EP0000102B1 - Immunologische Zusammensetzungen, Methoden zur Herstellung derselben und Methoden zur Durchführung von Hämagglutinationstesten - Google Patents

Immunologische Zusammensetzungen, Methoden zur Herstellung derselben und Methoden zur Durchführung von Hämagglutinationstesten Download PDF

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EP0000102B1
EP0000102B1 EP78300034A EP78300034A EP0000102B1 EP 0000102 B1 EP0000102 B1 EP 0000102B1 EP 78300034 A EP78300034 A EP 78300034A EP 78300034 A EP78300034 A EP 78300034A EP 0000102 B1 EP0000102 B1 EP 0000102B1
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Prior art keywords
erythrocytes
composition
antigen
stabilised
gonadotrophin
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EP78300034A
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French (fr)
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EP0000102A1 (de
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Michael Allen Hirsch
Douglas Stalker Irvine
John Krupey
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Wyeth LLC
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American Home Products Corp
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    • 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
    • G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
    • 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
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/554—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being a biological cell or cell fragment, e.g. bacteria, yeast cells
    • G01N33/555—Red blood cell
    • G01N33/556—Fixed or stabilised red blood cell

Definitions

  • This invention relates to immunologic compositions and antiserum compositions which may be used with the immunologic compositions.
  • compositions and methods already noted and numerous others suffer individually from various disadvantages such as instability, lack of reproducibility, false positive results, low haemagglutination titres, cost or time of preparation, and others.
  • compositions and methods of this invention demonstrate in part by use in haemagglutination tests, provide a superior grade of stabilised sensitized cells.
  • the compositions of this invention have higher haemagglutination titres, are stable for iong periods of time, and give reproducible patterns and results.
  • Another advantage of the herein disclosed invention is the stability of results obtained. For example, in utilizing a commercially available reagent the haemagglutination patterns change with time. Thus, in a pregnancy test, a negative result, i.e. complete haemagglutination within 2 hours, will change over 24 hours to show a quasi-inhibition pattern; results recorded from 2-24 hours at room temperature will therefore indicate a significant number of false positive results.
  • results are obtained within 2 hours of testing and will not change for about 11 days thereafter or more.
  • the present invention provides an immunologic composition of pyruvic aldehyde or dimethylsuberimidate stabilised erythrocytes sensitised with a polypeptide or glycoprotein antigen selected from chorionic gonadotrophin pregnant mare's serum gonadotrophin, carcino embryonic antigen, luteinising hormone, follicle stimulating hormone, human menopausal gonadotrophin and thyroid stimulating hormone, said stabilised erythrocytes being coupled to said antigen with a bifunctional molecule selected from glutaraldehyde, glyoxal, succinaldehyde, hexamethylene diisocyanate, toluene 2,4-diisocyanate, a bifunctional imido ester, (e.g.,
  • diethyl malonimidate dihydrochloride or dimethyl suberimidate diethyl malonimidate dihydrochloride or dimethyl suberimidate
  • cyanuric chloride tetrazotized o-anisidine
  • water soluble carbodiimide e.g. 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide
  • the bifunctional molecule is other than dimethyl suberimidate, and that when the erythrocytes are pyruvic aldehyde stablised the bifunctional molecule is glutaraldehyde, glyoxal, succinaldehyde, hexamethylene diisocyanate, toluene 2,4- diisocyanate or a bifunctional imido ester.
  • the erythrocytes are pyruvic aldehyde stabilised.
  • an immunologic composition comprising pyruvic aldehyde stabilised erythrocytes sensitized with a polypeptide or glycoprotein antigen selected from chorionic gonadotrophin, pregnant mare's serum gonadotrophin, carcino embryonic antigen, luteinizing hormone, follicle stimulating hormone, human menopausal gonadotrophin and thyroid stimulating hormone, said stabilised erythrocytes being coupled to said antigen with a bifunctional molecule selected from glutaraldehyde, glyoxal, succinaldehyde, hexamethylene diisocyanate, toluene-2,4-diisocyanate, bifunctional imido esters including diethylmalonimidate hydrochloride and dimethyl suberimidate. it is preferred to use as the bifunctional molecule for coupling glutaraldehyde, hexamethylene di
  • the coupling agent or bifunctional molecule can be glutaraldehyde, glyoxal succinaldehyde, hexamethylene diisocyanate, toluene 2,4-diisocyanate, a bifunctional imido ester other than dimethyl suberimidate, cyanuric chloride, tetrazotized o-anisidine or a water soluble carbodiimide such as 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide.
