersinia2308;YersiniaenterocoliticaenterocoliticaO:9O:9Culture medium ofTrypticasein SoyTrypticasein SoyTrypticasein SoyBroth (TSB)Broth (TSB)theBrucellaBroth (TSB)Broth (TSB)Agar (TSA)Trypticasein SoyTrypticasein SoyMethod of culture ofBroth:Broth:PLATE.Broth:Broth: incubation at 37° C.theBrucellaincubationincubationincubationincubationwith stirring 200 rpm48 hours at 37° C.48 hours at 37° C.2.3 days,48 hours at 37° C.with stirringwith stirringat 37° C.with stirringPre-harvest inactivation0.5% phenol at0.5% phenol at0.5% phenol at0.5% phenol atdoes not applymethod37° C. for37° C. for37° C. for37° C. for24 hours24 hours24 hours24 hoursHarvest method ofCentrifugationCentrifugation |Centrifugation atTangentialTangentialtheBrucellaat 12000 xg,at 12000 xg,12000 xg,filtrationfiltration30 minutes, 5° C.30 minutes, 5° C.30 minutes, 5° C.Harvest washing2 washes in2 washes in2 washes in2 washes in2 washes intechniquesaline solutionsaline solutionsaline solutionsaline solutionsaline solutionSolution to resuspendDistilled waterDistilled waterDistilled waterDistilled waterDistilled waterthe harvestPost-harvestSterilization atSterilization atSterilization atSterilization atPhenol 0.5% atinactivation method120° C.,120° C.,120° C.,120° C.,37° C. to 24 hours.30 minutes30 minutes30 minutes30 minutesExtraction anddoes not applydoes not applyHot water 120° C.does not applySterilization atPrecipitation Method120° C., 15 minutesCentrifugation atCentrifugation atdoes not specifyCentrifugation atCentrifugation12000 xg,12000 xg,12000 xg,30 minutes, 5° C.30 minutes, 5° C.30 minutes, 5° C.3X-Ethanol for3X-Ethanol for3X-Ethanol forReference R.3X-Ethanol1.8 hours at24 hours at 4° C.18 hours at 4° C.Diaz, 19815° C., withwith stiringstirringCentrifugation:Centrifugation:Centrifugation:Reference R.does not specify5000 g, for 155000 g, for 155000 g, for 15Diaz, 1981minutes, at 5° C.minutes, at 5° C.minutes, at 4° C.2X-Ethanol by2X-Ethanol by2X-Ethanol byReference R.2X-EthanolO/N at −20° C.O/N at −20° C.O/N at −20° C.Diaz, 1981Centrifugation:Centrifugation:Centrifugation:Reference R.does not specify5000 g, for 155000 g, for 155000 g, for 15Diaz, 1981minutes, at 5° C.minutes, at 5° C.minutes, at 5° C.Solution to resuspendDistilled waterDistilled waterdoes not specifydoes not specifydoes not specifythe NH antigen pelletPurificationDialysis againstFiltration in 40100 mg of theDigestion withDigestion withdistilled watermg chide NH in2nd precipitatenucleases,nucleases and2.5 ml Tris HCLin 50 ml of 4%DNAses andproteinase KSDSRNAse, 18 h37° C.ChromatographyWater bathDialysis againstUltra-with Sephacrylincubation 15 min0.1M sodiumcentrifugationS300acetatePM30Removed LPSDigestion with B-Phenolmembranecomplexes withD-glucosideExtractionfractionation5% potassiumglucohydrolaseacetate18 h 37° C.Dialysis againstIncubation 4° C.Proteinase K 1 hEthanoldistilled water 3for 12 h55° C. 2 timesprecipitationdays at 4° C.does not applyCentrifugation:Ultra-5000 g, for 10centrifugationminutes, at 4° C.6 h, 200,000 g5 volumes of1 volume of 70°ethanolphenolwas suspended9000 g 15 minin 150 mldistilled water.It was treated5 volumes oftwice with 4 g ofethanol −20° C.Dowex 1X4-200overnightDialysis7 volumes ofethanol −20° C.overnightLyophilizedDialysisdoes not applyDialysis7 volumes ofethanol −20° C.overnightLyophilizedDialysisSolution in 10mM borate-NaOHChromatography10 ml per h,column P300 biogel with boratebufferCollection ofhapten fractionsPrecipitationwith 5 volEthanoDialysisLyophilization ofYesYesYesYesdoes not specifythe antigenSensitizationAntigen concentrationdoes not apply20μg/ml1-2.5μg/mldoes not apply2.5μg/mlper ml of diluentDiluentBarbital acetate60mM CABI10mM PBSpH 4.6, with NapH 9.6pH 7.2azide 0.02%Volume of antigen100μldoes not specify100μldilution per wellAntigen concentration2μgdoes not specify0.25μgper well1st incubation12 h 37° C.Over Night 37° C.Over Night 4° C.1st wash solutionSol. Saline,does not specifyPSS Tween 20Tween 20(0.05%)(0.03%)Number of washes3does not specify4Volume of washdoes not specifydoes not specifydoes not specifysolution per wellBlocking solutiondoes not specifydoes not specifydoes not specify2nd Incubationdoes not specifydoes not specifydoes not specify2nd wash solutiondoes not specifydoes not specifydoes not specifyNumber of washesdoes not specifydoes not specifydoes not specifyVolume of washdoes not specifydoes not specifydoes not specifysolution per wellStoragedoes not specifydoes not specify4° C.Preparation of sampleType of sampleBlood serumBlood serumBlood Serum/Blood serumBlood serumMilk SerumSample treatmentdoes not applyinactivation ofdoes not applydoes not applydoes not applypositive sera at56° C. tor 30 minSample PredilutionYesYesYesSample quantitydoes not specifydoes not specifydoes not specifySample diluentPBS pH 7 2-does not specifyPBS Tweenalbumine 0.5%0.05%Amount of diluentdoes not specifydoes not specifydoes not specifyChallengeAmount of diluteddoes not apply100μl50μldoes not applydoes not specifysample per wellCompetitive50 μl ofdoes nto applySpecificationsheterologousof Competitive ELISAmolecule(concentration200 μg/ml)Final volume per well100μl100μldoes not specifyIncubation1 h 37° C.does not specify1 h 37° C.WashesWash solutiondoes