WO1999030161A1

WO1999030161A1 - Laboratory test for assaying incomplete antibodies in brucellosis and equipment for carrying out such test

Info

Publication number: WO1999030161A1
Application number: PCT/ES1998/000327
Authority: WO
Inventors: Almudena Rojas Gonzalez; Joaquín Mendoza Montero
Original assignee: Almudena Rojas Gonzalez; Mendoza Montero Joaquin
Priority date: 1997-12-09
Filing date: 1998-12-02
Publication date: 1999-06-17
Also published as: ES2131032B1; ES2131032A1; AU1435999A

Abstract

The invention relates to a laboratory test for assaying incomplete antibodies in brucellosis and the equipment used to carry out such test. The laboratory test is characterized in that in a series of ELISA wells a pH5 buffer is added; each of the sera is added to the first well; then a double dilution is carried out to pass liquid from each well to the next; finally, a certain quantity of the stabilized bacterial suspension is added in each well. A stove incubation is carried out during 24 hours at 37 °C in humid chamber and darkness conditions. The result can then be read by using an agglutination plate reader. The equipment necessary to implement such test is also disclosed. The test of the invention is used to assay incomplete antibodies in brucellosis and is both applicable to persons and animals.

Description

.LABORATORY TEST TO .Measure. INCOMPLETE ANTIBODIES IN

BRUCELOSIS AND EQUIPMENT TO PERFORM IT.

The laboratory test of the present invention (Brucellacapt) is used to measure incomplete antibodies in cases of brucellosis, and can be applied to both humans and animals.

Brucellosis or Malt Fever is a serious disease caused by infection with Brucella abortus or Brucella elitensis that is endemic to the countries of the Mediterranean basin, affecting both humans and goats, sheep and cows (Young EJ. Clin Infect Dis , 1995; 21: 283-290).

In humans, the disease is very common in Spain and causes disease to a large number of people annually. It also involves serious complications such as endocarditis, meningitis, orchitis, osteomyelitis, arthritis, etc. (CoJjnenero JD, Reguera JM, Fernández-Nebro A, Cabrera-Franquelo F.. Ann Rheu Dis, 1991; 50: 23-26). The consequences of the disease cause enormous economic damage because they cause disability in a large number of people. The most used tests in their serological diagnosis are agglutination (Bettelheim KA, Mas ill WJ, Pearce J, J Hyg Camb, 1983; 90: 33-39), Rose Bengal, Complement Fixation Reaction, test Coombs and ELISA tests (Alton GG, Jones LM, Pietz DE. Laboratory techniques in brucellosis. 2 ^nd ed. World Health Organization monograph ser. No 55. Geneva. World Health Organization. 1975; Bau M, Zamir 0, Bergan -Rios R, Katz E, Beider Z, Cohen A, Banai M,

SUBSTITUTE SHEET (RULE 26) J Clin Microbio !, 1995; 33: 2166-2170). While in the less evolved forms of the disease all serological tests are sensitive for the diagnosis of the disease, incomplete antibodies appear in the most evolved phases that are unable to bind the bruises, so to be able to highlight this type of antibodies, it is necessary to use techniques such as ELISA (Goldbaum FA, et al. J Clin Microbiol 1992; 30: 604-607; Goldbaum FA, Leoni J, Wallach JC, Fossati CA. J Clin Microbiol, 1993; 31: 2141 -2145), complement fixation reaction or Coombs Test (Foz A, Garriga J. Rev Immunol, 1954; 18: 288-298; Ariza J. Med Clin (Barc), 1992; 98: 494-496).

These techniques have the following disadvantages: The ELISA technique for measuring antibodies is not quantifiable at a single serum dilution, so a qualitative result is obtained that in endemic areas such as Spain has little value due to the frequency of people with antibodies. The complement fixation reaction is a quantitative test that is not very sensitive and therefore less useful in the initial stages of the disease and in addition to difficult execution, so only some laboratories perform it regularly (Sánchez-Sousa A, Torres C, Ca pello MG, Garcia C, Parras F, Cercenado E, Baquero F. J Clin Pathol, 1990; 43: 79-81).

