Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
  • G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
  • G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
  • G01N21/78—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator producing a change of colour
• • 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
  • G01N33/72—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving blood pigments, e.g. haemoglobin, bilirubin or other porphyrins; involving occult blood
  • G01N33/721—Haemoglobin
  • G01N33/725—Haemoglobin using peroxidative activity
• • 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
  • C12Q1/26—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase
  • C12Q1/28—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase involving peroxidase

Definitions

• the present invention relates to an assay and kit for
• the present invention relates to an assay for determining the level of haptoglobin which is readily adaptable for running in a dry format where the reagents are dried onto a surface prior to running the assay.
• Haptoglobin is one of a group of proteins, known as acute phase proteins, whose concentration increases significantly following infection, inflammation or trauma. Measuring the concentration of haptoglobin in plasma therefore provides valuable diagnostic information as to the health status of the human or animal from which the sample is obtained and there has been much interest in the art in developing an assay for haptoglobin in plasma, serum and other biological fluids.
• Immunoassays utilising antibody based methods with antiserum specific for haptoglobin have been developed and are known in the art. Commonly, such assays are based on immunoturbidimetric assays with the haptoglobin concentration being determined via measurement of the formulation of an antibody-haptoglobin precipitate in solution. Enzyme-linked immunosorbent assays (ELISA) are another common embodiment of such assays. Not only do such immunoassays require a continuing supply of antiserum, however, but tests have to be validated for each separate species under investigation, rending immunoassay based methods commercially less attractive.
• haemoglobin are now routinely used in veterinary diagnostic laboratories. These are economically more viable as they require less expensive reagents than antibody based methods and also can be performed on all species.
• haemoglobin present and hence the haptoglobin content of the sample, can be determined.
• reagents such as protein binding inhibitors and reducing agents effective against disulphide bonds and/or chaotropic agents are employed to suppress the peroxidase activity of serum albumin which interferes with the assay at low blood concentrations of haptoglobin.
• US 4695552 describes a method for determining the haemoglobin- haptoglobin complex in the presence of free haemoglobin which involves determining peroxidase activity.
• the substrate for the peroxidase activity is hydrogen peroxide.
• Hydrogen peroxide is also used as a reagent together with 3, 3', 5,5'- tetramethyl benzidine in the haptoglobin determining method described in JP 2208568.
• the present invention provides an assay method for determining a level of haptoglobin in a sample comprising the steps of:
• step (ii) contacting the product of step (i) with reagents for generating hydrogen peroxide and one or more chromogens which undergo an optically detectable change when
• peroxidase activity is present, in the presence of a buffer
• haptoglobin-haemoglobin complex by measuring the change in an optical property of the reaction mixture
• steps (i) and (ii) are carried out concurrently, for example by forming a reaction mixture containing all of the sample to be assayed, haemoglobin, reagents for generating hydrogen peroxide, one or more
• chromogens which undergoes a spectroscopically detectable change when peroxidase activity is present, and a suitable buffer.
• the invention also provides a kit for use in a haptoglobin assay according to the present invention comprising haemoglobin, reagents for generating hydrogen peroxide, one or more
• chromogens which undergo an optically detectable change when peroxidase activity is present and a buffer.
• an assay method for determining the level of haptoglobin in a sample which exploits the endogenous activity of haptoglobin to bind to haemoglobin by determining the peroxidase activity of haemoglobin complexed to the haptoglobin but which avoids the need to include hydrogen peroxide as a reagent in order to provide a substrate for this peroxidase activity.
• the assay method according to the present invention may be any assay method according to the present invention.
• the sample for use in the assay may comprise other biological fluids such as peritoneal fluid, synovial fluid, cerebrospinal fluid milk.
• the sample may be obtained from a mammal including a human .
• the haptoglobin concentration in the sample is in the range of from 0.02 mg/ml to 1.4 mg/ml.
• the haemoglobin for use in the assay method according to the invention may be obtained from the same animal from which the sample for assay is obtained or it may be obtained from a different species.
• the haemoglobin is met-haemoglobin .
• the haptoglobin in the sample and the added haemoglobin react together to form a haptoglobin-haemoglobin complex.
• the assay reaction mixture is incubated for up to 20 minutes, for example from 5 to 20 minutes.
• Formation of the haptoglobin-haemoglobin complex according to the method of the invention is detected by determining the peroxidase activity of the complex by measuring the change in an optical property of the reaction mixture.
• peroxidase activity of the haptoglobin-haemoglobin complex can then be correlated with the amount of haptoglobin in the sample.
• the peroxidase activity of the haptoglobin-haemoglobin complex is determined by generating hydrogen peroxide in situ in the assay to form a substrate for the peroxidase activity of the complex and detecting the peroxidase activity using a chromogen which undergoes a spectroscopically detectable change when peroxidase activity is present.
