(19)
(11)EP 3 608 672 A1

(12)EUROPEAN PATENT APPLICATION
published in accordance with Art. 153(4) EPC

(43)Date of publication:
12.02.2020 Bulletin 2020/07

(21)Application number: 18780939.7

(22)Date of filing:  05.04.2018
(51)Int. Cl.: 
G01N 33/569  (2006.01)
(86)International application number:
PCT/MX2018/050009
(87)International publication number:
WO 2018/186731 (11.10.2018 Gazette  2018/41)
(84)Designated Contracting States:
AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR
Designated Extension States:
BA ME
Designated Validation States:
KH MA MD TN

(30)Priority: 06.04.2017 MX 2017004490

(71)Applicant: Maroun Cortez, Victoria
Torreon, Coahuila 27250 (MX)

(72)Inventor:
  • Maroun Cortez, Victoria
    Torreon, Coahuila 27250 (MX)

(74)Representative: Temiño Ceniceros, Ignacio 
Calle Amador de los Rios 1-1°
28010 Madrid
28010 Madrid (ES)

  


(54)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


(57) 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 µg 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.


Description

OBJECT OF THE INVENTION



[0001] The indirect enzyme-linked immunosorbent assay of anti-NH ELISA is a test that is fundamentally based on the determination of IgG isotype antibodies against the Brucella antigen named Native Hapten (NH) in samples of blood serum, whole milk and/or whey, being either, individual or bulk milk samples, with the ability to distinguish infected animals from those animals with temporary exposure to Brucella spp. This product is exclusive for veterinary use for in vitro diagnostic in bovines.

[0002] The novelty of this invention is the development of a diagnostic kit based on the detection of antibodies against the Native Hapten antigen of Brucella, using the Indirect ELISA method. This kit includes microplates coated with the Native Hapten antigen in its raw form, the washing solutions, the sample diluent, the conjugate diluent, the substrate, the stop solution and the positive and negative controls, previously submitted to a lyophilization process, which are specific for blood serum and bovine milk.

BACKGROUND



[0003] The present invention is related to the biotechnological area, specifically for brucellosis detection in cattle through detection of reactive antibodies against the Native Hapten (NH) antigen of Brucella. Nowadays, brucellosis is an infectious disease that affects livestock and humans; it is caused by Gram-negative coccobacilli of the genus Brucella. It is cataloged as an important bacterial disease with a high impact on public health, since every year are reported about 500,000 new cases worldwide; it also decreases milk production, delays the growth of calves, causes abortions and produces reproductive problems in females, which is reflected in their production.

[0004] In Mexico, it is considered the most important bacterial zoonosis, being Brucella abortus the species that mainly affects cattle. The control of the disease depends on the NOM-041-ZOO-1995, National Campaign against Brucellosis in Animals application. The fundamental strategy of the campaign is based on diagnosis and vaccination.

[0005] In Mexico cattle immunization is carried out with strains S19 and RB51 of Brucella abortus, S19 induce the presence of antibodies in serum and milk that interfere with the tests remarked as officials, thus, strain RB51 has been used as an alternative (4, 5, 6, 7, 8, 9). Regarding serological diagnostic methods, the most commonly used are the Rose Bengal Test (RBT) and Rivanol test for blood serum and for milk samples is used the Milk Ring Test (MRT), which are considered as officials in NOM-041-ZOO-1995 (2, 10). These tests detect antibodies against the Brucella's outer membrane components, mainly directed against the O chain of Lipopolysaccharide (LPS) which is the most antigenic structure of smooth strains. Therefore, it is the first antigen against which antibodies are produced, however, these antibodies are present both in the infection and in vaccination, also, given the cross-reactivity present between Brucella's LPS and other Gram-negative bacteria's LPS, contact with brucellosis animals, productive stress and even the environmental factors, it is frequent the appearance of false positives results (11, 12, 13, 14, 15).

[0006] In the International Organization of Epizootics (OIE) manual on Bovine Brucellosis in terrestrial animals, the interferon gamma tests, the immunoprecipitation test that uses Native Hapten, and the indirect ELISA that used the rough lipopolysaccharide as antigen are highlighted as promising tests to differentiate between brucellosis infection and exposure to cross-reactive microorganisms; the manual also highlights the indirect ELISA test or the ring milk test, with whole milk samples, as effective tests to analyze and control brucellosis in dairy cows. However, no serological test is considered adequate for each and every epidemiological situation. Therefore, samples that are positive in the analysis tests must be confirmed using an established confirmatory strategy (16). For this reason, alternative tests with antigens different from LPS have been developed to decrease this reactivity and grant higher specificity levels.

