WO1990007118A2 - Polymerized antibodies directed against immunoglobulins, and their use in diagnostic tests - Google Patents
Polymerized antibodies directed against immunoglobulins, and their use in diagnostic tests Download PDFInfo
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- WO1990007118A2 WO1990007118A2 PCT/EP1989/001540 EP8901540W WO9007118A2 WO 1990007118 A2 WO1990007118 A2 WO 1990007118A2 EP 8901540 W EP8901540 W EP 8901540W WO 9007118 A2 WO9007118 A2 WO 9007118A2
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- antibodies
- polymerized
- labeled
- antigen
- specific
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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
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6854—Immunoglobulins
Definitions
- Antibodies circulating against several antigens of pathogenic parasites, bacteria or viruses are produced during the duration of an infection. Such antigens can be detected in whole blood, plasma or serum or other biological fluids by direct or specific agglutination tests.
- the lack of sensitivity can also be attributed to the presence of so-called “incomplete” and blocking antibodies which either do not agglutinate or inhibit agglutination.
- anti-i munoglobulin antibodies of the Ig type as a reagent for Coo bs, considerably improves the sensitivity.
- the reading of the test results is made more difficult because of the small size of the aggregates formed after the immunological reaction or of a prozone phenomenon well known in immunology which manifests itself in the presence of low dilutions of serum. (for example dilutions of the order of 1/2 or 1/10).
- agglutination with the anti-Ig is inhibited and the signal for the presence of the antibodies sought disappears.
- the implementation of infection detection test due to a parasite requires additional manipulations such as prior dilutions of the test sample.
- the inventors have demonstrated that an appropriate quantity of anti-Ig antibodies in polymerized form added to an agglutinable antigen, causes an increase in sensitivity during an immunological detection test involving agglutination reactions, and the reduction otherwise the abolition of the prozone phenomenon. Therefore, no additional manipulation is required prior to the implementation of the reactions, such as prior dilutions of the sample to be assayed.
- the present invention relates to polymerized Ig-antibodies and to their use in immunological tests for the detection, for example, of parasites or viruses.
- the present invention relates to the use of antibodies polymerized jointly with particulate antigens, of a contrasting color with respect to the support on which they are used or with respect to the biological fluid or vice versa to increase the sensitivity of the agglutination tests. by increasing the size of the aggregates.
- the present invention also relates to rapid tests for the detection of determined antibodies, for example due to infectious diseases in humans or animals thanks to the use of polymerized anti-Ig antibodies.
- This application includes all kinds of tests which allow the detection of aggregates of particles (constituted by multicomplexes of the anti-Ig type, antibody sought, specific antigen of said antibody sought) of sufficient size to be visible to the naked eye, taking into account the conditions for revealing these aggregates.
- the invention takes advantage of the capacity preserved by antibodies-Ig in poly erized form to non-selectively fix other antibodies and the immunological complexes containing them, among which the complexes formed between specific antibodies contained in a biological sample and corresponding antigens.
- Biological samples can be made up of biological fluids such as whole blood, serum, or plasma.
- Polymerized anti-Ig antibodies of the invention are further characterized by a rate of polymerization sufficient to allow the formation of aggregates visible to the naked eye, when these are formed during the contacting of a biological sample containing antibodies.
- polymerized anti-Ig antibodies of the invention are further defined by their ability to bind nonspecifically to the constant parts of specific determined antibodies present in a biological sample, these determined antibodies themselves being recognized by a specific antigen.
- polymerized anti-Ig antibodies composed of oligomers comprising at least 2 units, in particular 2 to 25 anti-Ig antibodies covalently coupled together. * It is remarkable that the polymers thus obtained retain the capacities of the antibodies in their unpolymerized form to form non-specific complexes with other antibodies among which the determined or chosen antibodies possibly present in the sample tested, these 4 antibodies are themselves recognized immunologically by a specific, labeled antigen.
- the polymerization rate of the polymerized anti-Ig antibodies does not exceed that which would cause the insolubilization of the polymer. This polymerization rate is however sufficient so that, in the presence of distinct antibodies, the polymerized anti-Ig antibodies are capable of producing, by agglutination with other antibodies, aggregates visible with the naked eye, if necessary after concentration of the medium. in which the operation is carried out.
- the polymerized antibodies are capable of binding to the constant part of the antibodies present in the medium, for example following an infection, to form the aforesaid aggregates, those of these aggregates in which the antibodies sought are then themselves recognized.
- homologous antigens labeled for example by staining normally capable of selectively forming with the desired antibody a complex of the antigen-antibody type; whereby those of the aggregates formed which include labeled antigens, can be easily identified by the marker, among the aggregates involving the polymerized anti-Ig and other antibodies, heterologous towards the labeled antigen.
- the invention also relates to a diagnostic test for the presence of determined antibodies in a biological sample comprising the following steps:
- the Ig immunoglobulins used for the production of anti-Ig antibodies must come from the same mammalian host as that providing the biological medium, in particular the serum or the plasma, in which the antibody sought is to be assayed. If the host is human, the anti-Ig antibodies are obtained by immunization of a non-human animal with immunoglobulins G obtained from a human serum.
- the preferred polymerization rate for anti-Ig antibodies is to be chosen according to the nature of the biological samples and of the antibodies sought. This rate of polymerization must be sufficient for the aggregates to be capable of forming and to individually retain a maximum rate of the antibodies sought. This rate will not exceed that which could lead to too massive agglutination in the presence of other antibodies.
- the diagnostic method carried out on a biological sample in solution optionally containing the antibodies to be assayed this is brought into contact with the labeled antigens and the polymerized anti-Ig antibodies and the medium is concentrated until '' to cause the insolubilization of the aggregates formed.
- Advantageously small quantities of the medium are deposited in spots on a support, in particular a paper and one causes a rotary agitation.
- the marker is colored, the presence of the desired antibody is manifested by staining points at the level of the then individualizable aggregates (concentration of the staining at several points of the sample). On the contrary, a uniform coloration of the sample shows that the antibodies sought are absent.
- the determined antibodies sought may be antibodies formed as a result of infection by a parasite or by a virus.
- a virus By way of example, mention may be made of the trypanosome, the vector of brucellosis, the virus
- the detection of the aggregates formed can be revised by means of the step of detecting the coloration of these aggregates, as soon as the specific antigen incorporated in the medium has been labeled.
- This marking can be obtained using a dye such as Coomassie blue or Bengal rose or a bifunctional dye such as Procion blue or also using a fluorescent agent. Markers enzymes are also usable.
- the revelation of the aggregates is in this case carried out by means of an enzyme activity indicator.
