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
12222353 ยท 2025-02-11
Inventors
Cpc classification
International classification
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 cross-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. 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 L 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 L of Negative Control in wells A1 and B1; and 15 L of Positive Control in wells C1 and D1, continuing adding 15 L of the sample (blood serum or milk) in the remaining wells; Taking 50 L 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 L of the previously diluted Washing Solution, and making a total of 4 washes; wherein the washing solution is PBS-TWEEN 20; Adding 50 L 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 L of Substrate (ABTS) to each well; Incubating the Microplate for 15 minutes at room temperature (20 C.-25 C.) in darkness; Distributing 50 L 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.
2. A kit for indirect ELISA test, wherein the kit comprises the following components: ten (10) microplates coated with 1 microgram per well concentration of antigen crude Native Hapten of claim 1; 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; 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 dodecyl sulfate (SDS) at 4% concentration; one (1) freeze dried positive blood serum vial; one (1) freeze dried negative blood serum vial; one (1) freeze dried positive milk serum vial; one (1) freeze dried negative milk serum vial; and wherein all control vials contain one (1) milliliter of freezed dried serum for reconstitution in one (1) milliliter of distilled water.
Description
DETAILED DESCRIPTION OF THE INVENTION
(1) 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.
(2) 1.Antigen Production
(3) Brucella melitensis 16M cultivation
(4) 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 the Brucella melitensis 16M 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 of B. melitensis strain. 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.
(5) Cell Harvest
(6) 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.
(7) Extraction
(8) 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.
(9) Lyophilization of Raw Native Hapten Antigen
(10) 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 in
(11) 2.Microplate Coating
(12) 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 g/ml), and it is mixed perfectly. To each well of the microplates is added 50 l (1 g of antigen) of this solution and sealed with parafilm. The microplates are incubated at 4 C. for 18 hours (Overnight).
(13) The microplates are washed with a 0.05% PBS-Tween 20 washing solution by adding 250 l 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 l 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 in
(14) 3.Procedure of the Indirect ELISA Anti-NH
(15) Preparation of Samples
(16) 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.
(17) Blood Serum:
(18) 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.
(19) Milk:
(20) 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.
(21) Preparation of ELISA Reagents
(22) 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.
(23) Sample Diluent Solution. Bicarbonate Carbonate Buffer (CABI):
(24) 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.
(25) Washing Solution. PBS-TWEEN 20 (0.05%):
(26) Dilute the 10 Wash Solution in a 1:10 rate. Example, to prepare 250 mL, dilute 25 mL in 225 mL of distilled water.
(27) Concentrated Conjugate:
(28) 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 l of Concentrated Conjugate in 4.9 mL of Conjugate Diluent Solution.
(29) Stop Solution. SDS 4%:
(30) 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.
(31) Positive and Negative Controls of Blood Serum and Milk Whey:
(32) 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.
(33) Indirect ELISA Process
(34) 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 L of Sample Diluent Solution to each well in the pre-dilution Microplate. Add 15 L of Negative Control in wells A1 and B1; and 15 L of Positive Control in wells C1 and D1. Continue adding 15 L of the sample (blood serum or milk) in the remaining wells. Take 50 L 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 L 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 L 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 L 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 L 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 in
(35) 4.Selection and Lyophilization of Positive and Negative Controls of Blood Serum and Milk Serum (Whey).
(36) Blood Serum and Milk Localization for Producing Controls
(37) 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.
(38) 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.
(39) Pre-Selection of Controls
(40) 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.
(41) Positive and Negative Control Pools Validation
(42) 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.
(43) Lyophilization of Positive and Negative Blood Serum and Milk Serum (Whey) Controls.
