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
  • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
  • G01N33/581—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with enzyme label (including co-enzymes, co-factors, enzyme inhibitors or substrates)
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
  • C12Q1/40—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving amylase
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/48—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase
  • C12Q1/50—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase involving creatine phosphokinase
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/54—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving glucose or galactose

Definitions

• THIS INVENTION relates to a method of determining a substrate in a sample, and has particular but by no means exclusive use in the determination of very small quantities of a substrate.
• the present invention has particular application in quantitative enzyme immunoassay techniques such as the known homogeneous or non-homogenous enzyme immunoassay (EIA) and enzyme-linked immuncsorbent assay (ELISA) described for example in 'Quantitative Enzyme Immunoassay', Ed. E.Engvall and A.J. Pesce, Blackwood Scientific Publications (Scand. J. Immunol. 8, Suppl. 7,1978).
• EIA enzyme immunoassay
• ELISA enzyme-linked immuncsorbent assay
• non-homogenous enzyme immunoassay is based on the competition for the active site of an antibody between a ligand and a covalently coupled derivative of that same ligand to an enzyme. With separation of the bound and free enzyme phases, enzyme assay of either fraction can then be made. Plotting of the percentage of the enzyme-ligand bound to the solid phase against the concentration of ligand results in a typical immunoassay curve.
• the enzyme immunoassay system is also capable of simple modification to provide for the measurement of serum antibody levels by making enzyme-antibody derivatives and using a solid phase ligand to assist in the separation step.
• the development has concentrated on the assay of serum digoxin as a working prototype.
• Therapeutic range of digoxin in serum is 0-6 nanomoles/litre and the working assay requires 50 microlitres of serum in a final volume of 500 microlitres.
• Results show that by simple modification the assav is capable of a 50-100 fold increase in sensitivity, and thus should be capable of measuring plasma ligands e.g. ACTH levels, in the range of 2-20 picomoles/litre.
• the present invention is not restricted to enzyme assay in non-homogeneous enzyme immuno-assays, and may equally be applied to enzyme assay in other known enzyme immuno-assay techniques. Further more, the method of the present invention also has application in fields entirely unrelated to enzyme immuno-assays. For convenience, however, the method of this invention will be described in detail with reference to enzyme assay in enzym immuno-assays.
• the present invention is directed to increasing the sensitivity of an enzyme immuno-assay by method of determining a substrate produced as a product in the course of an enzyme immuno-assay by the following steps:
• the present method may be schematically illustrated as follows: the present invention are discussed in more detail herein after.
• a first illustration of the application of the method of this invention is the ⁇ - galactosidase hydrolysis of a phenyl- ⁇ -galactoside, with the ⁇ -galactosidase covalent ly bound to an appropriate ligand as in, for example, digoxin- ⁇ -galactosidase.
• the phenol, or phenol derivative, produced by ⁇ nzymic action of the ⁇ -galactosidase is converted to o-catechol and o-quinone, catalysed by the enzyme tyrosinase, o-quinone may be reconverted to o-catechol in the cyclic reaction sequence, either non-enzymatically by use of a suitable redox acceptor such as NADH or an NADH analogue, or by use of a suitable enzyme such as lipoamide dehydrogenase , which uses NADH as cofactor.
• the indicator reaction may be determined by known means such as spectrophotometry or fluorometry.
• ether phenol derivatives that are substrates for the enzyme tyrosinase may similarly be used wi th system after forming an appropriate substituted phenyl- ⁇ -galactoside.
• Such derivatives include, for example, I-dopa and p-substit uted phenols with substituent, for example, -SCN, -COCH3, -CHO, -CN , -N02.
• substituent for example, -SCN, -COCH3, -CHO, -CN , -N02.
• dotted lines depicting the non reversible indicator reaction indicate that this reaction may be effected with either stage (1) or stage (2) of the main cyclic reaction sequence).
• the measurement of the coreaction may be made directly by nature of the inherent properties of the indicating substrate or indicating product e.g. in conversion of NADH to NAD, or it may require a subsequent measurement step e.g. in the measurement of ADP or ATP.