[0007] The LPS electrophoretic analysis allows the identification of a second component, the Native Hapten (NH), called second polysaccharide or poly-B, but for representing with certainty the equivalent in the Brucella genus of the native haptens of other Gram-negative bacteria, currently it is called as NH (17). It is chemically identical to the O chain, in B. melitensis, the O chain consists of repeated units of five N-formylperosamine residues, four linked to α-1,2 and one bound to α-1,3 (18). It is an intracellular antigen, located in the periplasmic space, of a non-proteic nature and low molecular weight; properties that make it less antigenic, the appearance of precipitating antibodies in animal serum depends on the intensity of its antigenic stimulus. That is, antibodies against NH will be produced only when the immune system is exposed to the antigen for a long time, as in the case of an infection and not by vaccination (4, 14, 19, 20, 21, 22).

[0008] Native Hapten has been studied over years, in which its efficiency has been described in several studies applied in different species and with different methods. In 1981, a Radial Immunodiffusion (RID) test was carried out on blood serum of cattle, describing the obtaining method of Native Hapten antigen from a B. melitensis 16 M strain cultured in Tripticasein Soy Broth for 48 h at 37 °C and inactivated with phenol 0.5%, in addition, it was washed with saline solution and resuspended in distilled water. In this method, the extraction was carried out with 3 and 2 volumes of ethanol in which the antigen obtained was purified and tested by the previously mentioned method in blood serum of bovines (23).

[0009] For brucellosis diagnosis in humans, an indirect ELISA with NH was developed in 1986. The NH extracted from B. melitensis 16 M by ethanol precipitation, the purified and freeze dried antigen was adhered to the ELISA plates (for 12 h at 37 °C) at a 2 µg per well concentration, known quantities of blood serum were placed (100 µl), anti-human (goat) IgG, IgA, IgM and 1N NaOH were used as conjugate and stop solution respectively. The results shown describe a 99% effectiveness in anti-NH IgG detection in patients positive for the disease (24). There are reports of studies made in Spain (1988) that uses native hapten in a competitive ELISA for bovine brucellosis where the antigen extraction is carried out as in the previous references, from B. melitensis 16 M cultured in Soy Tripticaseína agar (TSA) to then perform two ethanol precipitates to obtain the crude NH antigen which is then purified by a long process for its subsequent lyophilization. After coating the ELISA plates with antigen (over night 37 °C), 50 µl of blood serum or whey is added to each well, plus 50 µl of a heterologous molecule giving a total of 100 µl of sample per well for the formation of the antigen-antibody Ag-Ac complex by competition, which react with the secondary antibody (anti-rabbit IgG [goat]) and a substrate, the reading is made at 450 nm of the results allows to evaluate the efficacy of the antigen (25).

[0010] With the RID test, in 1993 the NH antigen extracted as described before was used by Díaz R, et al. (1981) and it was purified by a long process of dialysis and digestion, it was proved that it was possible to differentiate cattle and sheep infected from vaccinated with a sensitivity and specificity very similar to that of complement fixation test (CFT) 26. In 1994 the effectiveness of NH was once again demonstrated in blood serum of goats, through an indirect ELISA in which different strains of Brucella were used, such as B. melitensis 16 M, 115, Rev 1; Brucella abortus 2308. These strains were cultivated in broth with stirring and for its harvest; the cells were washed with saline solution and resuspended in distilled water. The extraction of phenol inactivated and sterilized antigen was performed with 3 and 2 volumes of ethanol. It was purified by digestion with nucleases and proteinase K, extracted with phenol and precipitated with ethanol, the crude extract was used to coat the ELISA plates at a concentration of 0.25 µg per well, incubating over night at 4 °C. The goat blood sera were tested by this method, placing 100 µl (diluted serum) per well, where polyclonal goat anti-goat (rabbit) and recombinant protein G were used as secondary antigen options. The reading at 405 nm of the samples showed that NH ELISA in goats presents 60% specificity (27).