(44) 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 in
PROCEDURES SIMILAR TO THE INVENTION
(45) A search was made to corroborate the existence of procedures similar to the present invention, finding the following:
(46) TABLE-US-00001 Summary of procedures found, similar to the invention. 2 3 4 5 1 1986 1988 1993 1994 Publication date 1981 Luis Fernndez B. Alonso Efren Diaz Efren Diaz Author (s) P. Diaz, etal. Lago, Ramn Urmeneta, et al Aparicio, et al. Aparicio, et al. Test method Radial Indirect ELISA Competitive Radial Indirect ELISA Imunodiffusion ELISA Imunodiffusion test test Species Bovine Human Bovine Bovine, Goat sheep-goat Bacteria for antigen Brucella Brucella Brucella Brucella Brucella production melitensis M16 melitensis M16 melitensis M16 melitensis M16; melitensis M16 B. abortus 2308; 115, Rev 1; B. abortus 19; Brucella abortus Yersinia 2308; Yersinia enterocolitica enterocolitica O:9 O:9 Culture medium of Trypticasein Soy Trypticasein Soy Trypticasein Soy Broth (TSB) Broth (TSB) the Brucella Broth (TSB) Broth (TSB) Agar (TSA) Trypticasein Soy Trypticasein Soy Method of culture of Broth: Broth: PLATE. Broth: Broth: incubation at 37 C. the Brucella incubation incubation incubation incubation with stirring 200 rpm 48 hours at 37 C. 48 hours at 37 C. 2.3 days, 48 hours at 37 C. with stirring with stirring at 37 C. with stirring Pre-harvest inactivation 0.5% phenol at 0.5% phenol at 0.5% phenol at 0.5% phenol at does not apply method 37 C. for 37 C. for 37 C. for 37 C. for 24 hours 24 hours 24 hours 24 hours Harvest method of Centrifugation Centrifugation | Centrifugation at Tangential Tangential the Brucella at 12000 xg, at 12000 xg, 12000 xg, filtration filtration 30 minutes, 5 C. 30 minutes, 5 C. 30 minutes, 5 C. Harvest washing 2 washes in 2 washes in 2 washes in 2 washes in 2 washes in technique saline solution saline solution saline solution saline solution saline solution Solution to resuspend Distilled water Distilled water Distilled water Distilled water Distilled water the harvest Post-harvest Sterilization at Sterilization at Sterilization at Sterilization at Phenol 0.5% at inactivation method 120 C., 120 C., 120 C., 120 C., 37 C. to 24 hours. 30 minutes 30 minutes 30 minutes 30 minutes Extraction and does not apply does not apply Hot water 120 C. does not apply Sterilization at Precipitation Method 120 C., 15 minutes Centrifugation at Centrifugation at does not specify Centrifugation at Centrifugation 12000 xg, 12000 xg, 12000 xg, 30 minutes, 5 C. 30 minutes, 5 C. 30 minutes, 5 C. 3X-Ethanol for 3X-Ethanol for 3X-Ethanol for Reference R. 3X-Ethanol 1.8 hours at 24 hours at 4 C. 18 hours at 4 C. Diaz, 1981 5 C., with with stiring stirring Centrifugation: Centrifugation: Centrifugation: Reference R. does not specify 5000 g, for 15 5000 g, for 15 5000 g, for 15 Diaz, 1981 minutes, at 5 C. minutes, at 5 C. minutes, at 4 C. 2X-Ethanol by 2X-Ethanol by 2X-Ethanol by Reference R. 2X-Ethanol O/N at 20 C. O/N at 20 C. O/N at 20 C. Diaz, 1981 Centrifugation: Centrifugation: Centrifugation: Reference R. does not specify 5000 g, for 15 5000 g, for 15 5000 g, for 15 Diaz, 1981 minutes, at 5 C. minutes, at 5 C. minutes, at 5 C. Solution to resuspend Distilled water Distilled water does not specify does not specify does not specify the NH antigen pellet Purification Dialysis against Filtration in 40 100 mg of the Digestion with Digestion with distilled water mg chide NH in 2nd precipitate nucleases, nucleases and 2.5 ml Tris HCL in 50 ml of 4% DNAses and proteinase K SDS RNAse, 18 h 37 C. Chromatography Water bath Dialysis against Ultra- with Sephacryl incubation 15 min 0.1M sodium centrifugation S300 acetate PM30 Removed LPS Digestion with B- Phenol membrane complexes with D-glucoside Extraction fractionation 5% potassium glucohydrolase acetate 18 h 37 C. Dialysis against Incubation 4 C. Proteinase K 1 h Ethanol distilled water 3 for 12 h 55 C. 2 times precipitation days at 4 C. does not apply Centrifugation: Ultra- 5000 g, for 10 centrifugation minutes, at 4 C. 6 h, 200,000 g 