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
  • G01N33/52—Use of compounds or compositions for colorimetric, spectrophotometric or fluorometric investigation, e.g. use of reagent paper and including single- and multilayer analytical elements
• • 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
  • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
  • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
  • G01N33/6827—Total protein determination, e.g. albumin in urine
  • G01N33/6839—Total protein determination, e.g. albumin in urine involving dyes, e.g. Coomassie blue, bromcresol green

Definitions

• This invention relates to an improved method for reduction of tetrazolium compounds to coloured formazans and to the use of this method in assays (ie detection and/or quantitative analysis) of analytes, in particular albumin.
• Tetrazolium salts contain the cationic tetrazolium nucleus:
• formazan will produce a colour change, as the formazan group is a strong coloured chromophore, and tetrazolium salts are often colourless. Therefore the presence and/or amount of formazan produced may conveniently be detected visually or
• Albumin is a protein, in one form having a relative molecular mass of 67000 and consisting of a single chain of 584 amino acids. It is present in human serum at a higher concentration than any other single protein.
• the primary functions of albumin in serum include the maintenance of osmotic pressure, and acting as a carrier protein for metabolites such as bilirubin, fatty acids, inorganic compounds such as calcium, hormones and many drugs; it also provides a source of amino acids. For several reasons albumin is often assayed in the clinical biochemistry laboratory.
• liver disease is used as an indicator of liver disease, is an essential requirement in albumin replacement therapy, it can indicate levels of unbound (unconjugated) bilirubin, it can indicate cases of myeloma and may be used in nutritional treatment regimes (Whicher and Spencer, Ann. Clin. Biochem.
• bromocresol purple does not have interspecies albumin specificity; many calibrators contain either bovine or equine albumin standards, which give an absorbance too far below that of human albumin to be of value. Further, heparin is reported to interfere (Perry, B.W. & Douman, B.T., Clin.Chem.
• Bilirubin is also believed to interfere.
• a method based on the formation of a coloured product in response to the presence and concentration of albumin which did not suffer interference from other components which may be present in the sample would be of great practical advantage. It is an object of the present invention to provide a new method which does not suffer the disadvantages described and can be used for the quantitation of albumin by a procedure generating a coloured product. Other objects and advantages of this invention will be apparent from the following description. According to a first aspect of this invention a method for the reduction of a tetrazolium compoun d containing the cationic nucleus:
• the invention derives from the unexpected discovery that although weak reducing agents can only reduce many tetrazolium salts at pH above 10 and in the absence of albumin, at the lower pH range of the invention the reduction of tetrazolium salts is substantially enhanced by interaction with albumin.
• the method of the invention is particularly applicable to reduction of the leuco form of 2-(2' - benzothiazolyl) - 5-styryl-3-(4' phthalhydrazidyl)-tetrazolium, (abbreviated herein to "BSPT”), especially the hydrochloride.
• BSPT 2-(2' - benzothiazolyl) - 5-styryl-3-(4' phthalhydrazidyl)-tetrazolium
• BSPT contains the tetrazolium cation:
• the interaction between the tetrazolium salt and the a lbumin is of a type that results in enhancement, acceleration or catalysis of the reduction of the tetrazolium salt to the coloured formazan dye.
• the precise nature of interaction between the tetrazolium salt and albumin in the method of the invention is not fully understood, it is believed that the interaction may involve formation of a complex between the salt and the albumin.
• the interaction and/or complex formation may result from the presence of the substituent groups on the tetrazolium nucleus.
• the method of the invention may be particularly applicable to tetrazolium salts in which the nucleus is substituted with one or more of the substituents present in BSPT ie benzothiazolyl, styryl or phthalhydrazidyl groups which may themselves carry substituent(s) on their ring systems, or a combination of such groups.
• Many reducing agents are suitable for use in the method of the invention, but preferably a weak reducing agent is used. Examples of those found to be suitable include dithiothreitol ("DTT”),
• mercaptoethanol eg betamercoptoethanol, cysteine, nicotinamide adenine dinucleotide and aminophenol .
• a particularly preferred reducing agent is NADH (reduced form of nicotinaminde adenine dinucleotide).
• An electron carrier may be used in the reaction medium of the method to enhance the speed of electron transfer between the reducing agent and the tetrazolium salt.
• a preferred electron carrier is a
• a preferred phenazinium salt is methoxy-N-methylphenazinium methyl sulphate (“MPMS”), ie
• phenazinium salts include phenazinium methosulphate (“PMS”) and phenazinium ethosulphate (“PES”)
• Tris- HCL tris hydroxymethylaminomethane HC1
• a surfactant in the reaction solution of the method, as this aids the solubilisation of the formazana produced.
• a preferred surfactant is a non-ionic surfactant
• the degree of reduction of the tetrazolium compound and consequent formation of formazan quantitatively relates to the quantity of albumin and/or reducing agent present in the reaction solution, and therefore when one of these two is the limiting factor in the reaction, ie all the other reactants are present in excess, the method of the invention may be used as the basis of assays for albumin or reducing agents.
• this invention provides a method for the assay of human or mammalian albumin in a sample comprises reacting a tetrazolium salt which is capable of interacting with albumin, with a reducing agent to produce a formazan in an aque ous solution at a pH between 6 and 10 in the presence of the sample, and relating the presence and/or amount of formazan produced to the presence and/or amount of albumin in the sample.
• preferred tetrazolium salts are those of BSPT, especially the hydrochloride, or other tetrazolium salts in which the nucleus is substituted with one or more of the substituents present in BSPT as mentioned above, which may themselves carry substituents on their ring systems or a combination of such groups.
• the method of assay of albumin is suitable for use with any sample believed to contain albumin provided that at least a proportion of any albumin contained therein can be dissolved to form the necessary aqueous solution if the solution does not already comprise an aqueous solution.
• the method is therefore most suited to the assay of aqueous samples believed to contain albumin, especially biological samples such as bodily fluids, and in particular blood serum, which may be used directly without any pretreatment, or urine etc.
• albumin especially biological samples such as bodily fluids, and in particular blood serum, which may be used directly without any pretreatment, or urine etc.
• the quantity of tetrazolium salt and reducing agent present in the reaction mixture should be in excess of that expected to interact with the albumin in the sample so that the quantity of albumin present is the limiting factor.
