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
  • G01N33/543—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
• • 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
  • G01N33/76—Human chorionic gonadotropin including luteinising hormone, follicle stimulating hormone, thyroid stimulating hormone or their receptors
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
  • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10S436/00—Chemistry: analytical and immunological testing
  • Y10S436/815—Test for named compound or class of compounds
  • Y10S436/817—Steroids or hormones
  • Y10S436/818—Human chorionic gonadotropin

Definitions

• erythrocytes from mammals such as cattle, horses, sheep, rabbits, as well as chickens and from human beings, can be used as an auxiliary in immunochemical determinations based on the reaction of an antigen with its antibody.
• the erythrocytes are applied, for instance, as carrier of antigens, for instance, human chorionic gonadotropin (HCG), serum gonadotropin, growth hormone, and further serum albumin, gamma globulins, insulin, luteinizing hormone and tetanus toxoid.
• HCG human chorionic gonadotropin
• serum gonadotropin serum gonadotropin
• growth hormone growth hormone
• serum albumin serum albumin
• gamma globulins insulin, luteinizing hormone and tetanus toxoid.
• tanning agents or mordants to improve the adsorptive properties of the erythrocytes is described in the literature: inulin, tannin, hydroquinone, bis-diazobenzidine, Formalin, acetaldehyde, pyruvic acid aldehyde and glyoxal. Also successive treatment of the erythrocytes with different tanning agents and mordants has already been applied, for example, a pre-treatment with Formalin, followed by reaction with tannin.
• erythrocytes sensitised after the pre-treatment have been stored as an aqueous suspension, but preferably in a freeze-dried form.
• erythrocytes are very unstable particles and the preservation of these carriers has already required much study.
• the erythrocytes pretreated with a mordant are less unstable and can be readily freeze-dried, while in a freeze-dried state they are stable for a pretty long time, There exists a need of a presentation form, however, in which the erythrocytes can be well handled and are at the same time stable and have kept all their properties essential for the performance of immunochemical determination methods.
• HCG human chorionic gonadotrophin
• Erythrocytes treated according to the invention have become strong enough to be compressed to, say, tablets.
• the conventional auxiliaries can be applied, which must not be aggressive against erythrocytes and the antigen attached to them. It stands to reason that the bond between the antigen and the erythrocytes must not be affected adversely by the auxiliaries used and that they must not have a perceptible influence on the determinations for which the erythrocytes are intended, so that the sensibility, the accuracy and the velocity of the determination reaction is not essentially changed.
• erythrocytes treated according to the invention do not only have greatly improved mechanical properties, but also a greater sensibility to agglutination with antiserum.
• complete agglutination could even have been obtained with a nontreated suspension of erythrocytes sensitised with HCG with a dilution of antiserum of while after treatment in accordance with the invention the same suspension caused the same reaction with a dilution of antiserum of from /7500 to A5000-
• the invention is not limited to application in the sensitisation of erythrocytes with HCG; it can also be applied in the sensitisation with any antigen that can stand up to the present treatment with formaldehyde.
• both the powder of sensitised erythrocytes obtained, for example, after freeze-drying or spray'drying of the suspension, can be applied and the tablets compressed therefrom.
• the powders obtained after freeze-drying of suspensions of erythrocytes are very hygroscopic, they are mixed and compressed with the other auxiliaries, preferably in an atmosphere with a slight relative degree of moisture, preferably to about It is advantageous to choose such ingredients and apply such conditions in compressing that the disintegration time of the tablets is short, for example, less than 3 minutes.
• auxiliaries to be applied are amongst others usable; saccharose, manitol, lactose and ureum; as lubricants for example: boric acid, starch, Carboway 4000 (polyethylene glycol) and magnesium stearate, as disintegration agents for example: starch and alginic acid.
• ingredients are for instance substances used in the said determination, such as phosphates, for example, monopotassium phosphate and disodium phosphate, serving as buffers for the suspension prepared from the tablets, further sodium chloride and a chelating agent, such as the disodium salt of ethylene-diamine tetra acetic acid (E.D.T.A.) and albumin.
• phosphates for example, monopotassium phosphate and disodium phosphate
• a chelating agent such as the disodium salt of ethylene-diamine tetra acetic acid (E.D.T.A.) and albumin.
• the required ingredients can be mixed in a dry condition with the treated erythrocytes before tabletting, but it is more advantageous to dissolve the soluble ingredients together and to dry them by spray-drying, followed by mixing with the treated erythrocytes to obtain a very homogeneous mixture and to prevent the erythrocytes from being damaged during the further treatment by coarse crystalline components of the auxiliaries.
• insoluble auxiliaries In the processing of insoluble auxiliaries special attention should be paid that they do not disturb the determinations performed with a suspension obtained from the tablets. For the insoluble components will be able easily to change the sedimentation pattern of the erythrocytes, thus influencing the accuracy of the observation unfavourably.
• EXAMPLE 1 A suspension of sheep erythrocytes treated in a known manner with Formalin and tannin was centrifuged and washed with a phosphate sodium chloride buffer of pH 6.4 and next incorporated in this buffer. To this mixture an equal volume was added of a solution of 50 I.U. HCG per ml. in a buffer of pH 6.4. This mixture was stored for 48 hours at 37 C., whereupon formaldehyde was added till a final concentration had been obtained of 0.25% (weight/volume) formaldehyde. Next this mixture was stored for 15 hours at a temperature of 37 C., whereupon the erythrocytes were again centrifuged, washed with a physiological salt solution, incorporated as 10% v./v. suspension in a physiological salt solution containing 0.1% of bovine serum albumin, and stored for a few days at 04 C.
• the powder is a good reagent on HCG.
• EXAMPLE II 300 ml. of the suspension of erythrocytes of pH 6.4 according to Example I were treated with 300 ml. of a solution of HCG with 50 I.U. HCG per ml. at pH 7.6 and next with formaldehyde in a final concentration of 0.5%. After freeze-drying the powder was tabletted. The tablets obtained gave excellent results as a reagent in immunochemical pregnancy tests.
• EXAMPLE III By the method of Example I erythrocytes were sensitised with HCG in the presence of formaldehyde in a final concentration of 0.25%, and that for the last 24 hours at 30 C. After freeze-drying and compression tablets were obtained which gave excellent results as a reagent in an HCG test.
• EXAMPLE IV Experiments were performed with treatment with formaldehyde for the last 15 hours of the sensitisation of erythrocytes with HCG in accordance with Example I, but this time at pH 5.5.
• the suspension was dried in a Niro spray-drier with an inlet temperature of C. and an outlet temperature of 75 C.
• the resulting powder was next mixed with 0.4% (weight-weight) potato starch and compressed to tablets of about 15 mg. They gave excellent results as a reagent in immunochemical pregnancy determinations.
• sensitized erythrocytes are admixed with an auxiliary for tableting selected from the group consisting of saccharose, mannitol, magnesium stearate, and alginic acid, and then compressed into tablet form.
• an auxiliary for tableting selected from the group consisting of saccharose, mannitol, magnesium stearate, and alginic acid