5 volumes of 1 volume of 70 ethanol phenol was suspended 9000 g 15 min in 150 ml distilled water. It was treated 5 volumes of twice with 4 g of ethanol 20 C. Dowex 1X4-200 overnight Dialysis 7 volumes of ethanol 20 C. overnight Lyophilized Dialysis does not apply Dialysis 7 volumes of ethanol 20 C. overnight Lyophilized Dialysis Solution in 10 mM borate- NaOH Chromatography 10 ml per h, column P300 bio gel with borate buffer Collection of hapten fractions Precipitation with 5 vol Ethano Dialysis Lyophilization of Yes Yes Yes Yes does not specify the antigen Sensitization Antigen concentration does not apply 20 g/ml 1-2.5 g/ml does not apply 2.5 g/ml per ml of diluent Diluent Barbital acetate 60 mM CABI 10 mM PBS pH 4.6, with Na pH 9.6 pH 7.2 azide 0.02% Volume of antigen 100 l does not specify 100 l dilution per well Antigen concentration 2 g does not specify 0.25 g per well 1st incubation 12 h 37 C. Over Night 37 C. Over Night 4 C. 1st wash solution Sol. Saline, does not specify PSS Tween 20 Tween 20 (0.05%) (0.03%) Number of washes 3 does not specify 4 Volume of wash does not specify does not specify does not specify solution per well Blocking solution does not specify does not specify does not specify 2nd Incubation does not specify does not specify does not specify 2nd wash solution does not specify does not specify does not specify Number of washes does not specify does not specify does not specify Volume of wash does not specify does not specify does not specify solution per well Storage does not specify does not specify 4 C. Preparation of sample Type of sample Blood serum Blood serum Blood Serum/ Blood serum Blood serum Milk Serum Sample treatment does not apply inactivation of does not apply does not apply does not apply positive sera at 56 C. tor 30 min Sample Predilution Yes Yes Yes Sample quantity does not specify does not specify does not specify Sample diluent PBS pH 7 2- does not specify PBS Tween albumine 0.5% 0.05% Amount of diluent does not specify does not specify does not specify Challenge Amount of diluted does not apply 100 l 50 l does not apply does not specify sample per well Competitive 50 l of does nto apply Specifications heterologous of Competitive ELISA molecule (concentration 200 g/ml) Final volume per well 100 l 100 l does not specify Incubation 1 h 37 C. does not specify 1 h 37 C. Washes Wash solution does not apply Sol. Saline, does not specify does not apply PBS Tween Tween 20 0.05% (0.05%) Number of washes 3 does not specify 4 Volume of wash does not specify does not specity does not specify solution per well Conjugate Conjugate does not apply IgG, IgA, IgM IgG anti-rabbit does not apply IgG anti-sheep anti-human (goat) (goat) policlonal (rabbit); Recombinant G protein Conjugate diluent does not specify does not specify PBS Tween (0.05%) Conjugate 1:600 does not specify IgG: 1:2000; G Concentration protein: 0.2 g/ml Final conjugate volume 100 l does not specify 100 l per well Incubation 1 h 37 C. does not specify 1 h 37 C. Washes Wash solution does not apply Sol. Saline, does not specify does not apply does not specify Tween 20 (0.05%) Number of washes 3 does not specify does not specify Volume of wash does not specify does not specify does not specify solution per well Substrate Substrate Name does not apply 5-AS 0.09% 5-amino-24 does not apply ABTS hydroxy benzoic acid Substrate Diluent H2O2 H2O2 0.05M citrate bufter (pH 4.0) and 0.004% H2O2 Amount per well 100 l does not specify 100 l Substrate incubation 1 h, Ambient does not specify 15 min, 20 C. conditions temperature in darkness Stop the reaction Stop solution does not apply NaOH 1N does not specify does not apply does not specify Quanity 0.025 ml does not specify does not specify Stability time does not specify does not specify does not specify Reading of Optical Densities Nanometers does not apply 450 450 does not apply 405 Lyophilization of controls Optical density for does not use it does not use it does not use it does not use it does not use it Positive control selection Optical density for does not use it does not use it does not use it does not use it does not use it Negative control selection Use of the lyophilization does not use it does not use it does not use it does not use it does not use it process for Negative Control and Positive Control Summary of procedures found, similar to the invention. 