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
  • G01N33/743—Steroid hormones
• • 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
  • G01N33/536—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase
  • G01N33/537—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase with separation of immune complex from unbound antigen or antibody
  • G01N33/539—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase with separation of immune complex from unbound antigen or antibody involving precipitating reagent, e.g. ammonium sulfate
  • G01N33/541—Double or second antibody, i.e. precipitating antibody
• • 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
  • Y10S435/00—Chemistry: molecular biology and microbiology
  • Y10S435/964—Chemistry: molecular biology and microbiology including enzyme-ligand conjugate production, e.g. reducing rate of nonproductive linkage
• • 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
  • Y10S435/00—Chemistry: molecular biology and microbiology
  • Y10S435/971—Capture of complex after antigen-antibody reaction
• • 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
  • Y10S435/00—Chemistry: molecular biology and microbiology
  • Y10S435/975—Kit
• • 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
  • Y10S436/807—Apparatus included in process claim, e.g. physical support structures
  • Y10S436/808—Automated or kit
• • 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 The invention relates to a process for the detection [30] Foreign Application Priority Data and determination of a component of the reaction be- Dec. 18, 1970 Netherlands 7018838 tween 3 Specific binding Protein and the correspond" ing bindable substance contacting a fluid containing 52 US. Cl. 195/1035 R, 424/12, 195/63 one of these Components with a conjugate Of the bind- 51 Int. Cl. G01n 31/14 able Substance with an enzyme and of antibodies [58] Field 01 Search 195/1035 R; 424/12; against the Specific binding protein which latter 23/230 B ponent is brought in an insoluble form, if necessary, in
• antibodies as specific binding proteins, and the corresponding antigens as bindable substances.
• Substances which can be determined with this system are, inter alia, protein hormones and their antibodies, or virus antigens and their antibodies;
• haptens as bindable substance.
• the haptens are defined as protein-free substances, which can react with antibodies, without being able to induce them.
• Substances which can be determined in such a system are, inter alia, steroid hormones and vitamines;
• proteins which in the body act as receptor or transport molecules as specific binding proteins, and substances which are bound by them as bindable substances.
• This system is e.g., suitable for the determination of steroid hormones, but also of thyroxine and triiodothyronine, vitamine 8, intrinsic factor, and adrenocorticotropic hormone.
• the separation methods can be divided as follows:
• conjugate and enzyme conjugate are used as synonyms for the coupling product of the bindable substance and the enzyme.
• the bindable substance can be detected and determined by bringing together the unknown sample or a dilution series of same with a known amount of a conjugate of the substance to be determined and an enzyme, and with an amount of specific binding protein, depending on the amount of enzyme conjugate that was added. Then an amount, preferably an excess, of the insoluble-made antibodies against the specific binding protein is added, so that all the enzyme conjugate that has reacted with the binding protein is coupled via this protein to these insoluble antibodies. 1n proportion as there is more bindable substance present in the sample, less enzyme conjugate will react with the specific binding protein and ultimately get into the insoluble phase; the result is that more unbound enzyme conjugate remains in the liquid phase, which can simply be determined there.
• the specific binding protein can be determined by incubating the sample or a dilution series of same with a known amount of enzyme conjugate and with an amount of the insoluble-made antibodies against the specific binding protein.
• the enzyme activity can only pass into the insoluble phase if the conjugate has reacted with the specific binding protein: the more specific binding protein there is in the sample, the less of bound enzyme conjugate remains in the liquid phase.
• the sensitivity of the test systems described can be varied by altering the quantities of the reagents (whether or not in the same ratio).
• the amount of enzyme conjugate that can be applied is limited at the lower end by the requirement that its enzyme activity can be measured reasonably, so that the sensitivity of the test systems has a limit.
• the minimum measurable enzyme activity depends inter alia on the nature of the enzyme used for the coupling and on the nature of the substrate and of the incubation period of the enzyme reaction.
• affinity of the specific binding proteins strongly influences the sensitivity of the determination. For a highly sensitive test system specific binding proteins with a higher affinity are required.