- Radioactive labeling of antigens can also be performed.
- the step of detecting aggregates is preceded by a step of physical separation of the non-aggregated complexes and of the sought-after aggregated complexes.
- the above separation can take place by ⁇ entrifugation or by filtration.
- the invention further relates to a kit for diagnosing the presence of determined antibodies comprising:
- this diagnostic kit can be used to detect specific antibodies due to infection by a parasite or a virus.
- the antigens in the kit are specific for this parasite or this virus.
- the antigens in this kit can be labeled as defined above.
- the invention also relates to a process for the preparation of polymerized anti-Ig antibodies, comprising the polymerization of anti-Ig antibodies in the presence of a polymerization agent, in particular under the standard conditions used for protein coupling reactions.
- Suitable polymerization agents are those usually used to polymerize or couple proteins. It is possible, for example, to react the antibodies in solution with ethyl chloroformate, glutaraldehyde or any polyfunctional reagent for coupling between active functions carried respectively by the proteins represented by these anti-Ig antibodies. Such active functions consist, for example, of inate, carboxyl or both functions. By adjusting the relative proportions of the antibodies and the polyfunctional reagent, the polymerization rate can be adjusted in a known manner.
- the invention also relates, as new reagents, to compositions containing both the polymerized anti-Ig antibody and the labeled antigen in relative proportions preferably adjusted as a function of the possible concentrations of the antibodies sought in the samples to be tested. Appropriate proportions must in each case be established experimentally, by using variable proportions of the two constituents and by reacting them with samples containing standard concentrations of the antibody immunologically homologous to the antigen, in particular at concentrations often encountered in clinically suspect subjects.
- EXAMPLE 1 Agglutination test on the Trypanozomoma evansi card.
- the principle of such a test is based on the detection of circulating antibodies against several antigen's infectious parasite using as particles agglutinate, whole trypanosomes carrying a variable antigen.
- This test detects agglutination of the antibodies which appeared during the first two weeks of infection.
- polymerized anti-Ig antibodies will sometimes be designated below by the term "anti-Ig adsorbent”.
- Antisera specific for buffalo, camel and pig immunoglobulins were obtained by immunization of rabbits with the specific immunoglobulins above, of each of the above animal species and respectively obtained from the seru s of these animals and isolated from protein A by complexation with a protein A immobilized on an appropriate support, in particular that known under the brand SEPHAROSE 4B. The technique used is that described in (3).
- the anti-immunoglobulins obtained were then polymerized with ethyl chloroformate and stored at 4 ° C. in a phosphate buffer (PBS) containing 1% bovine serum albumin with 0.1% acid as described, in ( 4). ⁇ Execution of the test.
- PBS phosphate buffer
- This test is carried out as follows: 45 ⁇ liters of serum diluted in the phosphate buffer at pH 7.4 and 45 ⁇ liters of the fixed antigen RoTat 1.2 colored with a blue dye and containing 10% of polymerized anti-Ig antibodies are deposited on ia reaction surface constituted by an agglutination card covered with a white plastic material, which can be substituted by photographic paper RC. The suspension is spread and subjected to reaction for 10 minutes on an agitator.
- test is significant for the following dilutions of the serum with or without the use of "polymerized anti-immunoglobulin " antibodies:
- the test detected antibodies circulating against parasites in whole blood, serum or plasma during the first two weeks of infection (1), in the absence of prozone phenomena constantly observed when using unpolymerized anti-Ig antibodies in place of polymerized antibodies. On the contrary, the use of anti-Ig antibodies leads to reliable results, even at low dilutions of serum (2).
- CATT Test for the search for Trypan some by agglutination on card.
- CFT Fixation test on the complement.
- D.O Optical Density: values greater than or equal to 0.050 are considered to be positive.
- the test includes the use of either a conventional Testryp (R) CATT antigen or RoTat 1.2 antigen as well as the use of a polymerized anti-Ig antibody (antibodies directed against human G immunoglobulins).
- R Testryp
- RoTat 1.2 antigen an antigen directed against human G immunoglobulins
- the preparation of 1 • anti-Ig adsorbent and the execution of the test are carried out as described in Example 1.
- the sensitivity of the test was demonstrated after titration of known sera from African patients infected with T gambiense and T rodhesiense and after titration of control sera obtained from healthy European people.
- This test is based on the use of an antigen in acid buffer, colored with Bengal pink and supplemented with polymerized anti-human Ig antibodies.
- a solid support is prepared, appropriately colored and consisting of particles of a polymerized substance such as a plastic or another organic polymer.
- the particles can also consist of insolubilized proteins which do not cross-react with the polymerized antibodies. These protein particles are prepared by controlled coupling.
- This example relates to a new method of synthesis of colored particulate antigen allowing detection by agglutination.
- a suspension of particles coated with antigens is prepared to carry out the agglutination test.
- Antibodies to IgM or IgG are detected using specific anti-IgM or anti-IgG polymerized adsorbents. The test is carried out as in Example 1.
- Agglutination tests using polymerized antibodies meeting the definitions of the invention have made it possible to detect circulating antibodies directed against parasites or viruses, from whole blood, plasma or serum. Such tests are very rapid (10mn), reliable, very sensitive and can be adapted for screening in large numbers. As a result, treatment of the infectious diseases described in the examples can be started immediately after a positive diagnosis.
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Abstract
Description
ANTICORPS POLYMERISES, DIRIGES CONTRE DES IMMUNOGLOBU¬ LINES - LEUR UTILISATION DANS DES TESTS DE DIAGNOSTIC. Des anticorps circulant contre plusieurs antigènes de parasites, bactéries ou virus pathogènes sont produits pendant la durée d'une infection. De tels antigènes peuvent être mis en évidence dans le sang total, le plasma ou le sérum ou d'autres fluides biologiques par des tests d'agglutination directs ou spécifiques. POLYMERIZED ANTIBODIES, DIRECTED AGAINST IMMUNOGLOBU¬ LINES - THEIR USE IN DIAGNOSTIC TESTS. Antibodies circulating against several antigens of pathogenic parasites, bacteria or viruses are produced during the duration of an infection. Such antigens can be detected in whole blood, plasma or serum or other biological fluids by direct or specific agglutination tests.
Ces tests spécifiques qui comprennent l'utilisation d'antigènes spécifiques permettent la détection d'agglutinines spécifiques. Leur utilité est cependant limitée par un manque de sensibilité. Ainsi, certaines infections ne sont pas détectables si l'antigène utilisé dans le test est exprimé seulement dans une phase tardive de l'infection ou si cet antigène n'est pas suffisamment i munogène.These specific tests which include the use of specific antigens allow the detection of specific agglutinins. Their usefulness is however limited by a lack of sensitivity. Thus, certain infections are not detectable if the antigen used in the test is expressed only in a late phase of the infection or if this antigen is not sufficiently munogenic.