6 7 8 9 10 1995 1990 1999 2005 2008 Publication date Efren Diaz B. Alonso Marin.C.M., Muoz, P.M , et Ricardo Fores, Author (s) Aparicio, et al. Urmeneta, et al et al al et al INVENTION Test method Radial Indirect ELISA Immunodifhision indirect ELISA; Polarized Indirect ELISA imunodiffusion on Agar Gel Radial fluorescence test (IDAG) Imunodiffusion test Species Sheep Bovine, Sheep Bovine Goat Bovine sheep-goat Bacteria for antigen Brucella Brucella Brucella Brucella Brucella Brucella melitensis production melitensis M16, melitensis M16, melitensis M16 melitensis M16 melitensis M16, M16 Rev 1; Brucella Brucella abortus Rev 1 abortus 2308; 2308 Yersinia enterocolitica O:9 Culture medium of Broth (TSB) 1.7% casein- Reference R. Trypticasein Soy Brucella Agar Trypticasein Soy the Brucella Trypticasein Soy 0.3% soy-0.5 % Diaz. 1981 Broth (TSB) Agar (TSA) yeast extract- 0.25% K2HPO4- 2% glucose, 0.5% NaCl-0.01% acetate A-butyl antifoam Method of culture Broth: 1.5 liters in Reference R. Broth: PLATE: PLATE: incubation of the Brucella incubation at Fermenter Diaz, 1981 incubation 48 incubation 48 3-5 days, at 37 C., with Biostat, 36 h, hours at 37 C. hours, at 37 C. 37 C. 5% CO2 stirring 35% O2 with stirring Pre-harvest inactivation does not apply 0.5% phenol at Reference R. does not specify 0.5% phenol at does not apply method 36 C. For 48 Diaz, 1981 37 C. for 24 hours hours Harvest method of Tangential Tangential Reference R. Reference R. Centrifugation Collection with hoes the Brucella filtration filtration Diaz, 1981 Diaz, 1981; Reference to B. Urmeneta, et al, 1998 Harvest washing does not apply 2 washes in Reference R. Reference R. does not apply 1-2 washes in technique saline solution Diaz, 1981 Diaz. 1981; saline solution Reference to B. (6000 rpm for Urmeneta, et al, 30 minutes) 1998 Solution to resuspend Distilled water does not specify Reference R. Reference R. Distilled water Saline solution the harvest Diaz, 1981 Diaz, 1981; Reference to B. Urmeneta, et al, 1998 Post-harvest Sterilization at does not specify Reference R. Reference R. Sterilization at Sterilization of inactivation method 120 C., 15 Diaz, 1981. Diaz, 1981; 120 C.,15 120 C., minutes Reference to B. minutes 15 pounds, Urmeneta, et al, 25 minutes 1998 Extraction and does not apply Hot water 100 C. Reference R. Reference K. does not apply does not apply Precipitation Method Diaz. 1981 Diaz, 1981; Reference to B. Urmeneta, et al, 1998 Centrifugation: does not specify Reference R. Reference R. Centrifugation Centrifugation: 12000 g, for 15 Diaz, 1981 Diaz, 1981; 6000 rpm minutes Reference to B. for 30 minutes Urmeneta, et al. 1998 3X-Ethanol for 3X-Ethanol for Reference R. Reference R. 3X-Ethanol for 18 3X -Ethanol for 18 hours at 4 C., 18 hours at 4 C., Diaz, 1981 Diaz, 1981; hours at 4-8 C., 18 hours of 4 C., with stirring with stirring Reference to B. with stirring with stiring Urmeneta, et al, 1998 Centrifugation: does not specify Reference R. Reference R. Centrifugation Centrifugation: 5000 g. for 10 Diaz, 1981 Diaz, 1981; 6000 rpm minutes, at 4 C. Reference to B. for 30 minutes Unmeneta, et al, 1998 2X-Ethanol for 2X-Ethanol by Reference R. Reference R. 2X-Ethanol for 2X-Ethanol 18 hours at O/N at 20 C. Diaz, 1981 Diaz. 1981; 18 hours at for 18 hours 20 C. Reference to B. 20 C. at 20 C. Unmeneta, et al, 1998 Centrifugation: does not specify Reference R. Reference R. Centrifugation Centrifugation: 5000 g, for 5 Diaz, 1981 Diaz, 1981; 6000 rpm minutes, at 5 C. Reference to B. for 30 minutes Urmeneta, et al, 1998 Solution to resuspend does not specify does not specify Reference R. Reference R. does not specify Saline solution the NH antigen pellet Diaz, 1981 Diaz, 1981; Reference to B. Urmeneta, et al. 1998 Purification