• the quantity of enzyme conjugate will be determined with the help of the enzyme activity; then this quantity is incubated with a dilution series of the specific binding protein to determine the required quantity of this protein.
• a quantity of specific binding protein is chosen, which binds 50-90% of the enzyme conjugate.
• the insoluble-made antibodies against the specific binding protein are preferably added in excess in the two types of determination; the dosing of same is determined in preliminary tests.
• radioisotopes can be replaced by working with enzymes. This requires considerably less laboratory facilities and apparatus, whilst the staff can be less highly qualified. Moreover, working with radioactive isotopes is highly limited because of legal regulations. Further the reagents according to the invention will keep long and the safety is increased;
• the combination ofDouble Antibody and Solid Phase methods offers several advantages over the existing methods.
• the performing of the found method viz.: addition of insoluble-made antibodies against the specific binding protein, incubation, centrifuging and measuring, is very simple.
• the closing is often easier than in the solid phase methods because it suffices to add an excess of insoluble material, whereas in the solid phase methods an accurately measured amount of material has to be used.
• the affinity of the binding protein for the bindable substance is not impaired by the binding to the carrier material, which can be the case in the solid phase method.
• a further advantage over the latter method is the rapid equilibrium adjustment of the reaction between specific binding protein and bindable substance (both in solution).
• a further advantage is that in the DASP method the insoluble-made antibody, called immunoadsorbent below, can be used in any system in which antibodies are used as specific binding protein, provided that these antibodies have been prepared in the same animal species.
• the antibodies have to be made insoluble.
• the double antibody method is very sensitive for relatively low variations in salt concentrations, pH and the like, which makes a rigid control of the conditions necessary.
• the method requires the addition of carrier y-globuline and consequently much second antibody to obtain an immune precipitate. In addition to being a simpler procedure.
• the DASP method which does not require "carrier” y-globuline, thus leads to material saving. Further it be added that a double antibody-like separation applied to transport or receptor proteins is not possible. as no suitable carrier protein is available for this purpose.
• the method according to the invention will therefore offer a unique opportunity in this respect.
• the method according to the present invention can easily be automatized. In principle it is possible to bring the reaction components together at once or to add them in any sequence. It was found, however, that the determination acquires a higher sensitivity if the immunoadsorbent is added after the incubation of the other components.
• the reagent required for the invention viz. the coupling product of antigen, hapten or bindable substance with an enzyme
• These methods can also be used to bind a hapten or a low-molecular bindable substance to an enzyme, provided that one substance possesses one or more aminogroups and the other one or more carboxyl-groups. If the latter is not the case then it is possible to introduce the desired group into the molecule to be coupled with the help of known organo-chemical processes.
• Methods are also known to bind amino or earboxyl-groups together, whether or not by introducing a bridge.
• the choice of the enzyme which is taken up in the coupling product is determined by a number of properties of that enzyme. It is, of course, essential that the enzyme should be resistant to the coupling with another molecule, i.e., modification of one or more aminoacid side chains. Also of great importance is the specific activity of the enzyme. As less enzyme conjugate needs to be added to reach a measurable enzyme effect, the test system grows more sensitive. Further those enzymes are to be preferred, of which the determination of the activity can be made in a simple manner. In the first place those enzymes are considered that can be determined colorimetrically, spectrophotometrically or fluorimetrically. This kind of determinations is suitable for automation, which is an additional advantage.
• catalase peroxidase
• ,B-glucoronidase ,B-D-glucosidase
• B-D- galactosidase urease
• glucose oxidase galactose oxidase
• alkaline phosphatase alkaline phosphatase
• the enzyme activity of the liquid or solid phase of the reaction mixture or of the two phases can be determined. Most simple is, however, to determine the enzyme activity of the liquid phase.
• the insoluble-made antibodies against the specific binding proteins which are also an essential reagent for the process of the invention, can also be prepared in a known way.