Le défaut de sensibilité peut être attribué également à la présence d'anticorps dits "incomplets" et bloquants qui, soit n'agglutinent pas soit inhibent l'agglutination.The lack of sensitivity can also be attributed to the presence of so-called "incomplete" and blocking antibodies which either do not agglutinate or inhibit agglutination.
L'utilisation d'anticorps anti-i munoglobuline du type Ig (anticorps anti-Ig) en tant que réactif de Coo bs, améliore considérablement la sensibilité. Cependant, dans ces conditions, la lecture des résultats du test est rendue plus difficile à cause de la petite taille des agrégats formés après la réaction immunologique ou d'un phénomène de prozone bien connu en immunologie qui se manifeste en présence de faibles dilutions de sérum (par exemple des dilutions de l'ordre de 1/2 ou 1/10). Dans ce cas, lorsque les anticorps à doser sont présents à haute concentration, l'agglutination avec les anti-Ig est inhibée et le signal de présence des anticorps recherchés disparait. La mise en oeuvre de test de détection d'infection due à un parasite nécessite des manipulations supplémentaires telles que des dilutions préalables de l'échantillon à tester.The use of anti-i munoglobulin antibodies of the Ig type (anti-Ig antibodies) as a reagent for Coo bs, considerably improves the sensitivity. However, under these conditions, the reading of the test results is made more difficult because of the small size of the aggregates formed after the immunological reaction or of a prozone phenomenon well known in immunology which manifests itself in the presence of low dilutions of serum. (for example dilutions of the order of 1/2 or 1/10). In this case, when the antibodies to be assayed are present in high concentration, agglutination with the anti-Ig is inhibited and the signal for the presence of the antibodies sought disappears. The implementation of infection detection test due to a parasite requires additional manipulations such as prior dilutions of the test sample.
Les inventeurs ont mis en évidence qu'une quantité appropriée d'anticorps anti-Ig sous forme polymérisée ajoutée à un antigène agglutinable, entraine un accroissement de la sensibilité lors d'un test de détection immunologique impliquant des réactions d'agglutination, et la réduction sinon l'abolition de phénomène de prozone. De ce fait, aucune manipulation supplémentaire n'est requise préalablement à la mise en oeuvre des réactions, comme par exemple des dilutions préalables de l'échantillon à doser.The inventors have demonstrated that an appropriate quantity of anti-Ig antibodies in polymerized form added to an agglutinable antigen, causes an increase in sensitivity during an immunological detection test involving agglutination reactions, and the reduction otherwise the abolition of the prozone phenomenon. Therefore, no additional manipulation is required prior to the implementation of the reactions, such as prior dilutions of the sample to be assayed.
La présente invention se rapporte à des anticorps-Ig polymérisés et à leur utilisation dans des tests immunologiques de détection par exemple de parasites ou de virus.The present invention relates to polymerized Ig-antibodies and to their use in immunological tests for the detection, for example, of parasites or viruses.
En particulier, la présente invention vise l'utilisation des anticorps polymérisés conjointement avec des antigènes particulaires, de couleur constrastante par rapport au support sur lequel ils sont employés ou par rapport au liquide biologique ou vice-versa pour augmenter la sensibilité des tests d'agglutinations par l'augmentation de la taille des agrégats.In particular, the present invention relates to the use of antibodies polymerized jointly with particulate antigens, of a contrasting color with respect to the support on which they are used or with respect to the biological fluid or vice versa to increase the sensitivity of the agglutination tests. by increasing the size of the aggregates.
La présente invention vise également des tests rapides pour la détection d'anticorps déterminés par exemple dus à des maladies infectieuses chez l'homme ou l'animal grâce à la mise en oeuvre des anticorps anti-Ig polymérisés. Cette application comprend toute sorte de tests qui permettent la détection d'agrégats de particules (constituées par des multicomplexes du type anti-Ig, anticorps recherché, antigène spécifique dudit anticorps recherché) de taille suffisante pour être visibles à l'oeil nu, compte tenu des conditions de révélation de ces agrégats.The present invention also relates to rapid tests for the detection of determined antibodies, for example due to infectious diseases in humans or animals thanks to the use of polymerized anti-Ig antibodies. This application includes all kinds of tests which allow the detection of aggregates of particles (constituted by multicomplexes of the anti-Ig type, antibody sought, specific antigen of said antibody sought) of sufficient size to be visible to the naked eye, taking into account the conditions for revealing these aggregates.
L'invention met à profit la capacité conservée par des anticorps-Ig sous forme poly érisée à fixer de façon non sélective d'autres anticorps et les complexes immunologiques les contenant, parmi lesquels les complexes formés entre des anticorps spécifiques contenus dans un échantillon biologique et des antigènes correspondants.The invention takes advantage of the capacity preserved by antibodies-Ig in poly erized form to non-selectively fix other antibodies and the immunological complexes containing them, among which the complexes formed between specific antibodies contained in a biological sample and corresponding antigens.
Les échantillons biologiques peuvent être constitués par des fluides biologiques tels que le sang complet, le sérum, ou le plasma.Biological samples can be made up of biological fluids such as whole blood, serum, or plasma.
Des anticorps anti-Ig polymérisés de 1'invention sont encore caractérisés par un taux de polymérisation suffisant pour permettre la formation d'agrégats visibles à l'oeil nu, lorsque ceux-ci sont formés à l'occasion de la mise en contact d'un échantillon biologique contenant des anticorps.Polymerized anti-Ig antibodies of the invention are further characterized by a rate of polymerization sufficient to allow the formation of aggregates visible to the naked eye, when these are formed during the contacting of a biological sample containing antibodies.
Les anticorps anti-Ig polymérisés de 1'invention sont encore définis par leur capacité à se fixer de façon non spécifique aux parties constantes d'anticorps déterminé spécifiques présents dans un échantillon biologique, ces anticorps déterminé étant eux-mêmes reconnu par un antigène spécifique.The polymerized anti-Ig antibodies of the invention are further defined by their ability to bind nonspecifically to the constant parts of specific determined antibodies present in a biological sample, these determined antibodies themselves being recognized by a specific antigen.