Dialysis against Digestion with Reference R. Reference R. Dialysis against RAW NATIVE distilled water nucleases and Diaz, 1981 Diaz, 1981; distilled water HAPTEN proteinase K Reference to B. (WITHOUT Urmeneta, et al, PURIFICATION 1998 PROCESS) Ultracentrifugation Reference R. Reference R. 200,000 g, Diaz, 1981 Diaz, 1981; 6 h, 10 C. Reference to B. Urmeneta, et al, 1998 Chromatography Reference R. Reference R. Diaz, 1981 Diaz. 1981; Reference to B. Urmeneta, et al, 1998 Dialysis Reference R. Reference R. Diaz, 1981 Diaz, 1981; Reference to B. Jrmeneta, et al, 1998 Reference R. Reference R. Diaz, 1981. Diaz, 1981; Reference to B. Urmeneta, et al, 1998 Reference R. Reference R. Diaz, 1981. Diaz, 1981; Reference to B. Urmeneta, et al, 1998 Reference R. Reference R. Diaz, 1981 Diaz, 1981; Reference to B. Urmeneta, et al, 1998 Reference R. Reference K. Diaz, 1981 Diaz, 1981; Reference to B. Urmeneta, et al 1998 Reference R. Reference R. Diaz, 1981 Diaz, 1981; Reference to B Urmeneta, et al, 1998 Reference R. Reference R. Diaz, 1981 Diaz, 1981; Reference to B. Urmeneta, et al, 1998 Reference R. Reference R. Diaz, 1981 Diaz, 1981; Reference to B Urmeneta, et al, 1998 Reference R. Reference R. Diaz, 1981 Diaz, 1981; Reference to B. Urmeneta, et al, 1998 Reference R. Reference R. Diaz, 1981 Diaz, 1981; Reference to B. Urmeneta, et al, 1998 Reference R. Reference R. Diaz, 1981 Diaz, 1981; Reference to B. Urmeneta, et al, 1998 Reference R. Reference R. Diaz, 1981 Diaz, 1981; Reference to B. Urmeneta, et al, 1998 Reference R. Reference R. Diaz, 1981. Diaz, 1981; Reference to B. Urmeneta, et al, 1998 Reference R. Reference R. Diaz, 1981. Diaz, 1981; Reference to B. Urmeneta, et al, 1998 Lyophilization of the Yes Yes does not specify does not specify Yes Yes antigen Sensitization Antigen concentration does not apply 2.5 g/ml does not apply 2.5 g/ml does not apply 20 g/ml per ml of diluent Diluent CABI pH 9.6; PBS CABI PBS 7.2 Volume of antigen does not specify does not specity 50 l dilution per well Antigen concentration does not specify does not specify 1 g per well Over Night 37 C.; Over Night 4 C. 1st incubation 4 C. Over Night 4 C. 1st wash solution 98S Tween 20 PBS Tween 20 PBS Tween (0.05%) (0.05%) 20 (0.05%) Number of washes 4 3 4 Volume of wash does not specify does not specify 250 l solution per well Blocking solution does not specify does not specify Sol 3% skim milk 2nd Incubation does not specify does not specify 1 h at 37 C. 2nd wash solution does not specify does not specify PBS Tween Number of washes does not specify does not specify 20 (0.05%) Volume of wach does not specify does not specify 250 l solution per well Storage 4 C. does not specify 2-8 C. Preparation of sample Type of sample Stood serum Blood serum Blood serum Blood serum Blood serum Blood serum and milk (serum or whole, tank or per animal) Sample treatment does not apply does not apply does not apply does not apply does not apply does not apply Sample Predilution Yes Yes Yes Sample quantity does not specify does not specify 15 l Sample diluent PBS Tween PBS Tween CABI 0.05% 0.05% Amount of diluent does not specify does not specify 285 l Challenge Amount of diluted does not apply 100 l does not apply 100 l does not apply 50 l sample per well Competitive does not apply does not apply does not apply Specifications of Competitive ELISA Final volume per well does not specify does not specify 50 l Incubation 1 h 37 C. 1 h 37 C. 1 h 37 C. Washes Wash solution does not apply PBS Tween does not apply PBS Tween does not apply PBS Twaan 0.05% 0.05% 0.05% Number of washes 4 3 4 Volume of wash does not specify does not specify 250 l solution per well Conjugate Conjugate does not apply IgG anti-sheep does not apply Recombinant G does not apply IgG anti-bovine (rabbit); IgG anti- protein (goat) bovine; IgG anti- ruminant; Recombinent G protein Conjugate diluent PBS Tween PBS Tween CABI (0.05%) (0.05%) Conjugate IgGs: 1:1000- 0.2 g/ml 1:2000 Concentration 1:2000; G protein: 0.2 g/ml Final conjugate 100 l 100 l 50 l volume per well Incubation 1 h 37 C. 1 h 37 C. 1 h 37 C. Washes Wash solution does not apply does not specify does not apply PBS Tween does not apply PBS Tween 0.05% Number of washes dices not specify 3 4 Volume of wash does not specify does not specify 250 l solution per well Substrate Substrate Name does not apply ABTS 0.1% does not apply ABTS does not apply ABTS Substrate Diluent 0.05M citrate 0.05M citrats does not apply buffer (pH 4.0) buffer (pH 4.0) and 0.004% and 0.004% H2O2 H2O2 Amount per well 100 l does not specify 50 l Substrate incubation 15 min, 20 C. 30 min, 20 C . 