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
  • G01N33/74—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
• • 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
• • 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

• ABSTRACT OF THE DISCLOSURE A novel method for the immunochemical determination of antigens or antibodies in a liquid sample in which they are present in low concentration or are attended with disturbing factors, comprising the steps of adsorbing one of the immunochemical reaction components on a carrier, reacting said component with the liquid to be tested, containing the other reaction component to be determined or, in case of an agglutination inhibition reaction, reacting said component with a solution of the other component and the test liquid, separating the carrier from the reaction mixture, suspending the carrier in a suitable liquid medium, and reading the sedimentation pattern of the carrier.
• Another example relates to the determination of the rheuma factor by the method of J. M. Singer and C. M. Plotz (Am. J. Med. 21 (1950) 888).
• 0.5 ml. of a suspension of polystyrene latex particles (diameter 0.8 micron) sensitized with human 'y-globulin is mixed with an equal volume of testserum in a dilute state, if desired.
• the tube with the reaction mixture is centrifuged for a certain time at a certain number of revolutions. Then the result is read from the turbidity of the supernatant liquid.
• the last example is the detection or estimation of HCG in urine by means of latex particles to which the hormone is adsorbed as described in Netherlands patent ice application No. 6504823, filed Apr. 15, 1965.
• this test system one drop of fluid to be tested, one drop of diluted antiserum to HCG, and one drop of a suspension of the sensitized latex are mixed on a glass slide. The result can be read after a few minutes of rocking the slide.
• antigens or antibodies which are present in the liquid to be tested in low concentration, or are attended with disturbing factors can be detected and estimated immunochemically without prior concentration or fractionation by an agglutination or agglutination inhibition reaction, comprising the steps of adsorbing one of the immunochemical reaction components on a carrier, reacting said component or both components of the immunochemical reaction and the liquid to be tested, separating the carrier from the reaction mixture, suspending the carrier in a suitable liquid medium, and reading the sedimentation pattern of the carrier thus separated from the other components of the reaction mixture.
• the method of the invention can be used both in the immunochemical determinations based on an agglutination reaction between antigen and antibody and in those based on an agglutination inhibition reaction, in which the component of the test liquid to be determined can inhibit the agglutination.
• the contaminants of the test liquid which can disturb the sedimentation pattern, are removed from the carrier particles prior to the reading of said pattern.
• the advantage of this procedure is illustrated by the following example.
• For the determination of HCG in serum of a pregnant woman 0.025 ml. of serum are mixed with 6 ml. of a suitable buffer. To this mixture are added 0.2 ml. of dilute antiserum and 0.3 ml. of a suspension of sensitized erythrocytes. After incubation of the reaction mixture the erythrocytes are isolated again and treated in a similar manner as described before.
• the sensitivity of the method according to the invention can be raised by inserting in the procedure an incubation of test fluid and antiserum solution prior to the addition of the sensitized carrier particles.
• An incubation overnight at refrigerator temperature, or 2 hours at room temperature is appropriate.
• pre-incubation the sensitivity can be raised by at least a factor two.
• the carrier may be separated from the reaction mixture in any conventional manner, for example, by filtration, but centrifugation is preferred as this process allows rapid and complete separation.
• the resulting carrier may be observed directly, for example, after it has been transferred to a slide, to determine whether agglutination has taken place, or resuspended in a small volume of liquid and allowed to settle in a round-bottomed tube, after which the result of the reaction can be read from the sedimentation pattern.
• erythrocytes and latex particles For application of the invention use can be made of all kinds of known carriers, especially erythrocytes and latex particles.
• the method is suitable for the demonstration of antibodies against various antigens, for example, antibodies against pathogenic bacteria which may be found in urine in low concentrations. It is also possible now to demonstrate antigens and antibodies occurring in serum in concentrations to that low degree that it has been impossible so far to demonstrate them in dilute sera, while dilution is necessary to prevent disturbance in agglutination.
• a test pack according to the invention comprises at least a test tube having a round transparent bottom, containing said antibodies or antigens or a mixture thereof, one of said components for the immunochemical determination being adsorbed on a carrier, and adapted to receive the liquid sample, to centrifuge the reaction mixture, and to observe the sedimentation pattern.
• a further simplification is obtained by lyophilizing antiserum andsensitized erythrocytes in the said tubes in a suitable quanity andconcentration.
• antiserum and sensitized erythrocytes can be put together in a tube by lyophilizing them separately. In the latter case only the urine to be tested has to be put in the tube before performing the test.
• the removable cap referred to above should protect the contents from moisture to ensure a satisfactory stability.
• This buffer is used for the preparation of a series of dilutions of HCG.
• 0.2 ml. of antiserum to HCG diluted to the desired strength with a citrate buffer of pH 6.5 containing 9 gm. of NaCl and 40 gm. of sodium citrate .2H O and 0.1% bovine serum albumin per litre, and 0.2 ml. of a 1.75% (v./v.) suspension of erythrocytes sensitized with HCG in the phosphate buffer. After incubating the mixture for 30 minutes at 37 C.
• HGH human growth hormone
• the activity of the examined growth hormone preparation was 1.5 u. per mg. It appeared that HGH could still be demonstrated and determined in a concentration of 15 mu. or 10 ugm. per litre.