Entrent en particulier dans le cadre de l'invention, des anticorps anti-Ig polymérisés composés d'oligomères comportant au moins 2 motifs, notamment 2 à 25 anticorps anti-Ig couplés entre eux de^ façon covalente. * Il est remarquable que les polymères ainsi obtenus, conservent les capacités des anticorps dans leur forme non polymérisée de former des complexes non spécifiques avec d'autres anticorps parmi lesquels les anticorps déterminés ou choisis éventuellement présents dans l'échantillon testé, ces 4 anticorps étant eux-mêmes reconnus immunologiquement par un antigène marqué, spécifique.Particularly within the scope of the invention are polymerized anti-Ig antibodies composed of oligomers comprising at least 2 units, in particular 2 to 25 anti-Ig antibodies covalently coupled together. * It is remarkable that the polymers thus obtained retain the capacities of the antibodies in their unpolymerized form to form non-specific complexes with other antibodies among which the determined or chosen antibodies possibly present in the sample tested, these 4 antibodies are themselves recognized immunologically by a specific, labeled antigen.
Le taux de polymérisation des anticorps anti-Ig polymérisés ne dépasse pas celui qui entraînerait 1'insolubilisation du polymère. Ce taux de polymérisation est cependant suffisant pour que, en présence d'anticorps distincts, les anticorps anti-Ig polymérisés soient capables de produire par agglutination avec d'autres anticorps des agrégats visibles à l'oeil nu, le cas échéant après concentration du milieu dans lequel l'opération est réalisée.The polymerization rate of the polymerized anti-Ig antibodies does not exceed that which would cause the insolubilization of the polymer. This polymerization rate is however sufficient so that, in the presence of distinct antibodies, the polymerized anti-Ig antibodies are capable of producing, by agglutination with other antibodies, aggregates visible with the naked eye, if necessary after concentration of the medium. in which the operation is carried out.
Les anticorps polymérisés sont capables de se fixer sur la partie constante des anticorps présents dans le milieu par exemple à la suite d'une infection, pour former les susdits agrégats, ceux de ces agrégats dans lesquels interviennent les anticorps recherchés étant alors eux-mêmes reconnus par les antigènes homologues marqués par exemple par coloration, normalement aptes à sélectivement former avec l'anticorps recherché un complexe du type antigène-anticorps ; ce grâce à quoi ceux des agrégats formés qui comprennent des antigènes marqués, peuvent être repérés aisément grâce au marqueur, parmi les agrégats faisant intervenir les anti-Ig polymérisés et d'autres anticorps, hétérologues vis-à-vis de l'antigène marqué.The polymerized antibodies are capable of binding to the constant part of the antibodies present in the medium, for example following an infection, to form the aforesaid aggregates, those of these aggregates in which the antibodies sought are then themselves recognized. by homologous antigens labeled for example by staining, normally capable of selectively forming with the desired antibody a complex of the antigen-antibody type; whereby those of the aggregates formed which include labeled antigens, can be easily identified by the marker, among the aggregates involving the polymerized anti-Ig and other antibodies, heterologous towards the labeled antigen.
L'invention vise également un test de diagnostic de _ la présence d'anticorps déterminés dans un échantillon biologique comprenant les étapes suivantes:The invention also relates to a diagnostic test for the presence of determined antibodies in a biological sample comprising the following steps:
- la mise en contact de 1'échantillon biologique avec un antigène marqué par exemple coloré, spécifique de l'anticorps recherché en présence d'anticorps anti-Ig polymérisés tels que définis plus haut, - la détection de ceux des agrégats d'anti-Ig qui portent également l'antigène marqué, par exemple coloré.bringing the biological sample into contact with a labeled, for example colored, antigen specific for the antibody sought in the presence of polymerized anti-Ig antibodies as defined above, - the detection of those of the anti-Ig aggregates which also carry the labeled, for example colored, antigen.
Il est à remarquer que les immunoglobulines Ig utilisées pour la production des anticorps anti-Ig doivent provenir du même hôte mammifère que celui fournissant le milieu biologique, notamment le sérum ou le plasma, dans lequel l'anticorps recherché est à doser. Si l'hôte est humain les anticorps anti-Ig sont obtenus par immunisation d'un animal non humain avec des immunoglobulines G obtenues à partir d'un sérum humain.It should be noted that the Ig immunoglobulins used for the production of anti-Ig antibodies must come from the same mammalian host as that providing the biological medium, in particular the serum or the plasma, in which the antibody sought is to be assayed. If the host is human, the anti-Ig antibodies are obtained by immunization of a non-human animal with immunoglobulins G obtained from a human serum.
Il est aisément concevable que le taux de polymérisation préféré pour les anticorps anti-Ig est à choisir selon les natures des échantillons biologiques et des anticorps recherchés. Ce taux de polymérisation doit être suffisant pour que les agrégats soient susceptibles de se former et de retenir individuellement un taux maximum des anticorps recherchés. Ce taux ne dépassera pas celui qui pourrait entraîner une agglutination trop massive en présence d'autres anticorps.It is easily conceivable that the preferred polymerization rate for anti-Ig antibodies is to be chosen according to the nature of the biological samples and of the antibodies sought. This rate of polymerization must be sufficient for the aggregates to be capable of forming and to individually retain a maximum rate of the antibodies sought. This rate will not exceed that which could lead to too massive agglutination in the presence of other antibodies.
D'une façon générale, on se placera dans des conditions permettant la production d'agglutinats multiples.Generally, we will place ourselves in conditions allowing the production of multiple agglutinates.
Du fait de l'affinité spécifique de l'antigène utilisé pour la détection avec l'anticorps recherché, on est encore amené à faire les constatations suivantes selon que l'échantillon biologique contenait ou ne contenait pas l'anticorps recherché : cas où l'échantillon contenait l'anticorps recherché : attraction sélective de l'antigène marqué vers les complexes que forment entre eux les anticorps anti-Ig et 1'anticorps recherché : d'où 1•individualisation et par conséquent visualisation des particules retenant le marqueur, en particulier lorsque celui-ci est coloré,Due to the specific affinity of the antigen used for detection with the antibody sought, we are still led to make the following observations depending on whether the biological sample contained or did not contain the antibody sought: case where the sample contained the desired antibody: selective attraction of the labeled antigen towards the complexes formed between the anti-Ig antibodies and the desired antibody: hence 1 • individualization and consequently visualization particles retaining the marker, in particular when the latter is colored,
- cas de l'absence de l'anticorps recherché : absence d'individualisation et par conséquent de visualisation d'agrégats particuliers parmi d'autres.- case of the absence of the antibody sought: absence of individualization and consequently of visualization of particular aggregates among others.
