USH1018H - Immunological method for the determination of free substances having hapten properties - Google Patents

Immunological method for the determination of free substances having hapten properties Download PDF

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Publication number
USH1018H
USH1018H US07/232,251 US23225188A USH1018H US H1018 H USH1018 H US H1018H US 23225188 A US23225188 A US 23225188A US H1018 H USH1018 H US H1018H
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substance
antibody
labelled antibody
affinity
conjugate
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US07/232,251
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Uwe Hantke
Rudy Thoma
Hartmut Rokos
Andre Gadow
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Henning Berlin GmbH Chemie- und Pharmawerk
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Henning Berlin GmbH Chemie- und Pharmawerk
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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
    • G01N33/78—Thyroid gland hormones, e.g. T3, T4, TBH, TBG or their receptors
    • 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
    • G01N33/54306—Solid-phase reaction mechanisms

Definitions

  • the invention relates to an immunological method for the determination of free substances having hapten properties, especially hormones, steroids, drugs or metabolites thereof, vitamins or toxins, in biological fluids in the presence of one or more physiological binding partners for the substances which are to be determined.
  • the total quantity of substances of this type in the biological fluid which is to be investigated is distributed over a free and a bound fraction.
  • the bound fraction is bound to one or more physiological binding proteins or similar binding partners for these substances which are capable of binding the particular substance more or less specifically with a defined affinity.
  • the bound and non-bound fractions are in mutual equilibrium, it being assumed on the basis of the theories currently valid that the non-bound fractions, that is to say the free substances, represent the physiologically active component, whereas the bound fractions form a type of reservoir for making this substance available.
  • the bound fractions form a type of reservoir for making this substance available.
  • transport proteins which act to distribute the substances in the organism and transport them to the site of action.
  • the binding proteins over which the T 4 circulating in the living human organism is distributed, are albumin (about 10%), thyroxine-binding prealbumin (TBPA, about 30%) and thyroxine-binding globulin (TBG, about 60%). Only 0.01 to 0.03% is in the form of physiologically active free T 4 (FT 4 ). This means that the normal range of concentration of FT 4 is from about 8 to 20 pg/ml of body fluid.
  • the recognized reference method for the determination of FT 4 is at present the equilibrium dialysis of serum (compare Clin. Invest. 45 (1966), pages 153 to 163). This entails radioactive T 4 being added to the serum, or the T 4 content in the dialysate being found directly in a T 4 radioimmunoassay. However, despite its high reliability, this method is unsuitable for routine clinical diagnosis because of the unacceptably long time needed for each determination, approximately 20 h.
  • immunological determination methods which can give direct information about the concentration of the free substances which are to be determined.
  • the immunological determination methods of this type which have hitherto been developed to the stage of practical application and which are, in particular, radioimmunoassays, operate on the known competition principle in which a labelled form of the substance which is to be determined is added to the sample which is to be investigated, and subsequently, after reaction with a suitable antibody, conclusions are drawn about the concentration of the substance which is to be determined from the fraction of the bound form established on the basis of the antibody labelling.
  • T 4 -analogue tracers are used as labelled forms of the substance which is to be determined (FT 4 ).
  • These tracers have by reason of their chemical structure, a distinctly reduced affinity for the binding proteins and thus are said to have a minimal effect on the free substance/bound substance equilibrium.
  • various authors have cast doubt on the clinical value and validity of these methods, at least for certain specific cases (Helenius, T., Liewendahl, K., Clin. Chem. 29(5) (1983), pages 816-822 Mardell, R., Gamlen, T.
  • the biological fluid is reacted with a defined quantity of a specific antibody against the substance which is to be determined, or of an antibody mixture, with a detectable tracer portion and, furthermore, with an excess of the substance which is to be determined, or of a derivative of this substance, in a form immobilized by binding to a solid phase, with the solid phase subsequently being separated from the liquid phase, and the content of labelled antibody in the liquid and/or solid phase being established by measuring the label, and then the content of the free substances which are to be determined in the biological sample being found by computational evaluation of the results of measurement obtained.
  • German Offenlegungsschrift 3,442,817 describes a modification of the principle of the method, which has just been described, for the quantitative determination of FT 4 , in which the sample is first incubated for not more than 10 min and in a 10- to 2000-fold excess, based on the total molar quantity of T 4 , with a labelled anti-T 4 antibody. Immediately thereafter excess immobilized T 4 is added, and renewed incubation is carried out for at least 1 min. The phases are then separated, and the label in one of the phases is measured.