15 min, 20-23 C. conditions in the dark Stop the reaction Stop solution does not apply does not specify does not apply does not apply does not apply SDS 4% Quanity does not specify 50 l Stability time does not specify 30 min Reading of Optical Densities Nanometers does not apply 405 does not apply does not specify does not apply 405 Lyophilization of controls Optical density for does not use it does not use it does not use it does not use it does not use it 1.0 Positive control selection Optical density for does not use it does not use it does not use it does not use it does not use it 0.20-0.28 Negative control selection Use of the lyophilization does not use it does not use it does not use it does not use it does not use it Use of the process Lyophilization for Negative Control process for Negative and Positive Control Conbol and Positive Control
Work Done on the KIT Application:
(47) 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 coli O: 157, Salmonella and Yersinia enterocolitica O: 9, being Yersinia the most frequent and significant false positive, since their levels of anti-LPS-O antibodies in serum and milk that cross with Brucella, are usually high, persistent and fluctuating. The cross reaction between Yersinia and Brucella is 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 .sup.(31).
(48) 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 Torren, 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.
(49) In half of these animals, this positivity to LPS-O was Fluctuating during the 12 months of length of the study.
(50) 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).
(51) 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.
(52) Results are shown in
(53) 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
(54) Raw Native Hapten
(55) 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.
(56) NH Concentration
(57) 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.
(58) 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 l 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.
(59) 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.
(60) 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.
(61) Use of Anti-Bovine IgG (Goat)
(62) 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.
(63) Native Hapten in Milk
(64) 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 to Brucella, 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.
(65) 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.
(66) Optical Density Range (DO) of Controls and their Lyophilization Process
(67) 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).
(68) 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.
(69) 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.
(70) 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.
(71) Kit Solutions Preparation
(72) Preparation of Culture Medium and Solutions
(73) The solutions to develop the kit are prepared with the procedure described below.
(74) 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.
(75) 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.
(76) 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.
(77) 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).
(78) 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.
(79) 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.
(80) 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.
(81) Substrate: A 2,2-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) commercial solution is used as substrate.