Selon un mode de réalisation avantageux du procédé de diagnostic réalisé sur un échantillon biologique en solution contenant éventuellement les anticorps à doser, on met celui-ci en contact avec les antigènes marqués et les anticorps anti-Ig polymérisés et l'on concentre le milieu jusqu'à provoquer l'insolubilisation des agrégats formés. Avantageusement de petites quantités du milieu sont déposées en tâches sur un support, notamment un papier et on provoque une agitation rotative. Dans le cas où le marqueur est coloré, la présence de l'anticorps recherché se manifeste par des points de coloration au niveau des agrégats alors individualisables (con¬ centration de la coloration en plusieurs points de l'échantillon). Au contraire, une coloration uniforme de l'échantillon montre que les anticorps recherchés sont absents.According to an advantageous embodiment of the diagnostic method carried out on a biological sample in solution optionally containing the antibodies to be assayed, this is brought into contact with the labeled antigens and the polymerized anti-Ig antibodies and the medium is concentrated until '' to cause the insolubilization of the aggregates formed. Advantageously small quantities of the medium are deposited in spots on a support, in particular a paper and one causes a rotary agitation. In the case where the marker is colored, the presence of the desired antibody is manifested by staining points at the level of the then individualizable aggregates (concentration of the staining at several points of the sample). On the contrary, a uniform coloration of the sample shows that the antibodies sought are absent.
Les anticorps déterminés recherchés peuvent être des anticorps formés à la suite, d'une infection par un parasite ou par un virus. A titre d'exemple on citera le trypanosome, le vecteur de la brucellose, le virusThe determined antibodies sought may be antibodies formed as a result of infection by a parasite or by a virus. By way of example, mention may be made of the trypanosome, the vector of brucellosis, the virus
HIV.HIV.
La détection des agrégats formés peut être révisée grâce à e étape de détection de la coloration de ces agrégats, dès lors que l'antigène spécifique incorporé dans le milieu a été marqué. Ce marquage peut être obtenu grâce à un colorant tel que le bleu de Coomassie ou le rose de Bengale ou un colorant bifonctionnel comme le bleu de Procion ou encore grâce à un agent fluorescent. Des marqueurs enzymatiques sont aussi utilisables. La révélation des agrégats est dans ce cas effectuée grâce à un révélateur d'activité enzymatique.The detection of the aggregates formed can be revised by means of the step of detecting the coloration of these aggregates, as soon as the specific antigen incorporated in the medium has been labeled. This marking can be obtained using a dye such as Coomassie blue or Bengal rose or a bifunctional dye such as Procion blue or also using a fluorescent agent. Markers enzymes are also usable. The revelation of the aggregates is in this case carried out by means of an enzyme activity indicator.
Un marquage radioactif des antigènes peut également être effectué. Dans ce cas, l'étape de détection des agrégats est précédée d'une étape de séparation physique des complexes non agrégés et des complexes agrégés recherchés.Radioactive labeling of antigens can also be performed. In this case, the step of detecting aggregates is preceded by a step of physical separation of the non-aggregated complexes and of the sought-after aggregated complexes.
La susdite séparation peut avoir lieu par σentrifugation ou par filtration.The above separation can take place by σentrifugation or by filtration.
L'invention vise en outre un kit de diagnostic de la présence anticorps déterminés comprenant :The invention further relates to a kit for diagnosing the presence of determined antibodies comprising:
- des anticorps polymérisés,- polymerized antibodies,
- des antigènes marqués spécifiques.- specific labeled antigens.
De façon avantageuse, ce kit de diagnostic est utilisable pour détecter des anticorps spécifiques dus à une infection par un parasite ou un virus. Dans ce cas les antigènes du kit sont spécifiques de ce parasite ou de ce virus.Advantageously, this diagnostic kit can be used to detect specific antibodies due to infection by a parasite or a virus. In this case, the antigens in the kit are specific for this parasite or this virus.
Les antigènes de ce kit peuvent être marqués de la façon définie précédemment.The antigens in this kit can be labeled as defined above.
L'invention concerne aussi un procédé pour la préparation d'anticorps anti-Ig polymérisés, comprenant la polymérisation d'anticorps anti-Ig en présence d'un agent de polymérisation, notamment dans les conditions standard utilisées pour les réactions de couplage de protéines.The invention also relates to a process for the preparation of polymerized anti-Ig antibodies, comprising the polymerization of anti-Ig antibodies in the presence of a polymerization agent, in particular under the standard conditions used for protein coupling reactions.
Des agents de polymérisation adaptés sont ceux habituellement utilisés pour polymériser ou coupler les protéines. On peut par exemple faire réagir les anticorps en solution avec le chloroformiate d'éthyle, la glutaraldéhyde ou tout réactif polyfonctionnel de couplage entre des fonctions actives respectivement portées par les protéines que représentent ces anticorps anti-Ig. De telles fonctions actives consistent par exemple en des fonctions a iné, carboxyle ou les deux à la fois. Par ajustement des proportions relatives des anticorps et du réactif polyfonctionnel on peut régler de façon en soi connue le taux de polymérisation.Suitable polymerization agents are those usually used to polymerize or couple proteins. It is possible, for example, to react the antibodies in solution with ethyl chloroformate, glutaraldehyde or any polyfunctional reagent for coupling between active functions carried respectively by the proteins represented by these anti-Ig antibodies. Such active functions consist, for example, of inate, carboxyl or both functions. By adjusting the relative proportions of the antibodies and the polyfunctional reagent, the polymerization rate can be adjusted in a known manner.
L'invention concerne aussi en tant que réactifs nouveaux des compositions contenant à la fois l'anticorps anti-Ig polymérisé et l'antigène marqué dans des proportions relatives de préférence ajustées en fonction des concentrations possibles en les anticorps recherchés dans les échantillons à tester. Des proportions appropriées doivent dans chaque cas être établies expérimentalement, en mettant en oeuvre des proportions variables des deux constituants et en les mettant à réagir avec des échantillons contenant des concentrations type de l'anticorps immu- nologiquement homologue de l'antigène, notamment à des concentrations souvent rencontrées chez des sujets cliniquement suspects.The invention also relates, as new reagents, to compositions containing both the polymerized anti-Ig antibody and the labeled antigen in relative proportions preferably adjusted as a function of the possible concentrations of the antibodies sought in the samples to be tested. Appropriate proportions must in each case be established experimentally, by using variable proportions of the two constituents and by reacting them with samples containing standard concentrations of the antibody immunologically homologous to the antigen, in particular at concentrations often encountered in clinically suspect subjects.
EXEMPLE 1 ; Test d'agglutination sur carte de Trypanozomoma evansi.EXAMPLE 1; Agglutination test on the Trypanozomoma evansi card.