• EXAMPLE 3 Determination of luteinizing hormone (LH) in urine from a woman with a normal menstrual cycle Of the urine to be examined the following series of dilutions is prepared: undilute, 1:2, 1:4, 1:8 and 1:16. The dilutions are prepared with a phosphate buffer in accordance with Example 1 containing 0.1% bovine serum albumin. Furthermore, a standard series is prepared of the Second International Reference Preparation for human menopausal gonadotripin (2nd IRP-HMG) in the same buffer in the concentrations 0, 12, 18, 25 and 35 International Units of LH per litre. Six ml. of test liquid with 0.2 ml.
• the LH-concentration of the urine is expressed in I.U. of LH per litre.
• LH can be determined in concentrations 25 I.U. of LH per litre.
• EXAMPLE 4 Determination of luteinizing hormone (LH) in urine from sterile patients
• the sensitivity of the estimation described in Example 3 can be raised by an incubation of the test fluid with antiserum solution prior to the addition of the suspension of sensitized erythrocytes. This so -called pre-incubation can be done overnight between 0 and C. The rest of the procedure is identical to that of Example 3.
• LH can be determined in concentrations greater than or equal to 10 LU. per litre taking the 2nd TRPHMG (see Example 3) as a standard. This method, with this level of sensitivity, can be used as a diagnostic tool to decide whether a case of sterility is of hypophyseal or of ovarian origin.
• HSA human serum albumin
• Example 1 After the addition of these erythrocytes the procedure is as described in Example 1.
• the attainable sensitivity in this set-up is 2.5 g per litre.
• the volume of the test fluid e.g. from 4 ml. of 8 ml. the sensitivity is raised accordingly.
• EXAMPLE 6 Determination of human chorionic gonadotropin (HCG) in serum To the serum from a sound woman are added 0, 1, 2, 3, and 6 LU. of HCG per ml. The serum is diluted 240x with phosphate buffer of Example. To 6 ml. of dilute serum are added 0.2 ml. of a solution of antiserum in citrate buffer (see Example 1) and 0.3 ml. of a suspension of 3.5% (v./v.) erythrocytes. After incubating the mixture for 30 minutes at 37 C. it is centrifuged, after which the erythrocytes are washed with phosphate buffer and finally taken up in the buffer to obtain a volume of 1.5 ml. Of this suspension 0.5 ml. is transferred to a round-bottomed ampoule. After 2 hours the reaction is read. By this method 3 I.U. HCG per ml. of serum can be detected.
• HCG human chorionic gonadotropin
• EXAMPLE 7 Application of a special test tube The determination described in Examples 1 to 4 inclusive are performed in a tube made of glass or synthetic material, with a perfectly regular, round transparent bottom, an inner diameter of 11 mm. and a height of 10 or 11 cm., which is strong enough to stand centrifugal forces and has such a shape as to allow of its being closed easily with a removable cap, so that it is not necessary to transfer the washed and resuspended erythrocytes.
• EXAMPLE 8 Application of a special test tube containing prelyophilized reagents Antiserum to purified luteinizing hormone prepared from human pituitaries diluted to the desired strength with the phosphate buffer of Example 1 is lyophilized in the test tubes described in Example 7 which are closed with a re movable cap in such a manner that no moisture can reach in the contents.
• the sensitized erythrocytes are lyophilized in a separate flask. Before the test the lyophilized erythrocycles are resuspended.
• the test liquid or a dilution thereof is mixed with lyophilized antiserum. To this mixture the desired quantity of sensitized erythrocytes is added, after which the test is performed further as described above.
• EXAMPLE 9 Determination of human chorionic gonadotropin (HCG) by means of a latex aggluatination inhibition reaction
• HCG human chorionic gonadotropin
• Bacto-latex of Difco Laboratories, Detroit, Mich. is sensitized with HCG as described in Example 1 of Netherlands patent application No. 6504823, filed Apr. 15, 1965.
• Method for the immunochemical determination, by means of an agglutination reaction, of antigens or antibodies in an aqueous solution in which they are present in a concentration too low to be reliably determined by conventional agglutination reaction procedure without prior fractionation or concentration or are attended with factors disturbing to conventional agglutination reactions arising from contaminants present in the aqueous solution to be tested, comprising (a) adsorbing one of the immunochemical reaction components selected from the group consisting of antigens and antibodies on carrier particles suitable for antigens and antibodies;
• Method for the immunochemical determination, by means of an agglutination inhibition reaction, of antigens or antibodies in an aqueous solution in which they are present in a concentration too low to be reliably determined by conventional agglutination inhibition reaction procedure without prior fractionation or concentration or are attended with factors disturbing to conventional agglutination inhibition reactions arising from contaminants present in the aqueous solutions to be tested, comprising (a) adsorbing one of the immunochemical reaction components selected from the group consisting of antigens and antibodies on carrier particles suitable for antigens and antibodies;

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

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Cited By (12)

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• 0
  • NL NL130516D patent/NL130516C/xx active
• 1967
  • 1967-10-20 US US676726A patent/US3565987A/en not_active Expired - Lifetime
  • 1967-10-25 CA CA003389A patent/CA937498A/en not_active Expired
  • 1967-10-30 GB GB49118/67A patent/GB1210819A/en not_active Expired
  • 1967-11-01 FI FI672944A patent/FI48510C/fi active
  • 1967-11-06 CH CH1548367A patent/CH512071A/de not_active IP Right Cessation
  • 1967-11-07 SE SE15220/67A patent/SE337494B/xx unknown
  • 1967-11-07 ES ES346848A patent/ES346848A1/es not_active Expired
  • 1967-11-08 DK DK556467AA patent/DK127200B/da unknown
  • 1967-11-08 DE DE1648999A patent/DE1648999C3/de not_active Expired
  • 1967-11-08 BE BE706213D patent/BE706213A/xx unknown
  • 1967-11-08 JP JP42071469A patent/JPS4937241B1/ja active Pending

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