  • German Offenlegungsschrift 3,442,817 is a typical "kinetic" method which is evidently based on the assumption that, during the first brief incubation with the labelled anti-T 4 antibody, initially only the quantities of T 4 which are present in the free form are captured, with there being no release, due to slow return to equilibrium, of previously bound T 4 , which would falsify the results of measurement. Since the duration of the preincubation may affect the result of the assay due to the known rapid attainment of equilibrium between free T 4 and bound T 4 , this assay ought, like all similar two-stage assays, to have the disadvantage that it is prone to interference from changes in the assay conditions, which makes it difficult to use the relevant method in clinical practice.
  • the object of this invention is to provide a method for the determination of free substances which is suitable for routine clinical diagnosis, is straightforward to carry out is relevant in terms of the clinical information, yielded has optimal sensitivity and furthermore allows a manufacturer to carry out optimal quality control of the materials and substances required for the determination method.
  • the present invention is based on the surprising recognition that, for the development of an immunological determination method based on a basic method which is known per se, as has been described by, for example, Ekins (loc. cit.), it is necessary for the antigens bound to the solid phase to have a greatly reduced cross-reactivity of distinctly less than 50%, preferably in the range from 8 to 25%, compared with the free antigen which is to be determined, if a determination method which has sufficiently high sensitivity and reproducibility for practical purposes is to be obtained. It has emerged, surprisingly, that higher cross-reactivities, in the region of or above the 100% cross-reactivity proposed by Ekins (loc.
  • the method according to the invention should be suitable generally for the determination of free substances having hapten properties, especially of hormones, steroids, drugs or metabolites thereof, vitamins or toxins, in biological fluids, it is particularly important in connection with the determination of FT 4 and FT 3 .
  • the method according to the invention has all the known advantages of determination methods using a reactant immobilized by binding to a solid phase.
  • the immobilization on a solid phase markedly simplifies the washing steps which are necessary, and the precision of the determination is improved.
  • Suitable solid phases to which is bound the immobilized form of the substance which is to be determined are all inert carrier materials which are known per se and which have sufficiently stable binding properties and an adequately high binding capacity, which include plastics such as polystyrene, polyethylene and Teflon, Suitable solid phases are also described in U.S. Pat. No. 657,873 and in a publication by Wood, W. G. and Gadow, A. in: J. Clin. Chem. Clin. Biochem. 21 (1983), pages 789-797.
  • the antibodies used are labelled by known methods using detectable tracer portions suitable for labelling. Labelling with relatively small markers which have relatively little effect on the reactivity of the antibodies is preferred within the scope of the present invention. These are, in particular, radioisotopes, especially iodine isotopes, and luminogens. However, assuming that the tracer portions allow the required cross-reactivities with respect to a suitable antibody to be maintained, also suitable as markers are, for example, enzymes, substrates, fluorescent labels, phosphorescent labels, biotin (detectable via labelled avidin) or cofactors. It is also possible to use indirect labelling methods in which the marker is detached again from the antibody before the measurement, as well as all substances which can be detected quantitatively on the basis of an optical, physical or chemical reaction.
  • Suitable antibodies for the method according to the invention are all antibodies known to be suitable for methods of this type.
  • Antibodies of this type preferably have an affinity for T 4 and T 3 which is no greater than the respective affinity of T 4 and T 3 for the physiological binding proteins.
  • Anti-T 4 (or T 3 ) anti-bodies of this type normally have an affinity constant of 10 10 l/mol or less.
  • the immobilized substance or its derivative is preferably immobilized on the solid phase in the form of a conjugate with a carrier substance.
  • Suitable carrier components for this are high molecular weight substances such as proteins, polypeptides or polysaccharides, towards which the labelled antibody and the labelled antibody mixture have a cross-reactivity of less than 0.5%.
  • Suitable and preferred carrier components are those which are not identical to the carrier component to which the substance to be determined was bound for the production of the antibodies used.
  • the incubation conditions for carrying out the method according to the invention depend, within certain limits, on the particular antibodies used, including consideration of the effects on their binding properties by the label used, and on the exact cross-reactivities within the range established by the present invention.
  • Suitable incubation conditions comprise incubation temperature of 17° to 37° C. and incubation times of 30 min to 3 h.
  • Preferred incubation conditions are the incubation conditions used in the example, which provide for incubation at 22° C. for two hours ( ⁇ 10 min), with shaking, preferably in a horizontal shaker.
  • Thyroxine ethyl ester was prepared by the modified method of Clayton, J. C. and Hems, B. A., J. Org. Chem., 1950, pp. 840-843.
  • reaction mixture was then allowed to warm to room temperature. 200 ml of water were added to the reaction mixture, and the resulting solution was saturated with sodium chloride. The organic phase was separated off, washed with a saturated sodium chloride solution and dried over dry magnesium sulphate. The dried phase was filtered and evaporated to dryness. The pure residue was used without further purification for the subsequent syntheses.
  • the conjugate was prepared by the active ester method.
  • the active ester mixture was used without further purification for preparing the conjugate.
  • rabbit IgG 100 mg of rabbit IgG (SIGMA, Kunststoff) were dissolved in 20 ml of water. 200 ⁇ l of the active ester were diluted in 800 ⁇ l of dry and amine-free DMF and added to the aqueous solution of rabbit IgG. After about 12 h, the reaction mixture was purified by ultrafiltration.