• the reagents for generating hydrogen peroxide for use according to the present invention suitably comprise an enzyme that catalyses a reaction which produces hydrogen peroxide as a reaction product together with a substrate for that enzyme.
• the reagents for generating hydrogen peroxide comprise the enzyme glucose oxidase and the substrate glucose.
• cholesterol /cholesterol oxidase or urea/urea oxidase cholesterol /cholesterol oxidase or urea/urea oxidase.
• Chromogens which undergo a spectroscopically detectable change when peroxidase activity is present are well known in the art and are described, for example, in EP 1031035B1 discussed above.
• haemoglobin levels may conveniently be used in the assay according to the present invention.
• the peroxidase activity of the haemoglobin-haptoglobin complex of the assay according to the invention is determined using a chromogen which undergoes a colour change which may be detected spectrophotometrically when peroxidase activity is present such as phenol, 4-iodophenol , 3-aminophenozone , 8-anilinonaphthalene sulphonic acid (ANS) , 4-aminoantipyrine (AAP) , 2-amino-4- hydroxybenzenesulphonic acid (AHBS) , tetramethyl benzidine (TMB) , O-phenylene diamine dihydrochloride , O-dianisidine , sodium-2-hydroxy-3, 5-dichlorobenzene sulphonate, 2,2'-azino- di (3-ethylbenzthiazoline-6-sulphonic acid (ABTS) or mixtures thereof
• the chromogenic substrate for use in the assay according to the present invention comprises a combination of phenol, 8-anilinonaphthalene sulphonic acid (ANS) and 4- aminoantipyrine .
• Detectable colour changes can be detected with chrornogens present in a variety of concentration ranges conventional in the art .
• Both the peroxidase activity of the haptoglobin-haemoglobin complex and the activity of the reagents generating the hydrogen peroxide in situ are pH dependent and the challenge addressed by the present invention is therefore to find a pH range in which both activities are operative.
• peroxidase activity of uncomplexed haemoglobin can be inactivated at low pH. As reported in Jpn. J Vet. Sci, 44, pl5-21 (1982), this activity can be inactivated at pH 4.1. It has been suggested, however, that peroxidase activity resulting from free haemoglobin may remain at such a pH and the assay described in EP 1031035B1 discussed above is preferably carried out at a pH below 4.1, especially at pH 3.8.
• the present inventors have however found that by performing the assay method of the present invention in the presence of a buffer at a pH in the range of from 3.9 to 4.5, the peroxidase activity of any uncomplexed haemoglobin remaining in the assay mixture can be substantially suppressed without inhibiting in situ hydrogen peroxide generation.
• the assay method according to the present invention is performed in the presence of a buffer at a pH in the range of from 4 to 4.5.
• the assay method of the invention is performed at a pH of 4.1.
• Any buffer conventional in the art may be employed to maintain the desired pH for the assay.
• a mixed citrate- phosphate buffer may be employed with the phosphate buffer (pH 7.4) being titrated to the desired pH with citrate buffer (pH 3.8) .
• the assay method of the invention is performed in the presence of one or more additional reagents for reducing the peroxidase effect due to any albumin or other proteins in the sample.
• additional reagent or reagents will be added in an amount which is insufficient to substantially inhibit formation of the haptoglobin-haemoglobin complex.
• Suitable additional reagents for reducing the peroxidase effect due to any albumin or other proteins in the sample are as described in EP 1031035B1 and include for example, protein binding inhibitors, reducing agents effective against disulphide bonds and chaotropic agents.
• the additional reagent or reagents may be present in a concentration of from 0.1 to 0.5mM.
• the assay method according to the present invention is performed in the presence of a protein binding inhibitor such as 8-anilinonaphthalene sulphonic acid (ANS) , protoporhyrin, bilirubin, taurodeoxycholic acids (bile),
• a protein binding inhibitor such as 8-anilinonaphthalene sulphonic acid (ANS) , protoporhyrin, bilirubin, taurodeoxycholic acids (bile
• the additional reducing agent effective against disulphide bonds is selected from dithiothreitol , dithioerythritol , cysteine, mercaptoethanol , glutathione, 4,4'- dithiopyridine or 5 , 5' -dithio ( 2-nitrobenzoic acid).
• the assay method according to the present invention is performed in the presence of a suitable chaotropic agent such as guanidine hydrochloride, potassium thiocyanate or sodium chloride.
• a suitable chaotropic agent such as guanidine hydrochloride, potassium thiocyanate or sodium chloride.
• the assay according to the invention suitably further comprises a detergent to help maintain the other components in solution.