• Suitable and preferred reaction conditions and reagents for the albumin assay method of this invention are as discussed above with reference to the first aspect of the invention, ie the choice of reducing agent, the use of an electron carrier, buffers and pH, and use of a surfactant etc. These parameters are summarised in Table 1 below:
• the method is sensitive to albumin levels below those of clinical significance and the colorimetic analysis has a linear relationship with initial albumin levels up to about 100mg/ml of original serum sample. Serum itself does not significantly absorb over the range of wavelengths used for measurement and so a sample blank is not normally required.
• the method appears to be specific to albumin, requires no pretreatment of a serum sample and is not significantly subject to interference.
• the method of estimating albumin of the present invention will have many applications, but will be
• the invention provides a method for the assay of reducing agents in a sample which comprises reacting a tetrazolium salt which is capable of interacting with albumin, with the sample in the presence of albumin in an aqueous solution at a pH between 6 - 10 and relating the presence and/or amount of formazan produced to the presence and/or amount of reducing agent in the sample, the quantity of tetrazolium salt used being in excess of the amount expected to be reduced by the reducing agent in the sample.
• This method of assay of reducing agents may be used to assay many different chemical reducing agents. It is particularly suitable for the assay of reducing agents which are of biochemical clinical or diagnostic importance, such as aminophenols, NADH or co-enzyme A (CO-A) and using this method they may be assayed in samples of bodily fluids such as serum, urine ctc ⁇ As a further modification, the method of assay of reducing agents may be used to assay compounds which can participate in a chemical reaction in which a reducing agent is formed which can be assayed by the method of the invention and related to the presence and/or quantity of the compound.
• biochemical clinical or diagnostic importance such as aminophenols, NADH or co-enzyme A (CO-A)
• CO-A co-enzyme A
• the method of assay of reducing agents may be used to assay compounds which can participate in a chemical reaction in which a reducing agent is formed which can be assayed by the method of the invention and related to the presence and/or quantity of
• the compound to be assayed may itself be converted into the reducing agent.
• the drug paracetamol p-hydroxy acetanilide
• the reducing agent para-aminophenol may be quantitatively converted into the reducing agent para-aminophenol using for example an aryl acylamidase enzyme in a well known reaction.
• the para-aminophenol formed may then be assayed and related to the amount of paracetamol.
• one or more of the participating reagents other than the compound may be converted into a reducing agent in an amount related to the quantity of the compound present.
• the antibiotics chloramphenicol and thiamphenicol, or gentanicins may be acetylated in a reaction in which acetyl Co-A is used as an acetylating reagent and which is consequently converted into Co-A.
• Such reactions proceed readily under the mediation of the enzymes chloramphenicol acetyl transferase ("CAT") or gentanicin acetyl transferace (“GAT”) respectively.
• CAT chloramphenicol acetyl transferase
• GAT gentanicin acetyl transferace
• the presence and/or amount of the Co-A produced may be quantitatively related to the presence and/or amount of antibiotic.
• an aqueous solution may be made up containing at least the sample, the tetrazolium salt and the reducing agent or albumin as appropriate at the indicated pH, optionally also containing the other reagents referred to above, and incubated at a suitable temperature. The colour produced is then detected and for example compared with a standard or measured colo rimetrically.
• the reaction solution is aqueous it may be necessary to include additional water-miscible solvents to assist in dissolution of all of the reagents, in particular the tetrazolium salt.
• One preferred example of such a solvent is dimethylformamide ("DMF").
• suitable reagents such as the tetrazolium salt, reducing agent or albumin as appropriate, buffer etc may be
• kits including in combination one or more ready made reagents as described above for the performance of the method.
• a kit may comprise separate solutions containing respectively the tetrazolium salt capable of interacting with albumin, buffer, and reducing agent or albumin as appropriate, at concentrations suitable to enable them to be easily used in the method of the invention, optionally together with the other reagents, eg surfactant, electron carrier referred to above.
• Such a kit may alternatively comprise a solid substrate having immobilised thereon suitable reagents.
• the kit may also include standards and instructions which may include a colour comparison chart.
• test kit the reagents have been found to be stable on storage for at least 30 days, but it is desirable to make up the solution of the tetrazolium salt in an acid solution, eg 0.01 - 0.2M hydrochloric, matic or especially citric acid, at a pH of 3 - 6 for stability on long term storage.
• acid solution eg 0.01 - 0.2M hydrochloric, matic or especially citric acid
• Fig 1 which shows the hyperchromic shift on reduction of a
• Fig 3 which shows the stability of the reagents on storage.
• Fig 4 which shows comparison between the method of the
• Fig 7 which shows absorbance versus Co-A concentration.
• Fig 8 which shows absorbance versus paracetamol concentration.
• Fig 9 which shows absorbance versus chloramphenicol
• Reagent B 50ml of Reagent B and 4.0ml of Reagent C. This mixture is stable for at least 12 hours.
• the reagents A, B and C as prepared and stored separately are stable for a minimum of 30 days as demonstrated in Fig 3 by the stability of the absorption at 590nm resulting from addition of various albumin concentrations to a working reagent prepared from reagents stored for the indicated periods.
• Table 2 below demonstrates the use of other reducing agents than NADH at indicated concentrations.
• the range of albumin concentrations over which the relationship between formazan production and albumin concentrations is linear is also shown, with NADH being used as a comparative standard.
• the tetrazolium salt was BSPT hydrochloride.
• the BSPT method also correlates well with the immunological technique as shown in Fig 6. (Results data not shown)
• a solution was prepared containing BSPT hydrochloride, albumin, MPMS, Tris-HCl buffer and Tween-80 at concentrations within the ranges indicated in Table 1. To aliquots of this was added Co-A over the concentration range 0.25 mM of Co-A final concentration. Fig 7 shows that over this range the absorbance at 590 nm was linearly related to Co-A concentration.
• Paracetamol was quantitatively cleared to form p-aminophenol using the enzyme aryl acylamidase in a known reaction.
• a solution was prepared containing BSPT hydrochloride, albumin, MPMS, Tris-HCl buffer and Tween-80 at concentrations within the ranges indicated in Table 1. To aliquots of this was added p-aminophenol.
• the graph of 4- aminophenol concentration against absorbance of 590 shows an
• Chloramphenicol was acetylated using acetyl Co-A and the enzyme CAT by a known reaction. This resulted in production of a quantity of Co-A which was quantitatively related to the amount of chloramphenicol originally present.
• the Co-A produced was assayed using the procedure of example 6. The absorbance at 590 nm against chloramphenicol concentration was linear over the chloramphenicol range 10 - 200 ⁇ M as shown in Fig 9.