  • the L-T 4 OEt incorporation rate determined by UV spectroscopy was 4.2 per mole of IgG.
  • Conjugate 2 IgG-L-T 4 conjugate (coupling at NH 2 and COOH)
  • the conjugate was prepared by the carbodiimide method.
  • the L-T 4 incorporation rate determined by UV spectroscopy was 4.3 per mole of IgG.
  • the conjugate was prepared by the carbodiimide method.
  • the L-T 4 incorporation rate determined by UV spectroscopy was 11.5 per mole of IgG.
  • Conjugate 4 IgG-L-T 4 OEt conjugate (coupling at NH 2 )
  • the conjugate was prepared by the carbodiimide method.
  • the L-T 4 OEt incorporation rate determined by UV spectroscopy was 1.5 per mole of IgG.
  • Conjugate 5 L-T 4 -IgG conjugate (coupling at NH 2 and COOH)
  • the conjugate was prepared by the carbodiimide method.
  • the L-T 4 incorporation rate determined by UV spectroscopy was 3.3 per mole of IgG.
  • the poly- or monoclonal antibodies, or mixtures of these antibodies, which were used were covalently bonded to microparticles which contained epoxy groups and had a uniform particle size of 1.055 ⁇ 0.032 ⁇ m.
  • the amount the antibodies/antibody mixtures coupled in each case was 350 ⁇ g of purified antibodies or antibody mixture per gramme of microparticles. Any further binding sites present after the coupling were saturated with inert substances.
  • the microparticles prepared in this way were taken up in 10 ml of 0.1M phosphate buffer, including 1 mol/l NaCl and 0.05% azide, pH 7.2, per gramme of microparticles.
  • the tracer used for the cross-reactivity tests was 125 I-labelled thyroxine with a specific activity of 6.22 MBq/ ⁇ g in a concentration of 27.6 mg/l.
  • conjugates to be investigated were made up fresh, for each assay procedure, in a buffer matrix composed of 20 mmol/l phosphate buffer containing 0.2% gelatin and free of binding proteins.
  • concentrations were chosen for this: FT 4 : 7.8, 16, 31, 62, 125, 250 and 500 ng L-T 4 /ml
  • conjugates concentrations of the conjugates were adjusted as follows: Conjugates: 0.01, 0.1, 1, 10 and 100 ⁇ g of conjugate/ml.
  • the dilution of the microparticle suspension coupled to the antibodies which was to be used for this was adjusted so that the range with the greatest discrimination was found to be a range between 30 and 40 ng of T 4 /ml.
  • the mixture was incubated at 22° C. for 1 h.
  • the last column in the table is a "50% intercept" (pg/ml) column which was determined on the basis of Examples 1 and 2 which follow. This column reveals whether a particular conjugate was suitable, in conjunction with the antibody investigated, for use in the method according to the invention.
  • 50% intercept which reflects the region of greatest slope of the standard plot, and thus the greatest discrimination between small differences in concentration, is in the normal physiological concentration range for FT 4 of 8 to 20 pg/ml, the corresponding conjugate is very well suited for use in the method according to the invention.
  • the antibody used was a monoclonal T 4 -specific mouse antibody.
  • the antibody was labelled with 125 I in a known manner.
  • the specific activity of the resulting labelled antibody was between 25 and 35 KBq/ ⁇ g of antibody.
  • the labelled antibody was taken up in 0.1 M phosphate buffer containing 1 mol/l NaCl and 0.05% azide, pH 7.2.
  • the concentration of the labelled antibody was between 1 and 1.25 ⁇ g/l in this.
  • the standard material used was a human serum matrix with the following concentrations: 0, 2.8, 5.6, 11.3, 22.5, 45 and 90 pg of FT 4 /ml.
  • the assay was carried out as follows:
  • the reactants were incubated-at 22° C. on a horizontal shaker for 2 h.
  • the immunological reaction was stopped by aspirating the incubation solution out of all the tubes.
  • washing solution (0.15 mol/l NaCl) was placed in each of the tubes, followed by decantation, times.
  • the activity remaining bound to the solid phase was measured in a gamma counter for 60 seconds.
  • the results obtained were evaluated by data reduction by known methods.
  • Coated polystyrene tubes as in Example 1 were used for the immobilization of the conjugate to be investigated.
  • the antibody used was a monoclonal T 4 -specific mouse antibody, but this time it was labelled not with a radionuclide but with a luminogen.
  • the luminogen had been coupled to the antibody in a manner known per se (compare U.S. Pat. No. 4,645,646; German Offenlegungsschrift 2,921,781 or 3,132,491).
  • a cyclic diacylhydrazide derivative was used as luminogen.
  • the antibody labelled with the luminogen was taken up in 0.1M phosphate buffer containing 1 mol/l NaCl and 0.05% azide, pH 7.2.
  • the concentration of the labelled antibody was between 1 and 1.25 ⁇ g/l in this.
  • the standard material used was the same human serum matrix as in Example 1.
  • the assay procedure also corresponded exactly to that in Example 1.
  • the activity remaining bound to the solid phase was measured in a luminometer suitable for measuring chemiluminescence and having at least one possibility for injection in the measuring position (Berthold LB 9502 or Hamilton LUMICON) for 4 s.
  • a luminometer suitable for measuring chemiluminescence and having at least one possibility for injection in the measuring position (Berthold LB 9502 or Hamilton LUMICON) for 4 s.
  • the measurement method and the reagents used for this are described in detail in, inter alia, the already cited U.S. Pat. No. 645,646.
  • the solid phases used in the two examples of the method, 1 and 2 were coated polystyrene tubes. However, it is possible without difficulty also to use as solid phases other plastics (for example polypropylene, nylon, Teflon and other suitable activated plastics) as well as glass.
  • the coupling of the conjugates in these cases is always carried out by methods known from the literature, for example by adsorption or covalently (compare Catt, K., Tregear, G. W., in: Science, 158 (1967), pages 1570-1572; U.S. Pat. No. 4,657,873 or Wood W. G. and Gadow, A. in: J. Clin. Chem. Clin. Biochem. 21 (1983), pages 789-797.
  • radionuclides 125 I
  • luminogens used in Examples 1 and 2