The Coombs test is the most appropriate test to measure this type of antibody, but it has two serious drawbacks: first it takes at least 24 hours in

TUCIÓN RULE 26 its execution and secondly it is very laborious to execute. If microtechnics are not used, the titration of a serum leads to the use of 8-12 tubes that have to be washed by centrifugation. It is easy to understand that the realization of a high number of sera is practically unfeasible, since for example analyzing 24 sera by this technique requires it to be used from 192 to 288. As the washing process has to be repeated three times this produces a huge manipulation which makes it almost unfeasible which has led despite its usefulness to not be done as widely as it should, so many cases of brucellosis can remain undiagnosed (Maravi-Poma E, Murie M, Gamboa J, Diaz R , Rivero-Puente A. Rev Clin Esp, 1982; 166: 101-105; Ariza J, Pellicer T, Pallares R, Foz A, Gudiol F. Clin Infect Dis, 1992; 14: 131-140). _f Brucellosis is endemic to countries with low technology and in which the execution of some of the techniques described above is hardly applicable.

Another problem frequently encountered in classical agglutination tests is the appearance of the so-called zone effect, which is that at high concentrations of positive sera the reaction is canceled by excess antibodies and is interpreted as negative.

With the serological techniques for brύcellosis available so far it is not possible to differentiate between an individual suffering from brucellosis and a person already cured of the disease but still maintaining antibodies to Brucella, which given the frequent occurrence of relapses in this disease makes the management of these patients very difficult.

In all infectious diseases the presence of antibodies of different affinity has been demonstrated in the different phases of the disease. At the beginning, antibodies with low affinity appear, antibodies that bind to weakly reactive antigens, are subsequently replaced by others that have a high affinity for the antigen and when new antigenic stimuli are not produced, the antibodies that are detected return to be low affinity, as was the case at the beginning of the infection.

In animals, the disease affects both cattle, goats and poultry, causing serious economic losses due to the frequent occurrence of abortions in affected cattle. Considering nothing more than sheep in Spain, more than 2,500 million pesetas are dedicated annually to the sanitation of Brucella-infected cattle, without considering the expenses caused by the vaccination of animals (Alonso-Urmeneta B, Moriyón I, Díaz R, Blasco JM J Clin Microbiol, 1988; 26: 2642-2646 .; Delgado S, Fernández M, Cármenes P. J Vet Diagn Invest 1995; 7: 206-209; Díaz-Aparicio E, et al. J Clin M.icrobiol 1994; 32: 1159-1165).

The laboratory tests used in the diagnosis in animals are the Bengal Rose test and the Complement Fixation Reaction (Díaz R, Levieux D. C R Acad Sci

Ser D, 1972; 274D: 1593-1596). The biggest diagnostic problem that appears in animals is that most of the herds are vaccinated against this microorganism and this causes the appearance of antibodies that are indistinguishable from those that appear after the disease. When screening campaigns are carried out it is necessary to sacrifice many animals that give positive laboratory tests, but are only vaccinated and not infected. A test capable of differentiating between the antibodies presented by the vaccinated and those of the infected animals would be of great utility because it would save a large number of unnecessary sacrifices (Sutherland SS. J Clin Microbiol 1985; 22: 44-47; Debbarh H , Cloeckaert A, Zyg unt M, Dubray G. Vet. Microbiol. 1995; 44: 37-48; Gómez-Miguel MJ, Moriyón I, Alonso-Urmeneta B, Riezu-Boj JI, Díaz R. Infec I mun 1988; 56 : 716-718).

An object of the present invention is a test that demonstrates, with little manipulation, both complete and incomplete brucellosis antibodies.

Another object of the invention is a test technique that incorporates an acid solution specially designed to prevent the occurrence of the zone phenomenon, which greatly facilitates the reading and correct identification of the results.

Another object of the invention is a test technique that incorporates a solution with a high molecular weight component in which the preparation of the bristle suspension incorporating the equipment is carried out and whose purpose is the stabilization thereof.