not applySol. Saline,does not specifydoes not applyPBS TweenTween 200.05%(0.05%)Number of washes3does not specify4Volume of washdoes not specifydoes not specitydoes not specifysolution per wellConjugateConjugatedoes not applyIgG, IgA, IgMIgG anti-rabbitdoes not applyIgG anti-sheepanti-human (goat)(goat)policlonal(rabbit);Recombinant GproteinConjugate diluentdoes not specifydoes not specifyPBS Tween(0.05%)Conjugate1:600does not specifyIgG: 1:2000; GConcentrationprotein: 0.2 μg/mlFinal conjugate volume100μldoes not specify100μlper wellIncubation1 h 37° C.does not specify1 h 37° C.WashesWash solutiondoes not applySol. Saline,does not specifydoes not applydoes not specifyTween 20(0.05%)Number of washes3does not specifydoes not specifyVolume of washdoes not specifydoes not specifydoes not specifysolution per wellSubstrateSubstrate Namedoes not apply5-AS0.09% 5-amino-24does not applyABTShydroxy benzoicacidSubstrate DiluentH2O2H2O20.05M citratebufter (pH 4.0)and 0.004%H2O2Amount per well100μldoes not specify100μlSubstrate incubation1 h, Ambientdoes not specify15 min, 20° C.conditionstemperature indarknessStop the reactionStop solutiondoes not applyNaOH 1Ndoes not specifydoes not applydoes not specifyQuanity0.025mldoes not specifydoes not specifyStability timedoes not specifydoes not specifydoes not specifyReading of Optical DensitiesNanometersdoes not apply450450does not apply405Lyophilization of controlsOptical density fordoes not use itdoes not use itdoes not use itdoes not use itdoes not use itPositivecontrol selectionOptical density fordoes not use itdoes not use itdoes not use itdoes not use itdoes not use itNegativecontrol selectionUse of the lyophilizationdoes not use itdoes not use itdoes not use itdoes not use itdoes not use itprocess forNegative Controland Positive ControlSummary of procedures found, similar to the invention.67891019951990199920052008Publication dateEfren DiazB. AlonsoMarin.C.M.,Muñoz, P.M , etRicardo Fores,Author (s)Aparicio, et al.Urmeneta, et alet alalet alINVENTIONTest methodRadialIndirect ELISAImmunodifhisionindirect ELISA;PolarizedIndirect ELISAimunodiffusionon Agar GelRadialfluorescencetest(IDAG)ImunodiffusiontestSpeciesSheepBovine,SheepBovineGoatBovinesheep-goatBacteria for antigenBrucellaBrucellaBrucellaBrucellaBrucellaBrucella melitensisproductionmelitensisM16,melitensisM16,melitensisM16melitensisM16melitensisM16,M16Rev 1;BrucellaBrucella abortusRev 1abortus2308;2308YersiniaenterocoliticaO:9Culture medium ofBroth (TSB)1.7% casein-Reference R.Trypticasein SoyBrucella AgarTrypticasein SoytheBrucellaTrypticasein Soy0.3% soy-0.5 %Diaz. 1981Broth (TSB)Agar (TSA)yeast extract-0.25% K2HPO4-2% glucose, 0.5%NaCl-0.01%acetate A-butylantifoamMethod of cultureBroth:1.5 liters inReference R.Broth:PLATE:PLATE: incubationof theBrucellaincubation atFermenterDiaz, 1981incubation 48incubation 483-5 days, at37° C., withBiostat, 36 h,hours at 37° C.hours, at 37° C.37° C. 5% CO2stirring35% O2with stirringPre-harvest inactivationdoes not apply0.5% phenol atReference R.does not specify0.5% phenol atdoes not applymethod36° C. For 48Diaz, 198137° C. for 24hourshoursHarvest method ofTangentialTangentialReference R.Reference R.CentrifugationCollection with hoestheBrucellafiltrationfiltrationDiaz, 1981Diaz, 1981;Reference to B.Urmeneta, et al,1998Harvest washingdoes not apply2 washes inReference R.Reference R.does not apply1-2 washes intechniquesaline solutionDiaz, 1981Diaz. 1981;saline solutionReference to B.(6000 rpm forUrmeneta, et al,30 minutes)1998Solution to resuspendDistilled waterdoes not specifyReference R.Reference R.Distilled waterSaline solutionthe harvestDiaz, 1981Diaz, 1981;Reference to B.Urmeneta, et al,1998Post-harvestSterilization atdoes not specifyReference R.Reference R.Sterilization atSterilization ofinactivation method120° C., 15Diaz, 1981.Diaz, 1981;120° C.,15120° C.,minutesReference to B.minutes15 pounds,Urmeneta, et al,25 minutes1998Extraction anddoes not applyHot water 100° C.Reference R.Reference K.does not applydoes not applyPrecipitation MethodDiaz. 1981Diaz, 1981;Reference to B.Urmeneta, et al,1998Centrifugation:does not specifyReference R.Reference R.CentrifugationCentrifugation:12000 g, for 15Diaz, 1981Diaz, 1981;6000 rpmminutesReference to B.for 30 minutesUrmeneta, et al.19983X-Ethanol for3X-Ethanol forReference R.Reference R.3X-Ethanol for 183X -Ethanol for18 hours at 4° C.,18 hours at 4° C.,Diaz, 1981Diaz, 1981;hours at 4-8° C.,18 hours of 4° C.,with stirringwith stirringReference to B.with stirringwith stiringUrmeneta, et al,1998Centrifugation:does not specifyReference R.Reference R.CentrifugationCentrifugation:5000 g. for 10Diaz, 1981Diaz, 1981;6000 rpmminutes, at 4° C.Reference to B.for 30 minutesUnmeneta, et al,19982X-Ethanol for2X-Ethanol byReference R.Reference R.2X-Ethanol for2X-Ethanol18 hours atO/N at −20° C.Diaz, 1981Diaz. 1981;18 hours atfor 18 hours−20° C.Reference to B.−20° C.at −20° C.Unmeneta, et al,1998Centrifugation:does not specifyReference R.Reference R.CentrifugationCentrifugation:5000 g, for 5Diaz, 1981Diaz, 1981;6000 rpmminutes, at 5° C.Reference to B.for 30 minutesUrmeneta, et al,1998Solution to resuspenddoes not specifydoes not specifyReference R.Reference R.does not specifySaline solutionthe NH antigen pelletDiaz, 1981Diaz, 1981;Reference to B.Urmeneta, et al.1998PurificationDialysis againstDigestion withReference R.Reference R.Dialysis againstRAW NATIVEdistilled waternucleases andDiaz, 1981Diaz, 1981;distilled waterHAPTENproteinase KReference to B.