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  • Health & Medical Sciences (AREA)
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  • Immunology (AREA)
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  • Urology & Nephrology (AREA)
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US07/232,251 1987-08-14 1988-08-15 Immunological method for the determination of free substances having hapten properties Abandoned USH1018H (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE3727238 1987-08-14
DE19873727238 DE3727238A1 (de) 1987-08-14 1987-08-14 Immunologisches bestimmungsverfahren zur bestimmung von hapteneigenschaften aufweisenden freien substanzen

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USH1018H true USH1018H (en) 1992-01-07

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US (1) USH1018H (ja)
EP (1) EP0303284B2 (ja)
JP (1) JP2661712B2 (ja)
AT (1) ATE77487T1 (ja)
DE (2) DE3727238A1 (ja)
ES (1) ES2042661T3 (ja)
GR (1) GR3005066T3 (ja)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5639670A (en) * 1992-05-06 1997-06-17 B.R.A.H.M.S. Diagnostica Gmbh Determination of free thyroid hormones by competitive immunoassay
US5672480A (en) * 1993-12-29 1997-09-30 Abbott Laboratories Immunoassays for prostate specific antigen

Families Citing this family (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB8800419D0 (en) * 1988-01-08 1988-02-10 Amersham Int Plc Method for measuring free fraction of ligands in biological fluids
DE19504198A1 (de) * 1995-02-09 1996-08-14 Behringwerke Ag Kompetitiver Immuntest unter Verwendung komplexierter Analytderivate
US5824478A (en) * 1996-04-30 1998-10-20 Vysis, Inc. Diagnostic methods and probes
US7004027B2 (en) 2003-03-03 2006-02-28 Yamaha Corporation Electrostatic-capacity-type acceleration sensor and acceleration measuring device therewith
US7150192B2 (en) 2003-03-03 2006-12-19 Yamaha Corporation Acceleration measurement method using electrostatic-capacity-type acceleration sensor