Dans la trypanosomiase chronique humaine, des recherches concernant le mécanisme de la variation antigénique et la réponse immune, ont conduit à la mise au point d'un diagnostic basé sur un simple test d'agglutination sur carte.In chronic human trypanosomiasis, research into the mechanism of antigenic variation and the immune response has led to the development of a diagnosis based on a simple card agglutination test.
Le principe d'un tel test est fondé sur la détection des anticorps circulants dirigés contre plusieurs antigène's du parasite infectieux en utilisant comme particules à agglutiner, les trypanosomes entiers portant un antigène variable.The principle of such a test is based on the detection of circulating antibodies against several antigen's infectious parasite using as particles agglutinate, whole trypanosomes carrying a variable antigen.
Lorsque ce test est employé dans les conditions connues pour le diagnostic de la trypanosomiase du chameau (5) et du buffle (2) , on observe une sensibilité faible. Des modifications ont été réalisées sur ce test dans le cadre de l'invention de façon à ce que la sensibilité soit portée à un niveau plus élevé par l'addition à l'antigène d'un anticorps anti-Ig polymérisé. L'utilisation d'un antigène coloré et d'un anti-IgG de couleur blanche conjointement avec une carte d'agglutination blanche permet de visualiser seule l'agglutination de l'antigène.When this test is used under the conditions known for the diagnosis of trypanosomiasis of camels (5) and buffaloes (2), low sensitivity. Modifications have been made to this test in the context of the invention so that the sensitivity is brought to a higher level by the addition to the antigen of a polymerized anti-Ig antibody. The use of a colored antigen and of a white anti-IgG together with a white agglutination map makes it possible to visualize the agglutination of the antigen alone.
Ce test détecte 1'agglutination des anticorps apparus dès les deux premières semaines de 1'infection.This test detects agglutination of the antibodies which appeared during the first two weeks of infection.
- Préparation des anticorps polymérisés anti-Ig. On désignera parfois dans la suite les anticorps anti-Ig polymérisés par le terme "adsorbant anti-Ig". Des antisérums spécifiques des immunoglobulines du buffle, du chameau, du porc ont été obtenus par immunisation de lapins avec des immunoglobulines spécifiques ci-dessus, de chacune des espèces animales ci-dessus et respectivement obtenues à partir des séru s de ces animaux et isolées sur protéine A par complexation avec une protéine A immobilisée sur un support approprié, notamment celui connu sous la marque SEPHAROSE 4B. La technique utilisée est celle décrite dans (3). Les anti-immunoglobulines obtenues ont ensuite été polymérisées par du chloroformiate d'éthyle et conservées à 4*C dans un tampon phosphate (PBS) contenant 1% de sérum albumine bovine avec 0,1% d'acide tel que décrit,-dans (4). ~ Exécution du test.- Preparation of polymerized anti-Ig antibodies. The polymerized anti-Ig antibodies will sometimes be designated below by the term "anti-Ig adsorbent". Antisera specific for buffalo, camel and pig immunoglobulins were obtained by immunization of rabbits with the specific immunoglobulins above, of each of the above animal species and respectively obtained from the seru s of these animals and isolated from protein A by complexation with a protein A immobilized on an appropriate support, in particular that known under the brand SEPHAROSE 4B. The technique used is that described in (3). The anti-immunoglobulins obtained were then polymerized with ethyl chloroformate and stored at 4 ° C. in a phosphate buffer (PBS) containing 1% bovine serum albumin with 0.1% acid as described, in ( 4). ~ Execution of the test.
Ce test est réalisé comme suit : 45μ litres de sérum dilué dans le tampon phosphate à pH 7,4 et 45μ litres de l'antigène fixé RoTat 1.2 coloré par un colorant bleu et contenant 10% d'anticorps anti-Ig polymérisés sont déposés sur ia surface de réaction constituée par une carte d'agglutination recouverte d'une matière plastique blanche, qui peut être substituée par un papier photographique RC. La suspension est étalée et soumise à réaction pendant lOmn sur un agitateur.This test is carried out as follows: 45 μ liters of serum diluted in the phosphate buffer at pH 7.4 and 45 μ liters of the fixed antigen RoTat 1.2 colored with a blue dye and containing 10% of polymerized anti-Ig antibodies are deposited on ia reaction surface constituted by an agglutination card covered with a white plastic material, which can be substituted by photographic paper RC. The suspension is spread and subjected to reaction for 10 minutes on an agitator.
Les résultats sont indiqués comme suit : +++ (très fortement positif) , ++ (fortement positif) , + (positif) , +/- (faiblement positif) (négatif) .The results are indicated as follows: +++ (very strongly positive), ++ (strongly positive), + (positive), +/- (weakly positive) (negative).
La plus forte dilution de sérum donnant un résultat positif (+) est considérée comme le titre limite de détection. - Spécificité du testThe highest dilution of serum giving a positive result (+) is considered to be the limit of detection. - Specificity of the test
Le test est significatif pour les dilutions suivantes du sérum avec ou sans l'utilisation d'anticorps "polymérisés anti-immunoglobulines" :The test is significant for the following dilutions of the serum with or without the use of "polymerized anti-immunoglobulin " antibodies:
- buffle (water-buffalo) : > 1/4 - bétail : 1/2- buffalo (water-buffalo):> 1/4 - cattle: 1/2
- porc : 1/2- pork: 1/2
- chameau : 1/2- camel: 1/2
Ce test est en très bonne corrélation avec la parasitémie et il est complémentaire des tests Elisa mettant en oeuvre des antigènes (voir le tableau I) basés sur des anticorps monoclonaux réagissant contre le T gambiense ou le T evansi.This test is in very good correlation with parasitaemia and it is complementary to the Elisa tests using antigens (see Table I) based on monoclonal antibodies reacting against T gambiense or T evansi.
- Sensibilité du test- Test sensitivity
.... La sensibilité du test a été mise en évidence par Q titration de 43 antisérums produits chez le lapin chez des bovins et des buffles expérimentalement infectés avec T evansi (voir la figure 1) et la titration de sérums obtenus à partir de 4 chameaux infectés et de deux chameaux non infectés, de 139 échantillons 5 collectés sur des porcs, du bétail et des buffles de différentes régions de Thaïlande.. . . . The sensitivity of the test was demonstrated by Q titration of 43 antisera produced in rabbits in cattle and buffaloes experimentally infected with T evansi (see Figure 1) and the titration of sera obtained from 4 infected camels and two uninfected camels, from 139 samples 5 collected from pigs, cattle and buffaloes from different regions of Thailand.