  • the invention provides a method for preparing immunologic compositions of animal erythrocytes sensitized with antigens useful in passive haemagglutination tests which comprises:
  • Immunologic compositions containing dimethyl suberimidate stabilised erythrocytes may be prepared in an analogous manner.
  • erythrocytes stabilised red blood cells sensitized with a polypeptide or glycoprotein such as, human chorionic gonadotrophin (hCG) is prepared as one component in a two component reagent system.
  • Stabilisation of the erythrocytes may be first accomplished by taking freshly drawn whole blood, generally from sheep, although turkeys, goats or horses can be used, and immediately mixing it with an isotonic sterile anticoagulant, such as Alsever's solution, so that a final ratio of blood to anticoagulant is from 0.8:1 to 1.2:1 preferably 1;1, of the resulting mixture.
  • the erythrocytes are then separated by centrifugation and washed with physiological saline.
  • Pyruvic aldehyde reagent is prepared in physiological saline.
  • a neutral buffered solution, preferably phosphate buffer, and a suspension of washed erythrocytes in saline are added to the pyruvic aldehyde reagent.
  • the suspension is then incubated at 30 to 55°C for one to five hours, preferably at 37°C for three hours, with occasional agitation. Subsequently the stabilised red blood cells are thoroughly washed with saline.
  • the pyruvic aldehyde stabilised cells may then be stored at 4°C as a 10% suspension in saline containing 0.1% sodium azide as a preservative, for at least a year, if desired.
  • PAC pyruvic aldehyde stabilised cells
  • the pyruvic aldehyde treated cells are washed with dilute saline and then suspended in a neutral buffered solution, preferably phosphate buffer, containing hCG. To this suspension is added glutaraldehyde in normal saline.
  • the mixture is then incubated, for example, at room temperature for about 2 hours under constant agitation and the cells are removed by centrifugation and washed not less than 3 times with neutral buffered saline, preferably phosphate buffered saline.
  • the sensitized cells are suspended in a neutral buffered saline, preferably phosphate buffered saline, containing as little as 0.2% normal rabbit serum in which complement had previously been fixed by heating at 56°C for about 30 minutes and from which non-specific agglutinins, interfering proteins, had previously been removed by serial adsorption with an equal volume of pyruvic aldehyde stabilised cells.
  • normal serum from goats, horses or sheep may be used.
  • Sensitized cells may be stored at 4°C. Again alternatively, in place of normal rabbit serum, one could use any properly treated stabilisation agent including properly adsorbed, complement fixed serum proteins.
  • stabilisation agents include gelatin, dextrans, polyvinyl pyrrolidone, albumins and water soluble carboxymethyl celluloses.
  • the hCG used for sensitization of the stabilised cells may be of various grades of purity.
  • material which assays, biologically, at approximately 2,500 LU./mg may be employed.
  • more purified hCG preparations for example, a preparation assaying at approximately 10,000 LUjmg or 14,000-16,000 1,Ujmg may be utilized in the sensitization of the aforementioned stabilised red blood cells.
  • beta sub-unit of hCG may also be utilized as the antigen on the red blood cell. It is generally preferable to use the more purified hCG in the stabilised, sensitized erythrocyte compositions of this invention.
  • mammalian erythrocytes such as those from sheep.
  • these stabilised, sensitized erythrocytes may also be prepared from avian or reptilian red blood cells. which being nucleated settle more rapidly than mammalian erythrocytes. In this way the results from haemagglutination tests may be obtained within 30 minutes or sooner.
  • the speed of the haemagglutination reaction is proportional to the buoyant density of the nucleated cell; thus as the buoyant density increases, less time is required to obtain results.
  • the invention was illustrated using Alsever's solution as the isotonic sterile anticoagulant, the freshly drawn whole blood may be optionally mixed with heparin sodium EDTA, or sodium oxalate.