[0011] Another study reported in 1996 the sensitivity of the RID test for the detection of anti-NH antibodies in sheep blood serum was tested, comparing the NH extracted from different species of Brucella and Yersinia (Brucella melitensis M16, Rev 1, Brucella abortus 2308, Yersinia enterocolitica O: 9) grown in TSB broth, in which it was concluded that the sensitivity of the test is greater when using the NH (purified and freeze dried) of B. melitensis 16M (28). In a comparative study between Brucella polysaccharides in an indirect ELISA in sheep and cattle using B. abortus 2308 and B. melitensis 16 M strains cultivated in a fermentor, it was concluded that for unspecified reasons, the Native Hapten is not the optimal antigen for the brucellosis diagnosis for this type of tests (29). In 1999, a comparative study was carried out in Spain between a competitive ELISA, an indirect ELISA and the Rose Bengal test (RBT) where all three tests used LPS, against the Radial Immunodiffusion Test using NH (AGID-NH) extracted from a B. melitensis 16M strain, based on the extraction method described by Diaz R, et al. (1981), based on the results obtained, it was concluded that using the RBT as a screening and with AGID-NH as confirmatory test, was an effective system to diagnose brucellosis in sheep, this same scheme was used for brucellosis eradication in cattle in the same country (30). In 2005, a group of researchers conducted an indirect ELISA study with NH in bovines, where favorable results were obtained using the NH extracted from B. melitensis 16M at a concentration of 2.5 µg / ml of antigen per well, taking as reference the methods described by Díaz R, et al. (1981) and Urmeneta B, et al (1998) (31). Likewise, in more recent studies, patent WO2008051065 A1, from May 2, 2008 reports a fluorescence polarization assay (FPA) using the NH of B. melitensis Rev 1, a strain that has been used previously, which was cultured on Brucella agar and inactivated with phenol, in which the NH was extracted with ethanol, dialyzed and purified for the detection of anti-NH antibodies in goats (32).

[0012] In order to make the confirmatory diagnosis of brucellosis more efficient and to avoid the "false positive" emitted by the existing tests based on the detection of anti-lipopolysaccharide antibodies (anti-LPS) and other inconveniences such as cross-reactions with enterobacteria, and post-vaccinal reactions, it was thought to develop the present test, which is intended to be protected by means of the present application, since it is an indirect ELISA test that is more efficient in its application and a suitable kit for detecting the presence of anti-Native Hapten antibodies, which only occur during a true brucellosis infection.

DETAILED DESCRIPTION OF THE INVENTION



[0013] 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 melitensis 16M cultivation



[0014] 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 10 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



[0015] 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



[0016] 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.

[0017] 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



[0018] 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 Figure 1.

2. - Microplate coating



[0019] 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.

[0020] 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).

[0021] 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 in Figure 2.

3. - Procedure of the indirect ELISA anti-NH


Preparation of Samples



[0022] 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:



[0023] 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:



[0024] 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



[0025] 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):



[0026] Dilute the 10X 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%):



[0027] Dilute the 10X Wash Solution in a 1:10 rate. Example, to prepare 250 mL, dilute 25 mL in 225 mL of distilled water.

Concentrated Conjugate:



[0028] 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%:



[0029] 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:



[0030] 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



[0031] 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 in Figure 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



[0032] 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.

[0033] 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



[0034] 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



[0035] 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.



[0036] 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 Figure 4.

PROCEDURES SIMILAR TO THE INVENTION:



[0037] A search was made to corroborate the existence of procedures similar to the present invention, finding the following:










Work done on the KIT application:



[0038] 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 (31).

[0039] 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.

[0040] In half of these animals, this positivity to LPS-O was Fluctuating during the 12 months of length of the study.

[0041] 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).

[0042] 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.

[0043] Results are shown in Figure 5.

[0044] 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



[0045] 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



[0046] 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.

[0047] 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.

[0048] 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.

[0049] 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)



[0050] 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



[0051] 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.

[0052] 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



[0053] 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).

[0054] 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.

[0055] 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



[0056] The solutions to develop the kit are prepared with the procedure described below.

[0057] 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.

[0058] 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.

[0059] 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.

[0060] Sample Diluent Solution (10x) and Conjugate Diluent Solution (1x). Carbonate-Bicarbonate Buffer (CABI): A CABI buffer at pH of 9.6 is used as diluent. To prepare the solution at a 10x concentration, (the amounts at 1X 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 (HCI) or sodium hydroxide (NaOH).

[0061] 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.

[0062] Washing Solution (10x). 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 (KCI) 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.

[0063] 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.

[0064] Substrate: A 2,2'-azino-bis(3- ethylbenzothiazoline-6-sulphonic acid) (ABTS) commercial solution is used as substrate.