L'addition des anticorps anti-Ig poly érisé à l'antigène, a conduit à un accroissement de 10 à 100 fois de la sensibilité. Le test a permis de détecter les anticorps circulants contre des parasites dans le sang total, le sérum ou le plasma lors des deux premières semaines de l'infection (1), en l'absence de phénomènes de prozone constamment observés lorsque l'on utilise des anticorps anti-Ig non-polymérisés à la place des anticorps polymérisés. Au contraire l'utilisation des anticorps anti-Ig conduit à des résultats fiables, même à des dilutions faibles de sérum (2) .The addition of poly erized anti-Ig antibodies to the antigen led to a 10 to 100 fold increase in sensitivity. The test detected antibodies circulating against parasites in whole blood, serum or plasma during the first two weeks of infection (1), in the absence of prozone phenomena constantly observed when using unpolymerized anti-Ig antibodies in place of polymerized antibodies. On the contrary, the use of anti-Ig antibodies leads to reliable results, even at low dilutions of serum (2).
Nombre Parasité- anticorps testés par: Ag-ELISA d'animaux mie CATT CFTParasite-antibody number tested by: Ag-ELISA of animals CATT CFT
CATT = Test pour la recherche de Trypan some par agglutination sur carte. CFT = Test de fixation sur le complément. D.O = Densité Optique : les valeurs supérieures ou égales à 0,050 sont considérées comme positives. EXEMPLE 2CATT = Test for the search for Trypan some by agglutination on card. CFT = Fixation test on the complement. D.O = Optical Density: values greater than or equal to 0.050 are considered to be positive. EXAMPLE 2
- Test d'agglutination sur carte pour la maladie du sommeil chez l'Homme.- Card agglutination test for human sleeping sickness.
Le test comprend l'utilisation soit d'un antigène conventionnel Testryp(R)CATT, soit d'antigène RoTat 1.2 ainsi que l'utilisation d'un anticorps anti-Ig polymérisé (anticorps dirigés contre des immunoglobulines G humaines) . La préparation de 1•adsorbant anti-Ig et l'exécution du test sont réalisées comme décrit dans l'exemple 1. La sensibilité du test a été mise en évidence après titration de sérums connus de patients africains infectés par T gambiense et T rodhesiense et après titration de sérums de contrôle obtenus chez des personnes européennes saines.The test includes the use of either a conventional Testryp (R) CATT antigen or RoTat 1.2 antigen as well as the use of a polymerized anti-Ig antibody (antibodies directed against human G immunoglobulins). The preparation of 1 • anti-Ig adsorbent and the execution of the test are carried out as described in Example 1. The sensitivity of the test was demonstrated after titration of known sera from African patients infected with T gambiense and T rodhesiense and after titration of control sera obtained from healthy European people.
L'addition d'un adsorbant anti Ig a conduit à un accroissement de 10 à 100 fois de la sensibilité. EXEMPLE 3The addition of an anti-Ig adsorbent led to a 10 to 100-fold increase in sensitivity. EXAMPLE 3
- Test de la brucellose.- Brucellosis test.
Ce test est basé sur l'utilisation d'un antigène en tampon acide, coloré avec du rose Bengal et complété avec des anticorps polymérisés anti-Ig humaines.This test is based on the use of an antigen in acid buffer, colored with Bengal pink and supplemented with polymerized anti-human Ig antibodies.
Ce test est réalisé sur des cartes d'agglutination jetables et ces réactions spécifiques ont lieu dans un intervalle de 5 minutes. La sensibilité du test a été mise en évidence par la titration de sérums de patients européens ayant des anticorps bloquants pour la brucellose ainsi qu'avec des sérums obtenus chez des européens sains. L'addition d'un adsorbant anti Ig à l'antigène conventionnel coloré par le rose Bengal a permis d'obtenir un accroissement de 2 à 8 fois de la sensibilité. EXEMPLE 4.This test is carried out on disposable agglutination cards and these specific reactions take place in an interval of 5 minutes. The sensitivity of the test was demonstrated by the titration of sera from European patients with antibodies blocking brucellosis as well as with sera obtained from healthy Europeans. The addition of an anti-Ig adsorbent to the conventional antigen colored with Bengal pink made it possible to obtain a 2-8 fold increase in sensitivity. EXAMPLE 4.
- Test d'agglutination pour HIV.- Agglutination test for HIV.
*-V- On prépare un support solide coloré de façon appropriée et consistant en des particules d'une substance polymérisée telles qu'un plastique ou un autre polymère organique. Les particules peuvent également être constituées par des protéines insolubilisées ne réagissant pas de façon croisée avec les anticorps polymérisés. Ces particules protéiques sont préparées par couplage contrôlé. Cet exemple porte sur une méthode nouvelle de synthèse d'antigène particulaire coloré permettant la détection par agglutination. Une suspension de particules recouvertes d'antigènes est préparée pour mettre en oeuvre le test d'agglutination. Des anticorps dirigés contre les IgM ou les IgG sont détectés en utilisant des adsorbants spécifiques anti IgM ou anti IgG polymérisés. La réalisation du test est conduite comme dans l'exemple 1.* -V- A solid support is prepared, appropriately colored and consisting of particles of a polymerized substance such as a plastic or another organic polymer. The particles can also consist of insolubilized proteins which do not cross-react with the polymerized antibodies. These protein particles are prepared by controlled coupling. This example relates to a new method of synthesis of colored particulate antigen allowing detection by agglutination. A suspension of particles coated with antigens is prepared to carry out the agglutination test. Antibodies to IgM or IgG are detected using specific anti-IgM or anti-IgG polymerized adsorbents. The test is carried out as in Example 1.
Les tests d'agglutination mettant en oeuvre des anticorps polymérisés répondant aux définitions de l'invention, ont permis la détection des anticorps circulant dirigés contre des parasites ou des virus, à partir du sang complet, du plasma ou de sérum. De tels tests sont très rapides (lOmn) , fiables, très sensibles et peuvent être adaptés pour des criblages en grand nombre. En conséquence, le traitement des maladies infectieuses décrites dans les exemples peut être immédiatement entrepris après un diagnostic positif. Agglutination tests using polymerized antibodies meeting the definitions of the invention have made it possible to detect circulating antibodies directed against parasites or viruses, from whole blood, plasma or serum. Such tests are very rapid (10mn), reliable, very sensitive and can be adapted for screening in large numbers. As a result, treatment of the infectious diseases described in the examples can be started immediately after a positive diagnosis.