• an initial enzyme immuno-assay is utilized as a substrate in the cyclising reaction to form a product which is itself reconverted to its original substrate form.
• the indicating coreaction is continuously being changed to a new form and thus is an effective multiplier in concentration terms of the original substrate.
• the effect of this cyclic process is, therefore, to increase the sensitivity of an enzyme immuno-assay by substantially reducing the quantity of the enzymic component able to be detected, and thus required for the enzyme immunoassay.
• the participating substrate in the method of the present invention may be produced directly or indirectly by the free or bound enzyme-ligand phases which are to be assayed. Enhancement of this process is possible by utilizing the indicator substance used in the cycling process as a new substrate in a second cyclising reaction with a different indicating reaction.
• a number of illustrations of this application of (b) is the use of a ligand ⁇ -galactosidase to produce p-catechol or p-catechol derivative by enzymatic hydrolysis of a p-catechol or p-catechol derivative ⁇ -galactoside in an enzyme immuno-assay. Conversion of the p-catechol so formed to p-quinone is catalysed by the enzyme laccase at a pH of 7-8 with the p-quinone being reconverted to p-catechol directly by a suitable redox acceptor such as NADH or NADH analogue, or with addition of a suitable enzyme such as menadione reductase.
• Other derivative B-galactosides which produce substrates that are similarly catalysed by the laccase include ⁇ -galactosides appropriately formed with p- ⁇ henylenediamine, N ,N-dimethyl-p-phenylenediamine, N-phenyl-p-phenylenediamine, o-phenylenediamine, and p-aminophenol.
• the indicator reaction in which NADH is converted to NAD the effect of which is multiplied due to the cyclic nature of the p-catechol/p-quinone reaction sequence, may be measured by known techniques, for example, a direct rate assay.
• the present invention is schematically illustrated as follows:
• the enzyme ⁇ -galactosidase may be covalently bound to a ligand and used to hydrolyse a p-OH-phenyl- ⁇ -galactoside and used with a fungal laccase at a pH of 4.5 in a similar manner.
• a third illustration of the present invention is the use of a ligand-pyruvatekinase covalent derivative in an enzyme immuno-assay to catalyse the production of pyruvate from phosphoenolpyruvate with ADP as cofactor. The pyruvate so formed is converted to lactate at pH 7.4 by the enzyme lactate dehydrogenase (E.C.
• This cyclising indicator system involving pyruvate kinase and the two lactate dehydrogenases may also be used as an additional second amplification assay involving ADP production or removal in a prior enzyme immuno-assay procedure.
• a suitable system is described in (d) below.
• a further illustration of the method of this invention is the use in an enzyme immuno-assay of a suitable glucose producing enzyme such as amylase covalently bound to an appropriate ligand.
• Glucose produced in an appropriate reaction is converted to glucose-6-phosphate catalysed by enzyme hexokinase.
• the cofactor ATP is converted to ADP as the indicator reaction sequence.
• the glucose-6-phosphate so formed is reconverted to glucose with liberation of inorganic phosphate by use of an appropriate glucose-6-phosphatase enzyme.
• the overall effect of this cyclic sequence is the conversion of the indicating substrate ATP to ADP at molarities which are greatly increased over the glucose concentration formed in the enzyme immune-assay reaction.
• the ATP loss or ADP gain may be assayed by appropriate means, either simultaneously (as by the measurement of ADP in (c) above), or subsequently (as by the known determination of ATP using luciferase).
• enzyme immuno-assay of biological products which could contain glucose or giuccse-6-phosphate
• prior incubation of the reaction mixture and biological sample with solid-phase glucose oxidase and solid-phase glucose-6- phosphate dehydrogenase would be required to remove these substrates before proceeding to the enzyme immuno-assay.
• the enzyme aidolase is covIERly bound to an appropriate ligand and used in an enzyme immuno-assay system together with the substrates dihydroxyacetone phosphate and gycerald ⁇ hyde-3-pho ⁇ phate to produce fructose-1,6-diphosphate.