(WITHOUTUrmeneta, et al,PURIFICATION1998PROCESS)UltracentrifugationReference R.Reference R.200,000 g,Diaz, 1981Diaz, 1981;6 h, 10° C.Reference to B.Urmeneta, et al,1998ChromatographyReference R.Reference R.Diaz, 1981Diaz. 1981;Reference to B.Urmeneta, et al,1998DialysisReference R.Reference R.Diaz, 1981Diaz, 1981;Reference to B.Jrmeneta, et al,1998Reference R.Reference R.Diaz, 1981.Diaz, 1981;Reference to B.Urmeneta, et al,1998Reference R.Reference R.Diaz, 1981.Diaz, 1981;Reference to B.Urmeneta, et al,1998Reference R.Reference R.Diaz, 1981Diaz, 1981;Reference to B.Urmeneta, et al,1998Reference R.Reference K.Diaz, 1981Diaz, 1981;Reference to B.Urmeneta, et al1998Reference R.Reference R.Diaz, 1981Diaz, 1981;Reference to BUrmeneta, et al,1998Reference R.Reference R.Diaz, 1981Diaz, 1981;Reference to B.Urmeneta, et al,1998Reference R.Reference R.Diaz, 1981Diaz, 1981;Reference to BUrmeneta, et al,1998Reference R.Reference R.Diaz, 1981Diaz, 1981;Reference to B.Urmeneta, et al,1998Reference R.Reference R.Diaz, 1981Diaz, 1981;Reference to B.Urmeneta, et al,1998Reference R.Reference R.Diaz, 1981Diaz, 1981;Reference to B.Urmeneta, et al,1998Reference R.Reference R.Diaz, 1981Diaz, 1981;Reference to B.Urmeneta, et al,1998Reference R.Reference R.Diaz, 1981.Diaz, 1981;Reference to B.Urmeneta, et al,1998Reference R.Reference R.Diaz, 1981.Diaz, 1981;Reference to B.Urmeneta, et al,1998Lyophilization of theYesYesdoes not specifydoes not specifyYesYesantigenSensitizationAntigen concentrationdoes not apply2.5μg/mldoes not apply2.5μg/mldoes not apply20μg/mlper ml of diluentDiluentCABI pH 9.6;PBSCABIPBS 7.2Volume of antigendoes not specifydoes not specity50μldilution per wellAntigen concentrationdoes not specifydoes not specify1μgper wellOver Night 37° C.;Over Night 4° C.1st incubation4° C.Over Night 4° C.1st wash solution98S Tween 20PBS Tween 20PBS Tween(0.05%)(0.05%)20 (0.05%)Number of washes434Volume of washdoes not specifydoes not specify250μlsolution per wellBlocking solutiondoes not specifydoes not specifySol 3% skim milk2nd Incubationdoes not specifydoes not specify1 h at 37° C.2nd wash solutiondoes not specifydoes not specifyPBS TweenNumber of washesdoes not specifydoes not specify20 (0.05%)Volume of wachdoes not specifydoes not specify250μlsolution per wellStorage4° C.does not specify2-8° C.Preparation of sampleType of sampleStood serumBlood serumBlood serumBlood serumBlood serumBlood serum andmilk (serum or whole,tank or per animal)Sample treatmentdoes not applydoes not applydoes not applydoes not applydoes not applydoes not applySample PredilutionYesYesYesSample quantitydoes not specifydoes not specify15μlSample diluentPBS TweenPBS TweenCABI0.05%0.05%Amount of diluentdoes not specifydoes not specify285μlChallengeAmount of diluteddoes not apply100μldoes not apply100μldoes not apply50μlsample per wellCompetitivedoes not applydoes not applydoes not applySpecificationsof Competitive ELISAFinal volume per welldoes not specifydoes not specify50μlIncubation1 h 37° C.1 h 37° C.1 h 37° C.WashesWash solutiondoes not applyPBS Tweendoes not applyPBS Tweendoes not applyPBS Twaan 0.05%0.05%0.05%Number of washes434Volume of washdoes not specifydoes not specify250μlsolution per wellConjugateConjugatedoes not applyIgG anti-sheepdoes not applyRecombinant Gdoes not applyIgG anti-bovine(rabbit); IgG anti-protein(goat)bovine; IgG anti-ruminant;Recombinent GproteinConjugate diluentPBS TweenPBS TweenCABI(0.05%)(0.05%)ConjugateIgGs: 1:1000-0.2μg/ml1:2000Concentration1:2000; Gprotein: 0.2 μg/mlFinal conjugate100μl100μl50μlvolume per wellIncubation1 h 37° C.1 h 37° C.1 h 37° C.WashesWash solutiondoes not applydoes not specifydoes not applyPBS Tweendoes not applyPBS Tween 0.05%Number of washesdices not specify34Volume of washdoes not specifydoes not specify250μlsolution per wellSubstrateSubstrate Namedoes not applyABTS 0.1%does not applyABTSdoes not applyABTSSubstrate Diluent0.05M citrate0.05M citratsdoes not applybuffer (pH 4.0)buffer (pH 4.0)and 0.004%and 0.004%H2O2H2O2Amount per well100μldoes not specify50μlSubstrate incubation15 min, 20° C.30 min, 20° C .15 min, 20-23° C.conditionsin the darkStop the reactionStop solutiondoes not applydoes not specifydoes not applydoes not applydoes not applySDS 4%Quanitydoes not specify50μlStability timedoes not specify30minReading of Optical DensitiesNanometersdoes not apply405does not applydoes not specifydoes not apply405Lyophilization of controlsOptical density fordoes not use itdoes not use itdoes not use itdoes not use itdoes not use it≥1.0Positivecontrol selectionOptical density fordoes not use itdoes not use itdoes not use itdoes not use itdoes not use it0.20-0.28Negativecontrol selectionUse of the lyophilizationdoes not use itdoes not use itdoes not use itdoes not use itdoes not use itUse of theprocessLyophilizationfor Negative Controlprocess for Negativeand Positive ControlConbol and PositiveControl
Work Done on the KIT Application:

In a study done in 2005 by a group of international researchers (including Dr. Bruno Garin-Bastuji, representative of the Reference Laboratory for Brucellosis in the European Union and of OIE/FAO) they used the Native Hapten ELISA with great success and recognizes that serological tests that detect anti-LPS-O antibodies could generate “False Positives” by cross-reactions with other Gram-negative bacteria including:Vibrio cholerae, Escherichia coliO: 157,SalmonellaandYersinia enterocolitica O:9, beingYersiniathe most frequent and significant “false positive”, since their levels of anti-LPS-O antibodies in serum and milk that cross withBrucella, are usually high, persistent and fluctuating. The cross reaction betweenYersiniaandBrucellais due to its strong similarity of their LPS-O chains. This study referenced 68 scientific articles, and recognizes up to 15% of “false positives” in free zones of the European Union and that, in advanced eradication programs, a differential diagnosis is required with a confirmatory test different from LPS-O(31).

The KIT, motive of the present application, has already been field tested in diverse and different statistical designs. One of these studies was a complete production cycle, with a length of twelve (12) months, at a stable of 2,533 milking cows (100%) in production located in Torreón, Coahuila, Mexico, a high brucellosis incidence region, all the conventional tests that measure anti-LPS-O, both in blood serum and milk, were performed. These conventional tests generated a total of 741 positive animals, that is, 29.25% of the cows in production.