The object of the invention is also a test that is capable

SUBSTITUTE SHEET (RULE 26) to differentiate between the antibodies presented by a sick individual and a healthy individual who passed the disease, but which is currently cured. Due to the pH of the diluent used in the technique of the invention, only high affinity antibodies are determined. With the data available, it can be affirmed that the test of the invention is able to differentiate between the antibodies presented by vaccinated animals from those presented by diseased animals. Finally, the object of the invention is also the equipment necessary to perform the test described above.

The rationale for the test of the invention is based on the attachment to the wells of a microplate of the type used in ELISA tests of goat antibodies antilgG and human antilgA (or in the case of tests intended for use in animals of the corresponding antiglobulins antioveja, anticabra, antivaca) (Desmonts G, Naot Y, Remington JS. J Clin Microbiol, 1981; 14: 486-491; Ong LY, Pang T, Lim SH, Tan EL, Puthucheary SD. J Med Microbiol, 1989; 29: 195-198; Cubel RCN, Ensign ACR, Cohen BJ, J Clin Microbiol, 1994; 32: 1997-1999). The sera of the patients are added and subsequently a Brucella suspension is added. After leaving the plate in incubation at 37 ° C for 24 hours, the test is read with the naked eye without the need for any type of equipment.

As can be seen, the test of the invention for measuring incomplete antibodies in brucellosis is based on the addition to ELISA type wells of antilgG antibodies and Human and animal antilgia adequately blocked. The kits include - positive and negative controls, Brucella abortus or Brucella mellitensis antigens, and buffer solution for sample dilution. 96-well ELISA plates or 8-well strips or 12-well strips are used, so that 12 sera and 8 dilutions or 8 sera and 12 serum dilutions can be analyzed per plate.

The contents of the equipment or kit necessary for carrying out the test of the invention have reagents to quantitatively investigate incomplete antibodies against Brucella abortus or mellitensis in 16 samples if 12 dilutions of each sample or 24 samples are analyzed if 8 dilutions are analyzed of each sample. It consists of the following:

96 U-bottom wells specially treated to capture immunoglobulins.

12 ml of Brucella abortus bacterial suspension or

Brucella mellitensis colored and killed by heat and treatment with 0.5% formaldehyde in saline solution with 1% dextran.

14 ml of diluent for sera composed of a saline solution of 100 mM acetates.

100 μl of positive control serum with 0.1% sodium azide.

100 μl of negative control serum with 0.1% sodium azide. This material should be stored at 4-8 ° C and check the expiration date.

As a necessary material, but not contained in the equipment, a precision pipette to dispense 5 μl and a multichannel precision pipette to dispense 50 μl must be provided.

The execution of the technique is simple, since it is based on the addition of 50 μl of dilution buffer in each of the wells and the serial dilution of the sera of the patients using a multichannel pipette; After this operation, the addition of a 50 μl multichannel antigen pipette and incubation until the next day to proceed with its reading is sufficient.

It should be noted that other secondary forms for the modification of the test that do not mean substantial modifications are also covered by the present invention.

The detailed test procedure is as follows:

1. Let the reagents reach room temperature. Remove the strips of wells needed for the number of sera to be processed, plus a strip for the positive and negative controls.

2. -Add 50 μl of diluent in well A [Phase a) of Figure 1].

3. Add 50 μl of diluent in the wells from A to H [Phase b) of Figure 1].

4. Put 5 μl of each of the sera and the positive and negative controls in well A. [Phase c) of the Figure 1] .

5. Doubledilute taking 50 μl of each well from A to H [Phase d) of Figure 1].

6. Add 50 μl of the bacterial suspension, previously agitated to ogenize it, to all the wells used [Phase e) of Figure 1].

7. Cover with adhesive plastic and incubate in an oven for 24 hours at 37 ° C, in a humid chamber in the dark, since the bacterial suspension once mixed with the diluent of the sera changes color, if subjected to a prolonged exposure to light.

8. Read the result with the help of an agglutination plate reader, taking into account that the titles obtained will be 1/40 for row A, 1/80 for row B, '1/160 for row C, 1 / 320 for row D, 1/620 for row E, 1/1280 for row F, 1/2560 for row G and 1/5120 for row H.