  • the described stabilised, sensitized erythrocyte compositions together with their antiserum compositions are most useful in haemagglutination and haemagglutination inhibition (passive haemagglutination) methods for determining the presence or absence of a particular antigen or antibody in the test fluid being examined, usually a body fluid such as urine or serum.
  • determining the presence or absence of a pregnant condition in a female animal can be based on a haemagglutination method for determining the presence of chorionic gonadotrophin which comprises mixing the erythrocyte composition of this invention with a chorionic gonadotrophin antiserum and with the urine of test subject and, following a suitable incubation period, visually observing the results, whereby if said gonadotrophin is not present agglutination of the erythrocytes occurs upon standing whereas if said gonadotrophin is present, no such agglutination occurs.
  • a method for determining the presence of luteinizing hormone in a woman and thereby the day of ovulation may also be done utilizing the compositions of this invention wherein a urine sample is concentrated by ultrafiltration and luteinizing hormone detected by immunological means, the antigen being human chorionic gonadotrophin, as described in U.S. Patent Specification No. 4,123,510.
  • pregnancy in a mare may be determined utilizing as the antigen pregnant mare's serum gonadotrophin and a composition utilizing carcino embryonic antigen would be useful in a blood test for cancer.
  • Other uses for the composition of this invention are described therein.
  • composition of the invention comprises a method for determining the presence or absence of human chorionic gonadotrophin and a human urine sample which comprises mixing the composition of the invention in which the antigen is human chorionic gonadotrophin and the erythrocytes are mammalian, avian or reptile pyruvic aldehyde stabilised erythrocytes with a highly purified hCG antiserum and with ultrafiltration concentrated human urine whereby if hCG is not present agglutination of the erythrocytes occurs upon standing whereas if hCG is present no such agglutination occurs.
  • compositions of this invention are due to the coupling of the stabilised erythrocytes to the hCG by means of a chemical reaction between the amino functions of the erythrocytes and the hCG and the difunctional glutaraldehyde.
  • the suggested course of reaction of glutaraldehyde is as follows:
  • an antiserum composition to be used in conjunction with the antigen sensitized erythrocytes previously described.
  • hCG which assays biologically as low as 2500 I.U./mg may be employed.
  • more purified hCG preparations that assay at approximately 10,000 I.U./mg, 15,000 I.U./mg or higher may be employed.
  • highly purified hCG and most preferred to use the 13- sub-unit of hCG in preparing the antiserum of this invention to obtain greater specificity.
  • An example of highly purified f3-hCG and a method of producing same is reported in our copending European application No. 0000103 filed concurrently herewith.
  • the antiserum compositions are generally prepared by immunizing a host animal with the desired antigen thereby producing antibodies to that antigen which may be obtained in the serum separated from the host animal.
  • a difficulty in the past has been that the hCG antigen has had other antigen impurities present, thus undesirable antibodies were also produced.
  • a haemagglutination method utilizing such an antiserum composition would thus possess undesirable cross reactivity.
  • the antisera composition of this invention is dilute diether in microtitre slides or in a tube system titration to determine the maximum dilution of antisera which can still agglutinate the red blood cell. The dilution is then adjusted when utilized in a method for determining pregnancy so that the anti-hCG serum has a sensitivity to hCG of about 100-150 m l.U./test.
  • the acceptable antisera will also have a cross reactivity of less than 25% particularly against other glycoprotein hormone antigens. Thus an antisera sensitive to 100 m ).U./test of hCG would at the same time have a sensitivity to 400 m LU./test or more of hLH or hMG.
  • compositions of this invention may be utilized in aqueous compositions, it is preferred to lyophilize these compositions for use in the particular haemagglutination method under consideration.
  • the compositions may be lyophilized into two separate pellets by methods known to those skilled in the art such as that described in U.S. Patent 3,862,302. That disclosure, however, does not teach how to make lyophilized compositions containing in either the antiserum or the stabilised erythrocytes enough buffer for complexing the interfering calcium ions present in urine. Rather it involves the use of a third lyophilized pellet containing the necessary buffers or as in a commercially available pregnancy test a separate buffer compositions which is reconstituted at the time of its use.