In half of these animals, this positivity to LPS-O was Fluctuating during the 12 months of length of the study.

This KIT was used as a confirmatory test, through which the response to a second specific brucellosis antibody, Native Hapten, was measured, resulting in 15.24% of “False Positives” (386 animals) generated by cross-reaction with other bacteria, and 14.02% of truly infected animals (355 animals positive to anti-NH ELISA KIT).

The 15% of “False Positives” emitted by all the conventional tests that measure anti LPS-O, in blood serum or milk, generates a great economic loss for cattle breeding in the world.

Results are shown inFIG.5.

During this twelve month of the study the milk tank of 2,533 cows in production was monitored weekly, using a commercial ELISA and the milk ring test (MRT), both tests measure antibodies against LPS-O, and give the same “False Positives” in a fluctuating way. The milk was also confirmed with the KIT of the present application, which allowed corroborating the presence of anti-Native Hapten antibodies. Therefore, using this KIT for milk tank monitoring allows us to identify milk truly infected with brucellosis, discarding “False Positive” milk due to cross reactions with other bacteria.

BEST METHOD FOR CARRYING OUT THE INVENTION

Raw Native Hapten

The extraction of NH was made with the traditional method mentioned in literature of the present application. The antigen pellet was re-suspended in saline and tested crude (without purification) by RID and indirect anti-NH ELISA tests with positive blood sera confirmed with the official tests of NOM-041-ZOO-1995. In this assay, favorable results were obtained, so it was decided to test the crude extract with milk samples in indirect anti-NH ELISA test, where a favorable result was also obtained. Therefore, it was concluded that antigen purification is not necessary for plate coating.

NH Concentration

Different antigen dilutions were made, microplates were coated with those antigens, sera were tested and it was found the best concentration to coating. The ELISA test standardization was carried out by titration with the Native Hapten antigen (NH) to determine the optimal concentration of the antigen with which the microplates should be coated.

The antigen titration was based on the optimal antigen concentration of Radial Immunodiffusion (RID) test, which is 2 mg/mL. From this concentration, a series of dilutions was performed (1:2, 1:4, 1:8, 1:16, 1:32, 1:64, 1:100 and 1:128) with CABI buffer. 50 μl of each dilution of the antigen were added to the plate, i.e. distributing one dilution per line, so that each dilution was present in 12 wells of the plate. Subsequently, the coating protocol was continued.

Four samples of positive sera and 4 samples of negative sera were selected, which were tested in ELISA for triplicate. From the absorbance obtained, the mean absorbance of the positive and negative sera were obtained to establish the threshold that allowed differentiating negative absorbance from positive ones.

The criteria used was the following: To consider a sample as positive, the optical density reading was above the range of the negative's average plus three standard deviations (0.5432+3 (0.1521)=0.9995). Taking this cut-off as a reference, it was determined that the optimal antigen concentration in the plate corresponds to the value that best approaches to the optical density of 0.9995, minus one antigen dilution. Thus establishing the dilution 1:100 in the case of the antigen which corresponds to 1 microgram per well.

Use of Anti-Bovine IgG (Goat)

To search for the best secondary antibody, we reviewed the literature cited above, where we found reports on different types of immunoglobulins used in ELISA methods in different animal species, including bovine. It was determined that to obtain a better efficiency of the test directed to bovines it is necessary to use an anti-bovine IgG.

Native Hapten in Milk

In order to determine the minimum point of detection of positive milk in a negative milk tank, 92 dilutions were made, starting with a ratio of 1:1 of positive milk diluted in negative milk. The positive milk sample came from a cow confirmed as positive toBrucella, with the highest positivity presented in the Ring Milk test, RBT, Rivanol (1:400), Radial Immunodiffusion (RID) and indirect ELISA with NH. These anti-NH ELISAs were performed in both blood serum and milk serum. The negative milk was obtained from a pool of nine cows negative to all the tests mentioned before.

