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WO1999030161A1 - Test de laboratoire permettant de mesurer les anticorps incomplets anti-brucellose et equipement pour sa mise en oeuvre - Google Patents

Test de laboratoire permettant de mesurer les anticorps incomplets anti-brucellose et equipement pour sa mise en oeuvre Download PDF

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Publication number
WO1999030161A1
WO1999030161A1 PCT/ES1998/000327 ES9800327W WO9930161A1 WO 1999030161 A1 WO1999030161 A1 WO 1999030161A1 ES 9800327 W ES9800327 W ES 9800327W WO 9930161 A1 WO9930161 A1 WO 9930161A1
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WO
WIPO (PCT)
Prior art keywords
test
well
brucellosis
laboratory test
sera
Prior art date
Application number
PCT/ES1998/000327
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English (en)
Spanish (es)
Inventor
Almudena Rojas Gonzalez
Joaquín Mendoza Montero
Original Assignee
Almudena Rojas Gonzalez
Mendoza Montero Joaquin
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Almudena Rojas Gonzalez, Mendoza Montero Joaquin filed Critical Almudena Rojas Gonzalez
Priority to AU14359/99A priority Critical patent/AU1435999A/en
Publication of WO1999030161A1 publication Critical patent/WO1999030161A1/fr

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
    • G01N2333/23—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Brucella (G)

Definitions

  • 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).
  • 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
  • TUCI ⁇ N 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.
  • zone effect 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.
  • 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.
  • 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:
  • a precision pipette to dispense 5 ⁇ l and a multichannel precision pipette to dispense 50 ⁇ l must be provided.
  • the execution of the technique is simple, since it is based on the addition of 50 ⁇ l 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 ⁇ l multichannel antigen pipette and incubation until the next day to proceed with its reading is sufficient.
  • 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.
  • 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.
  • 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.
  • 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).
  • 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.

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Immunology (AREA)
  • Engineering & Computer Science (AREA)
  • Urology & Nephrology (AREA)
  • Chemical & Material Sciences (AREA)
  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • Hematology (AREA)
  • Medicinal Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Biotechnology (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Virology (AREA)
  • Food Science & Technology (AREA)
  • Microbiology (AREA)
  • Analytical Chemistry (AREA)
  • Cell Biology (AREA)
  • Biochemistry (AREA)
  • Physics & Mathematics (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Investigating Or Analysing Biological Materials (AREA)

Abstract

L'invention concerne un test de laboratoire permettant de mesurer les anticorps incomplets anti-brucellose et l'équipement pour sa mise en oeuvre. L'examen ou test de laboratoire est caractérisé en ce qu'on ajoute dans une série de puits de type ELISA un tampon à pH5. On ajoute ensuite chacun des sérums dans le premier puits, on effectue une double dilution en faisant passer du liquide de chaque puits au suivant et on ajoute finalement dans chaque puits une certaine quantité de suspension bactérienne stabilisée. On effectue ensuite une incubation dans un four pendant 24 heures à 37 °C, dans une chambre humide dans l'obscurité. On peut par la suite lire le résultat à l'aide d'un lecteur de plaques d'agglutination. Fait aussi l'objet de cette invention l'équipement nécessaire pour mettre en oeuvre ce test. Le test sert à mesurer les anticorps incomplets lorsque les êtres humains ou les animaux sont atteints de brucellose.
PCT/ES1998/000327 1997-12-09 1998-12-02 Test de laboratoire permettant de mesurer les anticorps incomplets anti-brucellose et equipement pour sa mise en oeuvre WO1999030161A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU14359/99A AU1435999A (en) 1997-12-09 1998-12-02 Laboratory test for assaying incomplete antibodies in brucellosis and equipment for carrying out such test

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
ES9702551A ES2131032B1 (es) 1997-12-09 1997-12-09 Test de laboratorio para medir anticuerpos incompletos en brucelosis y equipo para realizarlo.
ESP9702551 1997-12-09

Publications (1)

Publication Number Publication Date
WO1999030161A1 true WO1999030161A1 (fr) 1999-06-17

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Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/ES1998/000327 WO1999030161A1 (fr) 1997-12-09 1998-12-02 Test de laboratoire permettant de mesurer les anticorps incomplets anti-brucellose et equipement pour sa mise en oeuvre

Country Status (3)

Country Link
AU (1) AU1435999A (fr)
ES (1) ES2131032B1 (fr)
WO (1) WO1999030161A1 (fr)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR2919391A1 (fr) * 2007-07-25 2009-01-30 Vacci Test Europ Soc Par Actio Methode immunologique pour la detection de la brucellose.
WO2018186731A1 (fr) * 2017-04-06 2018-10-11 Maroun Cortez Victoria Kit pour test elisa indirect à base de haptène natif brut et témoins lyophilisés pour le diagnostic de confirmation de la brucellose bovine dans le sérum sanguin et le lait d'animaux ou le lait de citernes

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3962413A (en) * 1974-05-14 1976-06-08 Cornell Research Foundation, Inc. Plate methods for diagnosing Brucella canis infection
WO1990007118A2 (fr) * 1988-12-13 1990-06-28 Bajyana Songa Emmanuel Anticorps polymerises, diriges contre des immunoglobulines - leur utilisation dans des tests de diagnostic

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3962413A (en) * 1974-05-14 1976-06-08 Cornell Research Foundation, Inc. Plate methods for diagnosing Brucella canis infection
WO1990007118A2 (fr) * 1988-12-13 1990-06-28 Bajyana Songa Emmanuel Anticorps polymerises, diriges contre des immunoglobulines - leur utilisation dans des tests de diagnostic

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
BEATON C.P. ET AL.: "A micromethod for agglutination and antiglobulin tests for antibody to brucella abortus", JOURNAL OF BIOLOGICAL STANDARIZATION,, vol. 12, no. 3, July 1984 (1984-07-01), pages 271 - 275 *
BROWN S.L. ET AL.: "Safranin O-Stained antigen microagglutination tests for detection of Brucella antibodies", JOURNAL OF CLINICAL MICROBIOLOGY,, vol. 13, no. 2, February 1981 (1981-02-01), pages 398 - 400 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR2919391A1 (fr) * 2007-07-25 2009-01-30 Vacci Test Europ Soc Par Actio Methode immunologique pour la detection de la brucellose.
WO2018186731A1 (fr) * 2017-04-06 2018-10-11 Maroun Cortez Victoria Kit pour test elisa indirect à base de haptène natif brut et témoins lyophilisés pour le diagnostic de confirmation de la brucellose bovine dans le sérum sanguin et le lait d'animaux ou le lait de citernes

Also Published As

Publication number Publication date
AU1435999A (en) 1999-06-28
ES2131032B1 (es) 2000-02-01
ES2131032A1 (es) 1999-07-01

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