BIBLIOGRAPHIEBIBLIOGRAPHY
(l)-Bajyana Songa E., et al, Ann. Soc. belge Méd. trop. 1987, 67, 51-57. (2)- Bajyana Songa E., et al, Ann. Soc. belge Méd. trop., 1987, 67, 137-148. (3)- Goding J.W. Journ. Imm. Meth., 1976, 13, 215-226. (4)- Van der Loo. W. , et al, European Journal of(1) -Bajyana Songa E., et al, Ann. Soc. Belgian Med. too much. 1987, 67, 51-57. (2) - Bajyana Songa E., et al, Ann. Soc. Belgian Med. too., 1987, 67, 137-148. (3) - Goding J.W. Journ. Imm. Meth., 1976, 13, 215-226. (4) - Van der Loo. W., et al, European Journal of
Im unology, 1977, 7(1), 15-22. (5)- Zweygarth E. et al, Ann. Soc. belge Méd. trop.Im unology, 1977, 7 (1), 15-22. (5) - Zweygarth E. et al, Ann. Soc. Belgian Med. too much.
1984, 64, 309-313. 1984, 64, 309-313.
Claims
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| FR88/16407 | 1988-12-13 | ||
| FR8816407A FR2640143B1 (en) | 1988-12-13 | 1988-12-13 | POLYMERIZED ANTIBODIES, DIRECTED AGAINST IMMUNOGLOBULINS - THEIR USE IN DIAGNOSTIC TESTS |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| WO1990007118A2 true WO1990007118A2 (en) | 1990-06-28 |
| WO1990007118A3 WO1990007118A3 (en) | 1990-07-26 |
Family
ID=9372893
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| PCT/EP1989/001540 WO1990007118A2 (en) | 1988-12-13 | 1989-12-13 | Polymerized antibodies directed against immunoglobulins, and their use in diagnostic tests |
Country Status (2)
| Country | Link |
|---|---|
| FR (1) | FR2640143B1 (en) |
| WO (1) | WO1990007118A2 (en) |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1992001472A1 (en) * | 1990-07-19 | 1992-02-06 | Celltech Limited | Multivalent anti-cytokine immunoglobulins |
| FR2676123A1 (en) * | 1991-05-02 | 1992-11-06 | Pasteur Diagnostics | AGGLUTINANT COMPLEX AND REAGENT FOR IDENTIFYING ANTIGENS ON CELL WALLS. |
| EP0516529A3 (en) * | 1991-05-28 | 1993-04-28 | Daiichi Pure Chemicals Co. Ltd. | Assay of specific antibody |
| WO1999030161A1 (en) * | 1997-12-09 | 1999-06-17 | Almudena Rojas Gonzalez | Laboratory test for assaying incomplete antibodies in brucellosis and equipment for carrying out such test |
| EP0955546A1 (en) * | 1998-05-07 | 1999-11-10 | Matsushita Electric Industrial Co., Ltd. | Indirect polymerized and labelled antibody and method for manufacturing the same |
| US6303757B1 (en) | 1996-11-08 | 2001-10-16 | Matsushita Electric Industrial Co., Ltd. | Indirect polymerized and labelled antibody and method for manufacturing the same |
| EP1462673A1 (en) | 2003-03-28 | 2004-09-29 | Pintsch Bamag Antriebs- und Verkehrstechnik GmbH | Procedure and device for monitoring an electomagnetically applied brake |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1239285A1 (en) * | 2001-03-07 | 2002-09-11 | Roche Diagnostics GmbH | Improved homogeneous immunoassay method |
Family Cites Families (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE2847877A1 (en) * | 1978-11-04 | 1980-05-22 | Behringwerke Ag | REAGENT FOR DETECTING INFECTIOUS MONONUCLEOSIS |
| BE900023A (en) * | 1984-06-27 | 1984-10-15 | Inst Internationale De Patholo | Immunoassay using specific and anti-idiotypic antibodies - and measuring inhibition of reaction, esp. agglutination, caused by the analyte |
| CA1256369A (en) * | 1985-02-26 | 1989-06-27 | W. Carl Saxinger | Detection of human t-cell leukemia virus type iii |
| DE3717401A1 (en) * | 1987-05-23 | 1988-12-08 | Behringwerke Ag | One-step immunoassay for the determination of antigen-specific antibodies from one of the immunoglobulin classes A, M, D or E and a suitable agent |
-
1988
- 1988-12-13 FR FR8816407A patent/FR2640143B1/en not_active Expired - Lifetime
-
1989
- 1989-12-13 WO PCT/EP1989/001540 patent/WO1990007118A2/en unknown
Cited By (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1992001472A1 (en) * | 1990-07-19 | 1992-02-06 | Celltech Limited | Multivalent anti-cytokine immunoglobulins |
| GB2261666A (en) * | 1990-07-19 | 1993-05-26 | Celltech Ltd | Multivalent anti-cytokine immunoglobulins |
| GB2261666B (en) * | 1990-07-19 | 1994-07-27 | Celltech Ltd | Multivalent anti-cytokine immunoglobulins |
| FR2676123A1 (en) * | 1991-05-02 | 1992-11-06 | Pasteur Diagnostics | AGGLUTINANT COMPLEX AND REAGENT FOR IDENTIFYING ANTIGENS ON CELL WALLS. |
| EP0512896A1 (en) * | 1991-05-02 | 1992-11-11 | Pasteur Sanofi Diagnostics | Agglutination complex for the determination of blood groups |
| EP0516529A3 (en) * | 1991-05-28 | 1993-04-28 | Daiichi Pure Chemicals Co. Ltd. | Assay of specific antibody |
| US6303757B1 (en) | 1996-11-08 | 2001-10-16 | Matsushita Electric Industrial Co., Ltd. | Indirect polymerized and labelled antibody and method for manufacturing the same |
| WO1999030161A1 (en) * | 1997-12-09 | 1999-06-17 | Almudena Rojas Gonzalez | Laboratory test for assaying incomplete antibodies in brucellosis and equipment for carrying out such test |
| ES2131032A1 (en) * | 1997-12-09 | 1999-07-01 | Gonzalez Almudena Rojas | Laboratory test for assaying incomplete antibodies in brucellosis and equipment for carrying out such test |
| EP0955546A1 (en) * | 1998-05-07 | 1999-11-10 | Matsushita Electric Industrial Co., Ltd. | Indirect polymerized and labelled antibody and method for manufacturing the same |
| EP1462673A1 (en) | 2003-03-28 | 2004-09-29 | Pintsch Bamag Antriebs- und Verkehrstechnik GmbH | Procedure and device for monitoring an electomagnetically applied brake |
Also Published As
| Publication number | Publication date |
|---|---|
| FR2640143A1 (en) | 1990-06-15 |
| FR2640143B1 (en) | 1991-03-15 |
| WO1990007118A3 (en) | 1990-07-26 |
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