Of the 92 dilutions tested, the indirect anti-NH ELISA was able to detect the positivity level until the dilution number 27 corresponding to 969,162 liters of positive milk within a milk tank containing a volume of 29,030,838 liters of negative milk, giving a total of 30,000 liters. The positivity of the 27 dilutions studied was confirmed by the Ring Milk test, demonstrating the efficiency of the test for this type of samples.

Optical Density Range (DO) of Controls and their Lyophilization Process

The controls obtained from samples of blood serum and milk from animals were preselected based on their clinical story and positive or negative result at the official tests of Rose of Bengal (RBT), Rivanol, and Ring Milk (RMT), as well as in; fluorescence polarization assay (FPA) and Radial Immunodiffusion (RID).

Indirect anti-NH ELISA tests were performed on the selected sera, and based on the results, we selected the samples that showed an absorbance ≥1,000 for positive control and 0.20-0.28 for negative control. Two pools of sera were formed, one pool for positive control and the other for negative control, to which the official tests, RID and indirect ELISA were made, to confirm their positivity and negativity. Once the control pools were approved, they were subjected to lyophilization.

Two vials of freeze dried controls were taken, one positive and the other negative. They were resuspended with 1 mL sterile tridestilated water, and homogenized perfectly. To check control's stability once resuspended, they were analyzed by the official serological tests established by the norm NOM-041-ZOO-1995 as well as RID and the indirect anti-NH ELISA tests. Those tests were performed every 15 days. Said resuspended controls were kept under refrigeration at 4° C.

The stability of the controls was confirmed observing a concordance of 1 (one) between the 5 tests, that is, the positive control was positive to the 5 tests and the negative control was negative to the 5 tests. Finding that the optical densities were stable.

Kit Solutions Preparation

Preparation of Culture Medium and Solutions

The solutions to develop the kit are prepared with the procedure described below.

Tripticase Soy Agar (TSA) Medium: To prepare 1 L of TSA medium. Weigh 40 g/L of TSA and dissolve in 1 L of distilled water, the medium must be dissolved by heat until boil, then sterilized in an autoclave at 120° C. and 15 lbs. For 15 minutes. Once sterilized the medium is allowed to cool and before solidifying it is poured into Petri dishes, pouring approximately 5 mm thick into each plate. The well-sealed plates are allowed to solidify and stored at 4° C. The latter must be worked in a laminar flow cabinet, or in the presence of a Fisher burner.

Saline solution (NaCl 0.9%): To prepare 1 L of saline solution. Weigh 9 g of sodium chloride and dissolve it in 1 L of distilled water, mixing perfectly. Sterilize in autoclave at 120° C. and 15 lb. for 15 minutes.

Blocking solution: A solution of 3% skim milk is prepared. Weighing 3 g of skimmed milk powder and dissolve in 100 ml of sterile distilled water, mix perfectly to dissolve completely.

Sample Diluent Solution (10×) and Conjugate Diluent Solution (1×). Carbonate-Bicarbonate Buffer (CABI): A CABI buffer at pH of 9.6 is used as diluent. To prepare the solution at a 10× concentration, (the amounts at 1× change in proportion): Weigh 35.6 g of sodium carbonate (Na2CO3) and dissolve with distilled water. Add 84 g of sodium bicarbonate (NaHCO3) and dissolve. Add 2 g of sodium azide (NaN3) as preservative and dissolve. Adjust the pH of the solution to 9.6, using hydrochloric acid (HCl) or sodium hydroxide (NaOH).

Gauge to 1 L using a volumetric flask, and filter the solution with filter paper. Sterilize in an autoclave at 120° C. and 15 lb for 15 minutes and label with name, batch number and date of manufacture.

Washing Solution (10×). PBS-Tween 20 (0.05%): Weigh 14.4 g of Sodium hydrogen phosphate (Na2HPO4) and dissolve in distilled water using a stirrer. Weigh 2.2 g of Potassium dihydrogen phosphate (KH2PO4) and add it, stir until it dissolves perfectly. Add more water if necessary. Add 2 g of potassium chloride (KCl) and dissolve perfectly. Add 80 g of sodium chloride (NaCl) to the solution and stir until it is perfectly dissolved, the solution should remain crystalline. Adjust the pH of the solution to 7.4, using hydrochloric acid or sodium hydroxide. Gauge to 1 L and shake the solution well. Filter the solution using filter paper and sterilize in an autoclave at 120° C. and 15 lb. for 15 minutes. Upon cooling, add 5 ml of tween-20, shaking gently. Pour into a bottle and label the bottle with name, batch number and date of manufacture.

Stop solution. SDS 4%: 4% sodium dodecyl sulfate (SDS) is used as a stop solution. Weigh 40 g of SDS. Mix with distilled water until it dissolves completely and gauge the solution at 1 L. If it does not dissolve completely, heat the solution until it is warm, at approximately 37° C. until it dissolves perfectly. Sterilize in an autoclave at 120° C. and 15 lb. for 15 minutes. Pour in a bottle and label with name, batch number and date.