  • compositions of this invention may also be lyophilized into a single layered cake, as described in U.S. Patent 3,269,905.
  • a suitable chelating agent may be included within the lyophilized compositions of this invention to remove quantities of calcium present in a urine test sample
  • the compositions of this invention without added chelating agent may be utilized with urine and/or ultra- concentrated urine which has been passed through a filter in which is placed a styrene divinyl benzene copolymer containing imide acetate functional groups (CHELEX 100 or DOWEX CHELATING RESIN A-1).
  • the Rbc concentration or packed cell vlume (pcv) of such a mixture should be in the range of 15-25% (i.e. a hematocrit of 30-50%) and preferably, 15 to 25% or more preferably 22%.
  • the cells from 100 ml of such a mixture were collected by centrifugation and washed three times with normal, saline solution, i.e. 0.9% (w/v) sodium chloride in distilled water.
  • the preferred volume of saline per wash is 320 ml but may be in the range of 100-500 ml.
  • the cells are suspended in normal saline solution at a concentration of 20%.
  • a mixture of pyruvic aldehyde (64 ml of 25% aqueous solution) and normal saline (120 ml) was adjusted to pH 7.0 using a 10% w/v aqueous sodium carbonate solution.
  • To this mixture of pyruvic aldehyde and saline was added the above-mentioned cell suspension (100 ml at 20%), followed by phosphate buffer (28 ml., 0.15 M, pH 8.0). This stabilization mixture was then incubated at 37°C for 3 hours, the mixture being shaken vigorously once every 30 minutes.
  • the cells were then collected by centrifugation and washed 4 times with normal saline solution utilizing preferably 320 ml of saline per wash.
  • the stabilised cells may then be stored as a suspension, preferably 10%, in normal saline containing, for example, 0.1 % sodium azide as a preservative. This is stable for at least a year at +4°C.
  • Stabilized cells as described in Example 1 (10 ml of about a 10% suspension) were collected by centrifugation and washed 3 times with normal saline solution, preferably 40 ml for each wash. Alternatively, one may of course use unstored cells directly from Example 1. These washed cells were then suspended in a solution of hCG (100 ⁇ g in 100 p, 1 of 0.5 M phosphate buffer) contained in 0.15 M phosphate buffer (8.65 ml, pH 7.4). To this suspension was added a glutaraldehyde reagent (1.75 ml), prepared by diluting a 25% aqueous solution of glutaraldehyde (1 ml) in normal saline (9 ml).
  • the sensitization mixture was thoroughly agitated and then gently mixed for 2 hours at room temperature.
  • the cells were then collected by centrifugation and washed, preferably 4-5 times, with 0.15 M phosphate buffered saline (pH 7.4).
  • the sensitized cells are then suspended in 0.15 M phosphate buffered saline (39 ml, pH 7.4) containing 0.2% of normal rabbit serum in which complement had previously been fixed by incubating at 56°C for 30 minutes and from which non-specific agglutinins had been adsorbed by triple treatment with stabilised cells (Example 1).
  • the removal of non-specific agglutinins is illustrated in Example 3.
  • stabilised cells Prior to use for the removal of non-specific agglutinins from normal rabbit serum, stabilised cells as prepared in Example 1, were washed with normal saline (5 volumes saline: 1 volume of 10% cell suspension) and resuspended in saline solution at 10%. Equal volumes of rabbit serum and a 10% suspension of stabilised red blood cells were then incubated at room temperature for 30 minutes. The cells were collected by centrifugation, discarded and the process repeated 2 more times, using collected cells as opposed to a 10% suspension.
  • the last adsorption may be allowed to proceed at +4°C for 18 hours.
  • the normal rabbit serum has been diluted 1:1 with saline. It should be understood, however, that for this adsorption it is possible to utilize collected cells as opposed to 10% suspension which would not result in a dilution of the normal rabbit serum.
  • Example 2 Utilizing the methods of Example 2, but with pregnant mare's serum gonadotrophin as the antigen, a stable sensitized composition was also prepared and satisfactorily evaluated.