• the latter is converted to fructose-6- ⁇ hos ⁇ hate and inorganic ohcsohate bv use of the enzyme fructose-1, 6-diphosphatase and the fructose-6-phosphate so formed reconverted to fructose-1, 6-dichosohate by use on an appropriate phospho-fructo-kinase.
• ATP is converted to ADP as the indicating reaction. It will be apparent that the overall effect of this cyclic reaction sequence is the conversion of high molarities of ATP to ADP in the presence of relatively small amounts of fructose-1,6-diphosphate formed in the enzyme immuno-assay step. The loss of ATP or gain of ADP is measured by techniques similar to those described in (d).
• Such further systems may include covalent ligand enzymes capable of producing substrates for use with the following: ascorbate oxidase (E.C. 1.10.3.3.) and oxidized asccrbate reductase (E.C. 1.5.1.4); and glutathione reductase (E.C. 1.6.4.2) and glutathione dehydrogenase (E.C. 1.8.5.1).
• covalent ligand enzymes capable of producing substrates for use with the following: ascorbate oxidase (E.C. 1.10.3.3.) and oxidized asccrbate reductase (E.C. 1.5.1.4); and glutathione reductase (E.C. 1.6.4.2) and glutathione dehydrogenase (E.C. 1.8.5.1).
• the animals used for the production of antibodies were goats and Droughtmaster cows. Both animals were amenable for obtaining large amounts of sera.
• the Droughtmaster cattle were chosen because they are a breed particularly resistant to ticks and tropical parasites. It was thought that this resistance was due to production of antibodies and therefore would be suitable for production of particular antibodies against a wide variety of immunogens.
• Digoxin haemocyanin was used as the immunogen in both goats and cattle.
• PBS phosphate buffered saline
• 2ml were injected into the animals intramuscularly and boosted with the same amount every month.
• a similar schedule was used with the cattle except the concentration of digoxin haemocyanin was increased to 10mg per animal.
• the digoxin specific antibodies were purified from goat and bovine serum using ammonium sulphate precipitation of the globulin fraction and preparation of the gamma globulin fraction was carried out using ion-exchange chromatography.
• the goat serum was found to contain titres of digoxin antibody such that 1 ml of serum contained sufficient antibody for 10,000 determinations under the conditions used in this laboratory.
• the serum was brought to 30% (w/v) with ammonium sulphate and the precipitated protein collected by centrifugation.
• the precipitate was dissolved in a minimum volume of 5 mM phosphate buffer pH 8.0 and dialysed exhaustively against this buffer. This material was applied to a DEAE cellulose (DE 52, Whatman) equilibrated against this buffer.
• the protein was eluted with a linear gradient from 5 mM to 100 mM phosphate buffer pH 8.0.
• the gamma globulin fraction was eluted off in the first two peaks. Digoxin binding activity was found to be associated with these two peaks. The appropriate tubes were combined and the protein precipitated out with ammonium sulphate.
• the antibody was stored at -20oC in this form and has been found to be extremely stable over a period of two years. This material has been used in routine digoxin assays in this laboratory in conjunction with an RIA method for some two years. For use in immunoassays the ammonium sulphate precipitate was dissolved in PBS, dialysed against this buffer and the appropriate dilutions required for the immunoassay prepared. The antibody stored lyophilised at -20oC in this form was also found to be quite stable over a similar period. 1.5
• the antidigoxin globulin prepared from both goats and cattle was found to have virtually identical characteristics.
• the binding constant for digoxin was found to be 1.8 x 10 11 litres/mole using a Scatchard plot.