Substrate: A 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) commercial solution is used as substrate.

REFERENCES

1. Brucellosis. Medical microbiology. INRA. 28:512-525.2. Arzola, V. E. A., Gonzalez, M. A., González, J. L. I., Hernández, M. L., Aparicio, E. D., Moorillón, G. V. N., & River, B. E. (2012). Diagnóstico rápido y efectivo de brucelosis bovina en sangre, mediante una reacción en cadena de la polimerasa doble. Revista Mexicana de Ciencias Pecuarias, 46(2), 147-158.3. Aparicio, E. D. (2013). Epidemiologia de la brucelosis causada porBrucella melitensis, Brucella suisyBrucella abortusen animales domésticos. Revue Scientifique et Technique, 32(1), 43-51.4. Esperanza Gonzalez, M., Hernández Andrade, L., & Díaz Aparicio, E. (2006). Prueba de inmunodifusión radial con hapteno nativo para diferenciar bovinos con revacunaciones repetidas con la cepa S19 deBrucella abortus,44, 269-276.5. E. Herrera López, L. Hernández Andrade, G. P. R. and E. D. A. (2007). Study of Brucellosis Incidence in a Bovine Dairy Farm Infected withBrucella abortus, Where Cattle Was Revaccinated with RB51. International Journal of Diary Science. 2(1): 50-57.6. Herrera, E., Palomares, G., & Diaz-Aparicio, E. (2008). Milk production increase in a dairy farm under a six-year brucellosis control program. Annals of the New York Academy of Sciences, 1149, 296-299.7. Leal-hernández, M., Jaramillo-meza, L., & Hernández-andrade, L. (2007). Producción de interferón gamma en cultivos de sangre completa en respuesta a antigenos deBrucella abortusen bovinos vacunados con RB51 Production of interferon gamma in whole blood cultures in response toBrucella abortusantigens in RB51-vaccinated cat, 45(2), 147-159.8. Maria Dolores Fuentes Delgado, Irene V. Vitela Mendoza, Beatriz Arellano-Reynoso, Rigoberto Hernández Castro, José Francisco Morales Álvarez, Carlos Cruz-Vazquez. (2007). Presence ofBrucella abortusvaccinal strain RB51 in Vaginal Exudates of abortaded cows. Research Journal of Dairy Sciences, 1 (1-4): 13-17.9. Aparicio-Bahena, A., Diaz-Aparicio, E., Hernández-Andrade, L., Pérez-González, R., Alfonseca-Silva, E., & Suárez-Güemes, F. (2003). Evaluación serológica y bacteriológica de un hato bovino con brucelosis y revacunado con dosis reducida deBrucella abortus cepa19. Técnica Pecuaria, 41(2), 129-140.10. A., Castro, A., González, H. A; Prat, S. R.; & Baldi, P. C. (2006). Detección de anticuerpos anti-Brucellaspp. en cerdos mediante técnicas de aglutinación y ELISA indirecto en las provincias de Buenos Aires y La Pampa, Argentina. Revista Argentina de Microbiologia, 38: 75-78.11. Kittelberger R, Hilbink F, Hansen M F, Penrose M, de Lisle G W, Letesson J J, et al. Serological crossreactivity betweenBrucella abortusandYersinia enterocolitica0:9. Immunoblot analysis of the antibody response toBrucellaprotein antigens in bovine brucellosis. (1995). Vet Microbiol; 47: 257-70.12. Rodriguez, A., Orduña, A., Ariza, X., Morivon, I., Diaz, R., Blasco, J., Almaraz, A., Martinez, F., Ruiz, C. y Abad, R. (2001). Manual de Brucelosis. Ed. Junta de Castilla y Leon. Copyright. Zamora, España.13. Abernethy D. A., Moscard-Costello J., Dickson E., Harwood R, Burns K., McKillop E., McDowell S, & Pftffer D. U. (2011).—Epidemiology and management of a bovine brucellosis cluster in Northern Ireland. Prev. vet. Med., 98 (4), 223-229.14. Bustamante Sanchéz, J., Hernández Salazar, I., Díaz, A. E., Mazano Cañas, C., Pérez González, R., & Andrade, L. H. (2000). Estudio Bacteriológico Y Serológico De Brucelosis en vacas revacunadas con dosis reducida decepa19 deBrucella abortus. Inifap, (5).15. Cantú, A., Díaz, E., Andrade, L. H., Adams, G. L., & Güemes, F. S. (2007). Estudio epidemiológico de un hato bovino con prevalencia media de brucelosis, vacunado con las mutantes rugosas de. Group, 38(2), 197-206.16. Diaz, R, P. Garatea, L. M. Jones, and I. Moriyon. (1979). Radial immunodiffusion test with aBrucellapolysaccharide antigen for differentiating infected from vaccinated cattle. J. Clin. Micro-biol. 10:37-41.17. Moriyón