  • Lyophilized cakes were prepared as follows. A suspension of the stabilised, sensitized red blood cells (S-PAGC) was washed about 3 times with a 0.15 M phosphate buffer solution adjusted to a pH of 7.4. The washed S-PAGC were then resuspended as a 0.415% v/v suspension in lyophilization buffer, LB, (LB used was 10 g sucrose, 10 ml of 1% merthiolate, 10 ml of NRS triply adsorbed, and q.s. to 1 litre with 0.15 M phosphate buffered saline containing about 0.2% EDTA to a pH of 7.0).
  • LB lyophilization buffer
  • the frozen reagents may then be stored at about -100°C until lyophilization or lyophilized immediately. Lyophilization of these reagents was then done in a freeze drier for at least 18 hours at about 75-200 microns Hg, while gradually reached ambient temperatures. The vacuum was not permitted to be less than 75 microns to avoid spontaneous haemagglutination.
  • the lyophilized reagents when stored at 4°C are stable for at least 9 months.
  • Lyophilized pellets were prepared as follows:
  • the antisera pellets were prepared similarly but may optionally have incorporated within the LB 2.5% of polyvinyl pyrrolidone or dextran.
  • the pellets may be stored as the cakes are stored in Example 4 or transferred immediately to the freeze drier. Pellets have been dried satisfactorily at about 50-200 microns Hg for about 18 hours, with the temperature finally reaching room temperature or in some cases 35°C.
  • the pellets were stored in a dessicator at room temperature until packing into siliconized vials. These pellets have been found to maintain their sensitivity and physical characteristics in long term stability studies at both 30°C and 4°C.
  • Pretreatment of tubes with silicone is most beneficial as exemplified in Example 8 below.
  • the method used for pretreatment with silicone is described under e), whilst other pretreatments, used for comparative purposes, are described in a) to d).
  • the antiserum is separated from the rabbit blood and complement may be fixed and the suspension adsorbed as described with the S-PAGC composition in Example 2 and Example 3, a single adsorption usually being sufficient. Undiluted antisera or diluted antisera may be stored preferably at -100°C in small aliquots.

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Claims (12)

1. Immunologisches Mittel aus mit Pyruvaldehyd oder Dimethylsuberinsäureimidat stabilisierten Erythrocyten, die mit einem Polypeptid- oder Glocyproteinantigen, ausgewählt unter Gonadotrophinium chorionicum, Gonadotrophinium sericum trächtiger Stuten, Embroykarzinomantigen, luteinisierendem Hormon, follikelstimulierendem Hormon, menschlichem Menopausen-gonadotropin und schilddrüsenstimulierendem Hormon sensibilisiert sind, wobei die stabilisierten Erythrocyten an die genannten Antigene mit einem bifunktionellen Molekül, ausgewählt unter Glutaraldehyd, Glyoxal, Bernsteinsäurealdehyd, Hexamethylendiisocyanat, Toluol-2,4-diisocyanat, einem bifunktionellen Imidoester, Cyanurchlorid, tetrazotiertem o-Anisidin und einem wasserlöslichen Carbodiimid, gekuppelt sind, wobei jeweils Voraussetzung ist, daß wenn die Erythrocyten mit Dimethylsuberinsäureimidat stabilisiert sind, das bifunktionelle Molekül nicht Dimethylsuberinsäureimidat sein kann, und wenn die Erythrocyten mit Pyruvaldehyd stabilisert sind, das bifunktionelle Molekül Glutaraldehyd, Glyoxal, Bernsteinsäurealdehyd, Hexamethylen - diisocyanat, Toluol - 2,4 - diisocyanat oder ein bifunktioneller Imidoester ist.
2. Mittel nach Anspruch 1, worin das Polypeptid- oder Glycoprotein-Antigen menschliches Gonadotrophinium chorionicum ist.
3. Mittel nach Anspruche 1 oder 2, worin die Erythrocyten mit Pyruvaldehyd stabilisiert sind.
4. Mittel nach einem der Ansprüche 1 bis 3, worin das bifunktionelle Molekül Glutaraldehyd, Hexamethylendiisocyanat oder Dimethylsuberinsäure-imidat ist.