REFERENCES
(82) 1. Brucellosis. Medical microbiology. INRA. 28:512-525. 2. Arzola, V. E. A., Gonzalez, M. A., Gonzlez, J. L. I., Hernndez, M. L., Aparicio, E. D., Moorilln, G. V. N., & River, B. E. (2012). Diagnstico rpido y efectivo de brucelosis bovina en sangre, mediante una reaccin 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 por Brucella melitensis, Brucella suis y Brucella abortus en animales domsticos. Revue Scientifique et Technique, 32(1), 43-51. 4. Esperanza Gonzalez, M., Hernndez Andrade, L., & Daz Aparicio, E. (2006). Prueba de inmunodifusin radial con hapteno nativo para diferenciar bovinos con revacunaciones repetidas con la cepa S19 de Brucella abortus, 44, 269-276. 5. E. Herrera Lpez, L. Hernndez Andrade, G. P. R. and E. D. A. (2007). Study of Brucellosis Incidence in a Bovine Dairy Farm Infected with Brucella 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-hernndez, M., Jaramillo-meza, L., & Hernndez-andrade, L. (2007). Produccin de interfern gamma en cultivos de sangre completa en respuesta a antigenos de Brucella abortus en bovinos vacunados con RB51 Production of interferon gamma in whole blood cultures in response to Brucella abortus antigens in RB51-vaccinated cat, 45(2), 147-159. 8. Maria Dolores Fuentes Delgado, Irene V. Vitela Mendoza, Beatriz Arellano-Reynoso, Rigoberto Hernndez Castro, Jos Francisco Morales lvarez, Carlos Cruz-Vazquez. (2007). Presence of Brucella abortus vaccinal 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., Hernndez-Andrade, L., Prez-Gonzlez, R., Alfonseca-Silva, E., & Surez-Gemes, F. (2003). Evaluacin serolgica y bacteriolgica de un hato bovino con brucelosis y revacunado con dosis reducida de Brucella abortus cepa 19. Tcnica Pecuaria, 41(2), 129-140. 10. A., Castro, A., Gonzlez, H. A; Prat, S. R.; & Baldi, P. C. (2006). Deteccin de anticuerpos anti-Brucella spp. en cerdos mediante tcnicas de aglutinacin 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 between Brucella abortus and Yersinia enterocolitica 0:9. Immunoblot analysis of the antibody response to Brucella protein antigens in bovine brucellosis. (1995). Vet Microbiol; 47: 257-70. 12. Rodriguez, A., Ordua, 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, Espaa. 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 Sanchz, J., Hernndez Salazar, I., Daz, A. E., Mazano Caas, C., Prez Gonzlez, R., & Andrade, L. H. (2000). Estudio Bacteriolgico Y Serolgico De Brucelosis en vacas revacunadas con dosis reducida de cepa 19 de Brucella abortus. Inifap, (5). 15. Cant, A., Daz, E., Andrade, L. H., Adams, G. L., & Gemes, F. S. (2007). Estudio epidemiolgico 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 a Brucella polysaccharide antigen for differentiating infected from vaccinated cattle. J. Clin. Micro-biol. 10:37-41. 17. Moriyn UI. Brucella cell 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 of Brucella abortus and Brucella melitensis. Internatl Microbiol, 19-26. 19. Alonso-Urmeneta, B., Moriyon, I., & Blasco, J. M. (1988). Enzyme-linked immunosorbent assay with Brucella native hapten polysaccharide and smooth lipopolysaccharide. Journal of Clinical Microbiology. 20. Dubray, G. (1984). Progrs rcents sur la biochimie et les pro-prits biologiques des antignes de Brucella. 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., Salv, M. D., & Pardo, M. L. (1981). A simple method for the extraction of polysaccharide B from Brucella cells 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 against Brucella melitensis 16M 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 with Brucella native 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 of Brucella serotype A and M and Yersinia 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., Aragn, V., Prez-Ortiz, S., Pardo, M., Moriyn, I. (1994). Evaluation of Serological Tests for Diagnosis of Brucella melitensis Infection 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). Diagnstico de Brucella melitensis en ovinos usando inmunodifusion radial con hapteno nativo. Tcnica Pecuaria En Mxico, 34(2), 99-103. 29. Alonso-Urmeneta B, Marin C, Aragn V, Blasco J M, Diaz R, Moriyn 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., Moriyn, 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. Muoz, P. M., C. M. Marn, D. Monreal, D. Gonzalez, B. Garn-Bastuji, R. Diaz, R. C. Mainer-Jaime, I. Moriyn, 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 to Yersinia enterolitica O:9 American Society for Microbiology. Clin Diagn Lab Immunol [Internet]. 2005; 12(1):141-51. 32. WO2008051065 A1, fecha de presentacin: Oct. 16, 2007. Fecha de publicacin: Mayo 2, 2008. Universidad Autnoma de Nuevo Len. https://www.google.com/patents/WO2008051065A1?cl=es
BRIEF FIGURES DESCRIPTION
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