• the drugs listed in the following table were tested for cross reactivity with this antibody. Over a period of two years two major digoxin antibody preparations have been carried out. The first was produced using disuccinyl-digoxigenin -BSA as the immunogen. The second was produced using digoxin haemocyanin as the immunogen. Different specificities for some of the closely related chemicals were observed. In particular the second preparation was found to be suitable for digitoxin determinations. % CROSS REACTIVITY
• Fab Derivative Preparation The Fab derivatives were prepared from the purified antidigoxin gamma globulin fraction by incubating the gamma globulin in 100 mM phosphate buffer pH 7 containing 10 mM cysteine, 2mM EDTA with papain (7% by wt. of gamma globulin). The papain digestion was carried out at 37o for 17 hours. Following this the digest was dialysed against PBS in the cold for two hours and then fractionated on a Sephadex G100 column equilibrated against this buffer. The tubes containing protein of molecular weight equivalent to 50,000 were pooled and the protein concentrated. 2. PREPARATION OF Fab ANTIBODY
• the Fab derivative prepared from the cow gamma globulin fraction was used as an immunogen to produce antibodies in goats.
• the Fab (2 mg) in 1 ml of PBS was diluted 1/1 with Freund's adjuvant and homogenised. This material was injected intramuscularly into each goat.
• the activated Sepharose was suspended in 0.1M NaHCO 3 solution (10 ml) at 5°.
• Fab antibody C 62.2 mg previously dialysed against 0.1M NaHCO 3
• the supernatant was filtered off, and the Sepharose was washed with 0.1M NaHCO 3 (20 ml) and was suspended in a solution of 0.1M NaHCO 3 , containing glycine (2 moles/1) and bovine serum albumen (50 mg/ml).
• the Sepharose was stirred at room temperature for 2 hrs. and stored overnight at 4o.
• the resultant Sepharose - Ab Fab was washed successively with 0.1M NaHC0 3 (200 ml), 0.5M NaCl (200 ml) and PBS (200 ml). The Sepharose was suspended and stored in PBS (7.5 ml).
• ⁇ -galactosidase digoxin conjugates have been prepared with the molar ratio of digoxin to enzyme varying between 1 and 10.
• Properties of ⁇ -galactosidase-digoxin Stability The enzyme preparation was stored in solution at 4 for 4 months in buffer but without ammonium sulphate or other protective agents and showed a slow loss of activity over the period being 60% of the original activity after this pe riod
• Enzyme-digoxin, serum digoxn, and antidigoxin antibody are incubated in buffer for thirty minutes at room temperature. 100 ul of solid chase precipitating antibody is then added, and the mixture incubated for 30 minutes. The mixture is then spun at 2000 rpm for 5 minutes on a bench centrifuge and the supernatant assayed for residual enzyme activity. Reagents: 1. Enzyme-ligand solution.
• Digoxin ⁇ -galactcsidase solution (50 ul) contains 240 f moles digoxin, 20 f moles protein in PBS buffer.
• Fab derivatives of anti-digoxin gamma globulin (100 ul) sufficient to give 60% binding of enzyme-ligand in assay in PBS buffer.
• Phenyl ⁇ -gaiactoside 0.6 mg/ml 0.05 M phosphate pH 6.0 - twice recrystallized.
• Tyrosinase 1.8 mg/ml buffer, 0.05 M phosphate pH 6.0.
• NADH/DOPA 6 mg/ml NADH, 30 um DOPA in 0.05 M phosphate pH 6.0.
• Serum containing 0,0.5, 1.0, 2.0, 4.0 u g/litre Set up in conical glass tubes:- 50 ul buffer (0.01 M phosphate pH 7.4, 0.15 M KCl containing 4 mg/ml BSA). 50 ul ⁇ -galactosidase-digoxin (150 nM) diluted 1/400 with buffer. 50 ul serum/standard. 100 ul Fab diluted 1/100 with buffer (suff icient to bind about 60 % ⁇ -galactos idase digoxin) .
• Applic- Ligands with serum concentrations 0-7 n ability moles/litre. Comment: The previous comments on how to reduce the incubation times of the assay apply here as well. Times of 15 mins. have been used manually and this should be able to be reduced to 5-10 minutes with precise automated control.

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• 1980
  • 1980-09-11 JP JP50199980A patent/JPS56501384A/ja active Pending
  • 1980-09-11 WO PCT/AU1980/000065 patent/WO1981000725A1/fr not_active Application Discontinuation
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