• the antibodies can be prepared by taking a purified preparation of the specific binding protein,
• the serum of the treated animal, or the gammaglobuline fraction thereof can be made insoluble by cross-linking with compounds such as glutaric aldehyde and chloroformic acid ethyl ester, or by binding to solid carrier particles, either physically by adsorption, or chemically by the formation of covalent bonds.
• solid carriers can be considered materials such as cellulose (modified or not), agarose, cross-linked dcxtran, polystyrene and the like.
• Covalent binding of the antibodies to these materials can be effected with the help of substances such as carbodiimides, diand tri-chloro-striazines, glutaric aldehyde, cyanogenbromide, and e.g., by diazotation.
• substances such as carbodiimides, diand tri-chloro-striazines, glutaric aldehyde, cyanogenbromide, and e.g., by diazotation.
• the forms in which the reagents can be used are manifold.
• the enzyme-conjugated component of the reaction system can be freeze-dried, or dissolved in a buffer.
• a solid carrier can be used, e.g., a strip of paper impregnated with the conjugate. This applies equally to the required specific binding proteins.
• the insoluble component can be brought in the form of particles of different dimensions, such as granules, flakes, rods, or in the form of a strip of some carrier material.
• test pack is applied by preference. This consists mainly of:
• test pack can further contain the reagents required for the enzyme determinations, and also auxiliary means for conducting the test, such as test tubes, pipettes and bottles with dilution liquid.
• auxiliary means for conducting the test such as test tubes, pipettes and bottles with dilution liquid.
• test pack An important embodiment of a test pack according to the present invention is a test pack to be used for the determination of gonadotropic hormones, and particularly for the determination of HCG (Human Chorionic Gonadotropin) as a means to diagnose pregnancy already in a very early stage, which test pack consists of an ampoule, tube, bottle or other container containing as essential ingredients, in separate lyophilized layers, pre-determined amounts of:
• HCG- peroxidase a conjugate of HCG with an enzyme, e.g., HCG- peroxidase.
• a preferably applied method for the determination of the enzyme activity consists in contacting an indicatorpaper impregnated with enzyme reagents, e.g., in case use is made of a peroxidase, a H O -supplier like urea- H ,O and a colour-reagent like o-tolidine.
• Example 1 Determination of human (HCG) a. Preparation of HCG-HRP. 5 mg HCG and 20 mg horse radish peroxidase (HRP) were dissolved in 2 ml 0.05 M phosphate buffer of pH 6.2. After addition of 40 ,ul 25% glutaric aldehyde solution the mixture was shaken for 2 hours at room temperature. After 5 minutes centrifugation at 250 g, the liquid was fractionated over Sephadex G-200 in 0.05 M phosphate buffer of pH 6.2. The fractions of which the highest percentage enzyme activity was bound by antibodies against HCG were used in the test system.
• HCG-HRP horse radish peroxidase
• the suspension was centrifuged, washed and the precipitate re-suspended in 43 ml 0.05 M sodium borate of pH 8.6. Then 7 ml of the prepared y-globuline solution was added. The mixture was stirred for 26 hours at 4C, then centrifuged and washed with 0.02 M phosphate buffer of pH 6.0.
• HCG-HRP conjugate a dilution series (32-16-8-4-2-1-05-0 lU/ml) of HCG in 0.02 M phosphate buffer with pH 6.0 was prepared, which contained 2% v/v normal sheep serum.
• 0.5 ml of each of the HCG-containing samples was incubated with 0.1 ml rabbit-(anti-HCG) serum and 0.1 ml HCG-HRP conjugate, both in suitable dilution, for half an hour at room temperature.
• 0.3 ml of the immunoadsorbent 10 mg/ml) prepared acccording to d) was added, and the resulting mixture was rotated at room temperature for 1 hour.
• Example 11 Determination of insuline and anti-insuline.
• a Preparation of insuline-(glucose oxidase). 5 mg pig insuline and 25 mg glucose oxidase were dissolved in 2 ml 0.05 M phosphate buffer of pH 6.5. To this, 5 ,ul 25% glutaric aldehyde solution was added, after which the mixture was shaken for 90 minutes at room temperature. The mixture was fractionated over Sephadex G-200 in 0.05 M phosphate buffer of pH 6.5. The fractions of which the highest percentage of enzyme activity could be bound by antibodies against insuline, were used in the test system.