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• Health & Medical Sciences (AREA)
• Life Sciences & Earth Sciences (AREA)
• Engineering & Computer Science (AREA)
• Immunology (AREA)
• Molecular Biology (AREA)
• Hematology (AREA)
• Cell Biology (AREA)
• Biomedical Technology (AREA)
• Urology & Nephrology (AREA)
• Chemical & Material Sciences (AREA)
• Microbiology (AREA)
• Analytical Chemistry (AREA)
• Biochemistry (AREA)
• Pathology (AREA)
• Food Science & Technology (AREA)
• Medicinal Chemistry (AREA)
• Physics & Mathematics (AREA)
• Biotechnology (AREA)
• Endocrinology (AREA)
• General Health & Medical Sciences (AREA)
• General Physics & Mathematics (AREA)
• Reproductive Health (AREA)
• Mycology (AREA)
• Medicinal Preparation (AREA)
• Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
• Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
• Medicines Containing Material From Animals Or Micro-Organisms (AREA)

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Cited By (10)

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Citations (2)

• 0
  • NL NL126572D patent/NL126572C/xx active
• 1966
  • 1966-03-24 NL NL6603909A patent/NL6603909A/xx unknown
• 1967
  • 1967-03-12 IL IL27578A patent/IL27578A/xx unknown
  • 1967-03-17 US US623825A patent/US3548051A/en not_active Expired - Lifetime
  • 1967-03-18 ES ES338201A patent/ES338201A1/es not_active Expired
  • 1967-03-20 DE DE19671617672 patent/DE1617672A1/de active Pending
  • 1967-03-21 CH CH412467A patent/CH508390A/de not_active IP Right Cessation
  • 1967-03-22 GR GR670136578A patent/GR36578B/el unknown
  • 1967-03-22 DK DK155467AA patent/DK126452B/da unknown
  • 1967-03-22 GB GB03515/67A patent/GB1179131A/en not_active Expired
  • 1967-03-23 SE SE04108/67A patent/SE335592B/xx unknown
  • 1967-03-23 AT AT284467A patent/AT270864B/de active
  • 1967-03-24 BE BE696050D patent/BE696050A/xx unknown

Patent Citations (2)

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