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4410633A (en) 1980-09-25 1983-10-18 Corning Glass Works Method for the measurement of free thyroxine or 3,5,3'-triiodothyronine in a liquid sample
US4434236A (en) 1982-10-20 1984-02-28 E. I. Du Pont De Nemours & Co. Immunoassay wherein labeled antibody is displaced from immobilized analyte-analogue
EP0155104A2 (en) 1984-02-24 1985-09-18 AMERSHAM INTERNATIONAL plc Free analyte assay
EP0143274B1 (en) 1983-10-03 1988-01-13 E.I. Du Pont De Nemours And Company Heterogeneous immunoassay for digoxin using ouabain as a separation means
EP0089806B1 (en) 1982-03-22 1989-06-28 AMERSHAM INTERNATIONAL plc Assay for the free portion of substances in biological fluids

Family Cites Families (9)

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Publication number Priority date Publication date Assignee Title
US4366143A (en) * 1979-09-24 1982-12-28 Amersham International Public Limited Company Assay for the free portion of substances in biological fluids
EP0073865B1 (en) * 1981-09-11 1986-01-29 AMERSHAM INTERNATIONAL plc Derivatives of iodothyronine compounds and their use in an assay for the free iodothyronine compounds
JPH0616046B2 (ja) * 1982-03-19 1994-03-02 エキンズ，ロジャー フィリップ 遊離リガンドアッセイ方法およびキット
US4595661A (en) * 1983-11-18 1986-06-17 Beckman Instruments, Inc. Immunoassays and kits for use therein which include low affinity antibodies for reducing the hook effect
DE3415818C1 (de) * 1984-04-27 1985-04-18 Henning Berlin Gmbh Chemie- Und Pharmawerk, 1000 Berlin Verfahren zur Bestimmung freier Substanzen in biologischen Fluessigkeiten
DE3442817A1 (de) * 1984-11-23 1986-05-28 Boehringer Mannheim Gmbh, 6800 Mannheim Verfahren und reagenz zur quantitativen bestimmung von freiem thyroxin in plasma, serum oder vollblut
FR2598514B1 (fr) * 1986-05-12 1988-07-01 Commissariat Energie Atomique Procede de dosage immunologique des hormones thyroidiennes t3 et/ou t4 utilisant la thyroglobuline
DE3624464A1 (de) * 1986-07-19 1988-01-28 Boehringer Mannheim Gmbh Verfahren zum nachweis eines analyten sowie hierfuer geeignetes mittel
GB8800419D0 (en) * 1988-01-08 1988-02-10 Amersham Int Plc Method for measuring free fraction of ligands in biological fluids

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4410633A (en) 1980-09-25 1983-10-18 Corning Glass Works Method for the measurement of free thyroxine or 3,5,3'-triiodothyronine in a liquid sample
EP0089806B1 (en) 1982-03-22 1989-06-28 AMERSHAM INTERNATIONAL plc Assay for the free portion of substances in biological fluids
US4434236A (en) 1982-10-20 1984-02-28 E. I. Du Pont De Nemours & Co. Immunoassay wherein labeled antibody is displaced from immobilized analyte-analogue
EP0143274B1 (en) 1983-10-03 1988-01-13 E.I. Du Pont De Nemours And Company Heterogeneous immunoassay for digoxin using ouabain as a separation means
EP0155104A2 (en) 1984-02-24 1985-09-18 AMERSHAM INTERNATIONAL plc Free analyte assay

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5639670A (en) * 1992-05-06 1997-06-17 B.R.A.H.M.S. Diagnostica Gmbh Determination of free thyroid hormones by competitive immunoassay
US5672480A (en) * 1993-12-29 1997-09-30 Abbott Laboratories Immunoassays for prostate specific antigen

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EP0303284B2 (de) 2000-05-03
JP2661712B2 (ja) 1997-10-08
DE3727238A1 (de) 1989-02-23
JPS6468661A (en) 1989-03-14
ES2042661T3 (es) 1993-12-16
DE3727238C2 (ja) 1989-05-18
EP0303284A1 (de) 1989-02-15
EP0303284B1 (de) 1992-06-17
GR3005066T3 (ja) 1993-05-24
ATE77487T1 (de) 1992-07-15
DE3872098D1 (de) 1992-07-23

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