References



[0065] 
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  24. 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. 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. 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. 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 of Brucella melitensis Infection of Goats. Journal of Clinical Microbiology, 32(5), 1159.
  28. 28. Diaz-Aparicio, E., Uria, I. M., Blasco-Martinez, J. M., Marin-Alcala, C., & Diaz, R. (1996). Diagnóstico de Brucella melitensis en ovinos usando inmunodifusion radial con hapteno nativo. Técnica Pecuaria En México, 34(2), 99-103.
  29. 29. Alonso-Urmeneta B, Marín C, Aragón V, Blasco JM, Díaz 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. 30. Marín, C. M., Moreno, E., Moriyon, I., Díaz, 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. 31. Muñoz, P.M., C.M. Marín, D. Monreal, D. Gonzalez, B. Garín-Bastuji, R. Díaz, 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 to Yersinia enterolítica O:9 American Society for Microbiology. Clin Diagn Lab Imunol [Internet]. 2005; 12(1):141-51.
  32. 32. WO2008051065 A1, fecha de presentación: OCT16, 2007. Fecha de publicación: Mayo 2, 2008.Universidad Autónoma de Nuevo León. https://www.google.com/patents/WO2008051065A1?cl=es

BRIEF FIGURES DESCRIPTION



[0066] 

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

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

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

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

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




Claims

1. KIT for indirect ELISA test based on Raw Native Hapten for confirmatory diagnosis of bovine brucellosis in blood serum and individual milk (per animal) and bulk milk (tank), characterized by ten (10) microplates coated with crude Native Hapten, each plate containing 96 wells (distributed in 12 strips of 8 wells each strip) of flat and clear bottom, with a surface treated specially for a high capacity of adhesion of the antigen, with a maximum capacity of 360 microliters per well. It includes five (5) microplates of 96 wells, without treatment, to perform the predilution of samples; four (4) bottles of sixty (60) milliliters each with PBS-Tween 20 Washing Solution, 0.05%, at (10 X) concentration; one bottle (1) of forty (40) milliliters of Sample Diluent Solution, based on CABI (carbonate bicarbonate) Buffer at (10 X) concentration; one (1) bottle of fifty (50) milliliters of Conjugate Diluent, based on CABI (carbonate bicarbonate) buffer at concentration (1 X); one (1) 50 milliliter bottle of Substrate, which is ABTS (commercial product); one (1) vial of one (1) milliliter consisting of 25 microliters of Concentrated Conjugate, which is an Immunoglobulin G - anti Bovine, conjugated with horseradish peroxidase produced in goat (commercial product) which is prediluted in a preservative HRP Protector, which is a peroxidases stabilizer; one (1) bottle of fifty (50) milliliters of Stop Solution, which is sodium Duodecyl Sulfate (SDS) at 4% concentration. One (1) freeze dried positive blood serum vial, one (1) freeze dried negative blood serum vial, and one (1) freeze dried positive milk serum vial and one (1) freeze dried milk serum vial. All control vials contain one (1) milliliter of freezed dried serum for reconstitution in one (1) milliliter of distilled water.
 
2. Indirect ELISA Test Procedure based on Raw Native Hapten for confirmatory diagnosis of bovine brucellosis in blood serum and individual milk (per animal) and bulk milk (tank), using this crude antigen obtained from B. melitensis 16M strain, without applying any purification treatment to it, and with an effective adhesion capacity to perform an indirect ELISA for brucellosis diagnosis in blood serum and milk.
 
3. Indirect ELISA test procedure based on a crude Native Hapten for confirmatory diagnosis of bovine brucellosis in blood serum and individual milk (per animal) and bulk milk (tank), that stands out for as it was claimed in the previous clause, the coating of ELISA plates with Native Hapten antigen at a 1 microgram per well concentration for the diagnosis of brucellosis in blood serum and milk.
 
4. Indirect ELISA Test Procedure based on the use of freeze dried controls for confirmatory diagnosis of bovine brucellosis in blood serum and individual milk (per animal) and bulk milk (tank), that stands out for as it was claimed in the previous clause, with the use of blood serum and milk controls freeze dried as a conservation method to avoid contamination and degradation of the antibodies present, guaranteeing the stability of the optical densities in positive and negative controls for a correct results interpretation in an indirect ELISA.
 
5. Procedure for the Selection and Freeze-drying of Positive and Negative Controls for use in other diagnostic tests.
 




Drawing































REFERENCES CITED IN THE DESCRIPTION



This list of references cited by the applicant is for the reader's convenience only. It does not form part of the European patent document. Even though great care has been taken in compiling the references, errors or omissions cannot be excluded and the EPO disclaims all liability in this regard.

Patent documents cited in the description




Non-patent literature cited in the description