Landscapes

• Health & Medical Sciences (AREA)
• Life Sciences & Earth Sciences (AREA)
• Engineering & Computer Science (AREA)
• Hematology (AREA)
• Immunology (AREA)
• Molecular Biology (AREA)
• Urology & Nephrology (AREA)
• Biomedical Technology (AREA)
• Chemical & Material Sciences (AREA)
• Physics & Mathematics (AREA)
• Cell Biology (AREA)
• Pathology (AREA)
• Food Science & Technology (AREA)
• Medicinal Chemistry (AREA)
• Biotechnology (AREA)
• Analytical Chemistry (AREA)
• Biochemistry (AREA)
• General Health & Medical Sciences (AREA)
• General Physics & Mathematics (AREA)
• Microbiology (AREA)
• Bioinformatics & Cheminformatics (AREA)
• Bioinformatics & Computational Biology (AREA)
• Biophysics (AREA)
• Proteomics, Peptides & Aminoacids (AREA)
• Investigating Or Analysing Biological Materials (AREA)
• Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
• Steroid Compounds (AREA)
• Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)

Applications Claiming Priority (2)

Publications (1)

Family

ID=10654837

Family Applications (1)

Country Status (9)

Families Citing this family (7)

Family Cites Families (2)

• 1989
  • 1989-04-12 GB GB898908182A patent/GB8908182D0/en active Pending
• 1990
  • 1990-04-11 HU HU903644A patent/HU903644D0/hu unknown
  • 1990-04-11 AU AU54279/90A patent/AU5427990A/en not_active Abandoned
  • 1990-04-11 JP JP2506014A patent/JPH04504467A/ja active Pending
  • 1990-04-11 CA CA002050884A patent/CA2050884A1/en not_active Abandoned
  • 1990-04-11 WO PCT/GB1990/000552 patent/WO1990012318A1/en not_active Application Discontinuation
  • 1990-04-11 EP EP90906274A patent/EP0467938A1/en not_active Withdrawn
• 1991
  • 1991-10-03 GB GB9121010A patent/GB2250026B/en not_active Expired - Fee Related
  • 1991-10-11 KR KR1019910701311A patent/KR920701821A/ko not_active Application Discontinuation
  • 1991-10-11 FI FI914812A patent/FI914812A0/fi unknown

Non-Patent Citations (1)

Also Published As

Similar Documents

Legal Events