• guinea pigs were given a weekly intramuscular injection with 1 mg pig insuline in complete Freunds adjuvans over a period from 4-8 weeks. After two weeks rest the animals were given 1 mg additional insuline by intravenous injection without adjuvans. 2 weeks after that the animals were bled. Hypoglycaemia occurring at times was counteracted by intraperitoneal administration of glucose.
• Guinea pig y-globuline was prepared by adding 1 volume saturated ammonium sulphate solution to 2 volumes guinea pig serum. The precipitate formed was twice washed with 33% saturated ammonium sulphate solution, and then taken up in a physiological salt solution.
• a sheep was immunized with increasing doses of the prepared 'y-globuline: 0.5, 1 and 2 mg. The injections were given every two weeks, whilst the immunogen was mixed with complete Freunds adjuvans. Two weeks after the last injection an additional 2 mg y-globuline in a physiological salt solution were given, and 1 week later the animal was bled.
• ml insuline-(glucose oxidase), in a suitable dilution was incubated with 0.4 m1 of a dilution series of a guinea pig anti-insuline serum for 4 hours.
• the dilution series was made with 0.05 M phosphate buffer of pH 6.0.
• 0.3 ml immunoadsorbent (15 mg/ml) and 0.2 ml buffer were added and the mixture was rotated during the night at 4C. After centrifuging the enzyme activity of the supernatant was determined by incubating 0.5 ml of same with 2.5 ml substrate for 30 minutes, and then measuring the extinction at 460 nm.
• the substrate con tained 50 mg glucose, 10 ug peroxidase and 1 mg S-aminosalicyclic acid per 2.5 ml 0.05 M phosphate buffer of pH 6.0.
• the antibody content of the different serums could be intercompared.
• the serum dilution at which 50% of the total combinable enzyme activity is bound.
• sheep was injected once in four weeks with 4 mg oestradiol-l7-succinyl-BSA in complete Freunds adjuvans. At regular intervals blood was taken from the sheep. The serum was absorbed with BSA that was made insoluble.
• Sheep-y-globuline was prepared as described in example I, but now with 16% w/v sodium sulphate. Rabbits were immunized with this sheepy-globuline according to the following schedule:
• Example lV Determination of cortisol and corticoid-binding globuline.
• CBG Corticoid-binding globuline
• the y-globuline fraction of anti-CBG serum was coupled to m-aminobenzyloxymethylcellulose, as described in example lll.
• CBG CBG oxidase
• a suitable dilution series of transcortine ranging from 0-1280 ng/ml
• 0.5 ml was incubated for 15 minutes with 0.2 ml cortisol-21- (galactose oxidase) in a suitable dilution.
• 0.3 ml immunoadsorbent suspension (5 mg/ml) was added, and the mixture rotated for 15 minutes. The two incubations were conducted at 4C. Then the enzyme activity in the supernatant was measured as under d).
• the sensitivity of the test system proved to be 50 ng/ml.
• Example V In a bottle the following reagents were subsequently 65 lyophilized in separate layers:
• step (b) separating the resulting mixture into a liquid phase and a solid phase by adding an insolubilized antibody against the antibody of step (b);
• a test pack for the detection and determination of a bindable substance selected from the group consisting of an antigen and a hapten in a fluid sample comprising:
• a a known amount of a conjugate of said bindable substance with an enzyme
• b a corresponding known amount of an antibody against said antigen or hapten
• c a known amount of an insolubilized antibody 15 against said antibody
• d a stabilizer
• e a substrate for the determination of the enzyme activity and thus of the quantity of the antigen or hapten to be determined.
• a test pack for the detection and determination of an antibody in a fluid sample, utilizing the reaction between said antibody and an antigen or hapten to said antibody, comprising:
• a stabilizer for the determination of the enzyme activity and thus of the quantity of antibody to be determined.
• a a predetermined amount of a conjugate of human chorionic gonadotropin and an enzyme

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