The interpretation of the results is as follows: The test is positive when an agglutination is observed that occupies most of the well (*). A test is negative when a bacterial button is observed in the center of the well (* *). A titer equal to or greater than 320 is strong evidence of brucellosis, but the other clinical tests should always be evaluated together before issuing a diagnosis. In endemic areas of the disease it is common to find positive results at titers below 320. For an evaluation of the test, 206 human sera were studied internally, 85 belonging to healthy controls, and the rest to brucellosis patients at different stages of the disease, comparing the (+) (Position 1 of Figure 2) (++ ) (Position 2 of Figure 2) results obtained with the test of the invention and those obtained using the Coombs test, using microtechnics for this. The results obtained are shown in Table I. It is first necessary to clarify that in serological tests the presence of antibodies is determined quantitatively by dilution of the sera in a suitable diluent, that is, when it is indicated that a serum is positive for 320, means that putting a part of serum in 319 of diluent the test is positive. Dilute dilutions are used, that is, each time it is diluted twice. The numbers that appear in the table represent until that dilution of the sera that these were positive in each of the techniques, that is, after diluting the patient's serum x times in its corresponding diluent, that the result continues to be positive in this dilution and Don't be when it is diluted once more. TABLE I

For another evaluation of the test, 53 sheep sera vaccinated with B. melitensis and analyzed on days 0, 30 and 360 of vaccination were studied internally. These animals and others were infected with B. melitensis and analyzed at day 1, 28, 70 and 336 of the infection. The results can be seen in table II

TABLE II

** NOT APPLICABLE; R.B. TEST OF ROSA DE BENGALA; P: POSITIVE; N: NEGATIVE; DAYS P.V. : POST VACCINATION DAYS; DAYS P.I .: DAYS POST INFECTION.

The results obtained indicate that the test of the invention is capable of pointing out infected animals and that they have manifested symptoms, discriminates vaccinated animals that resist infection and finally indicates vaccinated animals that do not resist infection.

In an external test conducted by the Department of Microbiology of the Faculty of Medicine of the University from Navarra (Professor Dr. D. Ramón Díaz) with sera from humans, the test of the invention was compared with the Coombs test using microtechnics. For this, samples of patients with brucellosis were used at the beginning of the symptoms of the disease (first sample) and when they were cured of it (final sample).

TA.BLA III

In these results it can be seen that the test is very effective for the diagnosis of the disease at the beginning of the same and what is more important that is able to differentiate when it is cured, because while the Coombs test continues to be positive in 6 of the thirteen patients, the test of the invention is only positive in two of them to titles over 320.

Claims

1. Laboratory test to measure incomplete brucellosis antibodies, applicable in both humans and animals, characterized in that in a series of ELISA type wells 50 μl of a dilution buffer are added, then adding 5 μl of each of the sera in the first well, and then proceeding to a double dilution by placing in each well 50 μl of the previous well, then adding in each well 50 μl of a homogenous bacterial suspension of Brucella abortus or B. melitensis, stirring and finally incubating in wet chamber and in the dark, then proceeding to read the result with the help of an agglutination plate reader.

2. Laboratory test according to claim 1, characterized in that the dilution buffer is a solution of 100 mM sodium acetate with a pH of 5.

3. Laboratory test according to claim 1, characterized in that the suspension of Brucella abortus or B. melitensis is colored and killed by heat and treatment with 0.5% formaldehyde and suspended with 1.% Dextran.

4. Laboratory test according to claim 1, characterized in that the incubation phase is carried out, in a humid and dark chamber, for 24 hours at 37 ° C.

5. Laboratory equipment or it used to perform the test described in the preceding claims, characterized in that it consists of: 24 strips of 8 U-bottom wells or 12 strips of 16 U-bottom wells, or a 96-plate U-shaped wells, specially treated for capture immunoglobulins; 12 ml of bacterial suspension of Brucella abortus or B. melitensis colored and killed by heat and treatment with 0.5% formaldehyde; 14 ml of diluent for sera; 100 μl of positive control serum with 0.1% sodium azide; 100 μl of negative control serum with 0.1% sodium azide.