UI.Brucellacell structure. In: Memory to 50th Anniversary Meeting of Brucellosis Research Conference. Chicago ILL, USA Nov. 8-9, 1997: 3-18.18. Moriyon, I. et al. (1998). Structure and properties of the outer membranes ofBrucella abortusandBrucella melitensis. Internatl Microbiol, 19-26.19. Alonso-Urmeneta, B., Moriyon, I., & Blasco, J. M. (1988). Enzyme-linked immunosorbent assay withBrucellanative hapten polysaccharide and smooth lipopolysaccharide. Journal of Clinical Microbiology.20. Dubray, G. (1984). Progrès récents sur la biochimie et les pro-priétés biologiques des antigènes deBrucella. Dev. Biol. Stand. 56:131-150.21. Stemshorn, B. W. (1984). Recent progress in the diagnosis of brucellosis. Dev. Biol. Stand. 56:325-340.22. Organizacion Mundial de Sanidad Animal. (2000). Brucelosis bovina, 1-8.23. Diaz, R., Toyos, J., Salvó, M. D., & Pardo, M. L. (1981). A simple method for the extraction of polysaccharide B fromBrucellacells for use in the radial immunodiffusion test diagnosis of bovine brucellosis. Annales de Recherches Veterinaires. Annals of Veterinary Research, 12(1), 35-39.24. Fernandez-Lago, L., & Diaz, R. (1986). Demonstration of antibodies againstBrucella melitensis16M lipopolysaccharide and native hapten in human sera by enzyme-linked immunosorbent assay. Journal of Clinical Microbiology.25. Alonso-Urmeneta, B., Moriyon, I., & Blasco, J. M. (1988). Enzyme-linked immunosorbent assay withBrucellanative hapten polysaccharide and smooth lipopolysaccharide. Journal of Clinical Microbiology, 26(12), 2642-2646.26. Diaz-Aparicio E, Aragon V, Marin C, Alonso B, Font M, Moreno E, et al. Comparative analysis ofBrucellaserotype A and M andYersinia enterocolitica O:9 polysaccharides for serological diagnosis of brucellosis in cattle, sheep, and goats. J Clin Microbiol. 1993; 31(12):3136-41.27. Diaz-Aparicio, E., Marin, C., Alonso-Urmeneta, B., Aragón, V., Pérez-Ortiz, S., Pardo, M., Moriyón, I. (1994). Evaluation of Serological Tests for Diagnosis ofBrucella melitensisInfection of Goats. Journal of Clinical Microbiology, 32(5), 1159.28. Diaz-Aparicio, E., Uria, I. M., Blasco-Martinez, J. M., Marin-Alcala, C., & Diaz, R. (1996). Diagnóstico deBrucella melitensisen ovinos usando inmunodifusion radial con hapteno nativo. Técnica Pecuaria En México, 34(2), 99-103.29. Alonso-Urmeneta B, Marin C, Aragón V, Blasco J M, Diaz R, Moriyón I. Evaluation of lipopolysaccharides and polysaccharides of different epitopic structures in the indirect enzyme-linked immunosorbent assay for diagnosis of brucellosis in small ruminants and cattle. Clin Diagn Lab Immunol [Internet]. 1998; 5(6):749-54.30. Marin, C. M., Moreno, E., Moriyón, I., Diaz, R., & Blasco, J. M. (1999). Performance of competitive and indirect enzyme-linked immunosorbent assays, gel immunoprecipitation with native hapten polysaccharide, and standard serological tests in diagnosis of sheep brucellosis. Clinical and Diagnostic Laboratory Immunology, 6(2), 269-272.31. Muñoz, P. M., C. M. Marín, D. Monreal, D. Gonzalez, B. Garín-Bastuji, R. Diaz, R. C. Mainer-Jaime, I. Moriyón, and J. M. Blasco. (2005). Efficacy of Several Serological Tests and Antigens for Diagnosis of Bovine Brucellosis in the Presence of False-Positive Serological Results Due toYersiniaenterolitica O:9 American Society for Microbiology. Clin Diagn Lab Immunol [Internet]. 2005; 12(1):141-51.32. WO2008051065 A1, fecha de presentación: Oct. 16, 2007. Fecha de publicación: Mayo 2, 2008. Universidad Autónoma de Nuevo León. https://www.google.com/patents/WO2008051065A1?cl=es

BRIEF FIGURES DESCRIPTION

FIG.1shows the flow diagram of the Native Hapten antigen obtaining process. From theB. melitensis16M strain cultivation, the cell harvest and the antigen extraction until the lyophilization process of the antigen obtained.

FIG.2shows 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.

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

FIG.4shows 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.

FIG.5shows 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.