5. Mittel nach einem der Ansprüche 1 bis 4, wobei das Mittel lyophilisiert ist.
6. Mittel nach einem der Ansprüche 1 bis 5, worin die Antigen-sensibilisierten Erythrocyten mit phosphatgepufferter Kochsalzlösung, die ein Serumprotein enthält, in dem das Komplement zuvor fixiert wurde, und aus dem unspezifische Agglutinine vorher durch Reihenadsorption mit mit Pyruvaldehyd stabilisierten Erythrocyten entfernt wurden, behandelt sind.
7. Mittel nach einem der Ansprüche 1 bis 5, worin die Antigen-sensibilisierten Erythrocyten mit phosphatgepufferter Kochsalzlösung, enthaltend einen Bestandteil, ausgewählt unter Gelatine, Dextran, Polyvinylpyrrolidin und wasserlöslichen Carboxymethylcellulosen, die zuvor einer Adsorptionsbehandlung unter Verwendung von mit Pyruvaldehyd stabilisierten Erythrocyten unterworfen worden waren, behandelt sind.
8. Verfahren zur Herstellung immunologischer Mittel aus tierischen Erythrocyten, die mit Antigenen sensibilisiert sind und zum passiven Hämagglutinationstest brauchbar sind, wobei man:
a) das Tierblut direkt in einem isotonischen, sterilen Antikoagulans aufnimmt, so daß sich ein Endverhältnis von Blut zu Antikoagulans von 0,8:1 bis 1.2:1 in der erhaltenen Mischung ergibt, wobei die Erythrocytenkonzentration, bzw. das Volumen der gepackten Zellen des Blutes dieser Mischung 15 bis 25% beträgt;
b) die Erythrocyten durch Zentrifugieren aus dieser Mischung abtrennt;
c) die Erythrocyten mit normaler Kochsalzlösung wäscht und dann bei einer Konzentration von 10 bis 30% die Erythrocyten in normaler Kochsalzlösung suspendiert;
d) die Erythrocytensuspension mit einer Mischung von Pyruvaldehyd, neutraler gepufferter Lösung und normaler Kochsalzlösung mischt, um eine Stabilisierungsmischung zu erhalten;
e) Die Stabilisierungsmischung aus Stufe (d) bei 30 bis 55°C 1 bis 5 Stunden lang inkubiert und die stabilisierten Erythrocyten durch Zentrifugieren und anschließendes Washen mit normaler Kochsalzlösung sammelt;
f) die stabilisierten Erythrocyten in einer neutralen gepufferten Lösung eines Antigens, ausgewählt unter Gonadotrophinium chorionicum, Gonadotrophinium sericum trächtiger Stuten, Embryokarzinomantigen, luteinisierenden Hormonen, follikelstimulierenden Hormonen, menschlichem Menopausen-Gonadotrophinium und schilddrüsenstimulierenden Hormonen, suspendiert;
g) eine sensibilisierende Mischung herstellt, indem man die Suspension von stabilisiertem Erythrocyten-Antigen grundlich mit einer Lösung eines bifunktionellen Moleküls in Kochsalzlösung rührt, wobei das bifunktionelle Molekül ausgewäht ist unter Glutaraldehyd, Glyoxal, Bernsteinsäurealdehyd, Hexamethylen-diisocyanat, Toluol-2,4-diisocyanat und -einem bifunktionellen Imidoester;
h) die sensibilisierten Erythrocyten durch Zentrifugieren sammelt und anschließend mit neutraler, gepufferter Kochsalzlösung wäscht;
i) die sensibilisierten Erythrocyten in einer neutralen gepufferten Kochsalzlösung suspendiert, welche normales Tierserum enthält worin das Komplement dieses Serums zuvor fixiert worden war, wobei die Erythrocyten von Sägetieren, Vögeln oder Reptilien stammen.
9. Verfahren nach Anspruch 8 zur Herstellung eines Mittels wie in Anspruch 3 beansprucht, wobei das Antigen menschliches Gonadotrophinium chorionicum ist.
10. Verfahren nach Anspruch 8 oder 9, wobei das Mittel lyophilisert ist.
11. Verfahren zur Bestimmung der An- oder Abwesenheit von menschlichem Gonadotrophinium chorionicum in einer Urinprobe von Menschen, dadurch gekennzeichnet, daß man das Mittel nach Anspruch 3, worin das Antigen menschliches Gonadotrophinium chorionicum ist und die Erythrocyten von Säugetieren, Vögeln oder Reptilien stammen, mit einem hochreinen menschlichen Gonadotrophinium chorionicum Antiserum (hCG) und mit ultrafiltriertem konzentriertem menschlichen Urin mischt, wobei wenn kein menschliches Gonadotrophinium chorionicum anwesend ist, die Erythrocyten beim Stehen agglutinieren, während wenn menschliches Gonadotrophinium chorionicum anwesend ist, keine derartige Agglutination eintritt.
12. Antiserummittel für menschliches Gonadotrophinium chorionicum zur Verwendung in einem Verfahren nach Anspruch 11, welches hergestellt wurde unter Verwendung eines stark gereinigten menschlichen Gonadotrophinium chorionicum als immunisierendem Antigen, wobei die "cross-reactivity" dieses Mittels gegenüber anderen Glycoproteinhormon-Antigenen geringer als 25% ist.
EP78300034A 1977-06-14 1978-06-12 Immunologische Zusammensetzungen, Methoden zur Herstellung derselben und Methoden zur Durchführung von Hämagglutinationstesten Expired EP0000102B1 (de)

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JPS5665044A (en) * 1979-10-31 1981-06-02 Dainippon Ink & Chem Inc Carbon fiber-reinforced resin composition
JPS56135549A (en) * 1980-03-26 1981-10-23 Dainippon Ink & Chem Inc Polyarylene sulfide resin composition
EP0039195B1 (de) * 1980-04-28 1986-06-18 Montefiore Hospital and Medical Center Verfahren zur Feststellung von Antikörpern
US4543339A (en) * 1982-03-16 1985-09-24 Oneill Christopher Early pregnancy detection by detecting enhanced blood platelet activation
GB2138132B (en) * 1983-04-12 1987-02-18 Robin Royston Amos Coombs Storage-stable antibody-linked erythrocytes
EP0134868A1 (de) * 1983-08-26 1985-03-27 Anda Biologicals Glutaraldehyd-aktivierte einzellige Organismen als feste Träger für Sensibilisierungsmittel und Gebrauch von sensibilisierten einzelligen Organismen
DE3428984A1 (de) * 1984-08-07 1986-02-20 Bayer Ag, 5090 Leverkusen Verfahren zur herstellung von hochmolekularen, gegebenenfalls verzweigten polyarylensulfiden
CA1239346A (en) * 1985-06-04 1988-07-19 Gursaran P. Talwar Birth control vaccine
US5494800A (en) * 1987-01-29 1996-02-27 Cytosignet, Inc. Analyte detection in particulate-containing samples
US4900685A (en) * 1987-01-29 1990-02-13 Cytosignet, Inc. Analyte detection in particulate-containing samples
JPH01107154A (ja) * 1987-10-20 1989-04-25 Seitetsu Kagaku Co Ltd 加工赤血球およびその製造方法
US4910294A (en) * 1988-06-20 1990-03-20 Idemitsu Petrochemical Company Limited Two-stage process for production of polyarylene sulfides with lithium compound
US11722295B2 (en) 2020-04-30 2023-08-08 Musarubra Us Llc Methods, apparatus, and articles of manufacture to securely audit communications

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3639558A (en) * 1968-02-19 1972-02-01 Louis Csizmas Immunological reagent particles having proteinaceous materials covalently bonded thereto
US3991174A (en) * 1970-07-20 1976-11-09 Rafa Laboratories Ltd. Method of determining concentration of luteinizing hormone in body fluid
FR2187909A1 (en) * 1972-06-08 1974-01-18 Merieux Inst Stable antigens - by freeze-drying antigen bonded to red blood cells
US3914400A (en) * 1973-05-21 1975-10-21 Us Health Stable antigen-erythrocytes for measuring antibodies against toxoplasma organism

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CA1108048A (en) 1981-09-01
IT1097807B (it) 1985-08-31
EP0000102A1 (de) 1978-12-20
GB1589107A (en) 1981-05-07
DE2861050D1 (en) 1981-11-26
JPS548719A (en) 1979-01-23
IT7824572A0 (it) 1978-06-14

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