• a detergent is added to the assay mixture in a low concentration, suitably in an amount of up to 0.3% (v/v)
• Suitable detergents which may be employed include non-ionic and ionic surfactants conventional in the art.
• the detergent for addition to the assay mixture comprises a non-ionic surfactant such as a polyoxylene sorbitol ester (for example TweenTM 20, 40, 60, 80) , a
• polyoxyethylene alcohol for example BrijTM 35,36
• a mixture thereof e
• the detergent comprises one or more ionic surfactants such as sodium dodecyl sulphate or cetrimide.
• the assay reagents may be added together in any sequence provided that the spectroscopically detectable change in the chromogen does not occur prior to initiation of the assay.
• the reagents may be pre- mixed in various stable combinations and brought together with the sample to perform the assay.
• An antibacterial agent may be included with one or more of the reagents or reagent mixtures to act as a preservative. Suitable ingredients include, for example, triclosan or thiomersalate .
• first and second reagent mixtures are provided and the assay is performed by forming a mixture of the sample to be assayed and the first and second reagent mixtures.
• the first reagent mixture comprises
• haemoglobin and an enzyme that catalyses a reaction which produces hydrogen peroxide as a reaction product and the second reagent mixture comprises one or more chromogens and a substrate for the enzyme of the first mixture.
• the enzyme comprises glucose oxidase and the enzyme substrate comprises glucose .
• one or more of the chromogens is included in the first reagent mixture together with the haemoglobin and an enzyme that catalyses a reaction which produces hydrogen peroxide as a reaction product.
• some or all of the component assay reagents can be combined in dry form and then added to the aqueous sample in order to perform the assay.
• assay reagent combinations may be prepared in solution and freeze-dried onto a solid surface such as paper or an assay stick .
• a dry format such as this is particularly convenient for storage and portability and this represents a significant advantage for the method of the invention compared to prior art methods as this cannot be achieved where hydrogen peroxide is required as one of the reagents.
• the level of peroxidase activity determined for the haptoglobin- haemoglobin complex formed in the assay method according to the invention can be correlated with the level of haptoglobin in the sample by reference to a standard curve generated using known concentrations of haptoglobin.
• kit for use in the method according to the invention may suitably comprise further components such as standards of serum with known haptoglobin concentrations.
• the assay method according to the invention may suitably be performed using equipment such as test tubes or microtitre plates.
• the assay may be performed using an automated biochemical analyser.
• any feature disclosed herein may be replaced by an alternative feature serving the same or a similar purpose.
• Equine haemoglobin at 30 mg/ml prepared according to Makimura & Susuki, (1982. Jap J Vet Sci 44:15-21) in ultrapure water Glucose oxidase (Sigma) lOmg/ml in phosphate buffer
• Reagent B chromogen/glucose
• DTT Dithiothreitol
• FCS Fcetal calf serum
• BSA Bovine serum albumin 2% (w/v)
• BSA Bovine serum with haptoglobin at 1.4 mg/ml
• Bovine and canine serum samples Bovine and canine serum samples.
• a 50 ⁇ 1 aliquot of equine haemoglobin stock solution was added to 25ml of phosphate buffer. 1.25 ml of glucose oxidase stock solution was added to 10ml of the diluted haemoglobin solution.
• haemoglobin/glucose oxidase reagent A and ⁇ of chromogen reagent B incubated 10 min at room temperature and the
• Haptoglobin in serum produced a dark blue colour in the reaction while FCS and water gave minimal reaction.
• Example 2 Comparison of DTT to Cys as reducing agent DTT is known to be the most unstable of the reagent mix and an alternative reagent capable of reducing SS double bonds would enhance stability of the haptoglobin assay.
• Buffers for reagent B were prepared by mixing citrate buffer and phosphate buffer as below (Table 3) and adding other chemicals as for Bi DTT in Example 2 with DTT as the reducing agent.
• the pH of the buffer mixes (B2-B5) was determined after addition of all chemicals. Samples (5yl)were placed in wells followed by 90 ⁇ 1 of haemoglobin/glucose oxidase reagent A and 90 ⁇ 1 of chromogen B3 - B6, incubated 20 min at room temperature and the absorbance was measured on EL I SA reader at 600nm Table 3: pH of reaction buffers
• Buffers (B6-B10) of differing pH as listed in Table 5 were prepared by titrating phosphate buffer pH 7.4 with citrate buffer pH 3.8 and adding reagents as under Example 2 Reagent Bi DTT . Samples (4 ⁇ 1) were placed in wells followed by 75 ⁇ 1 of haemoglobin/glucose oxidase reagent A and 75 ⁇ 1 of chromogen B2 - B5, incubated 30 min at room temperature and absorbance measured on ELISA reader at 600nm
• the reagents optimised on microtitre plate were used on the automated biochemistry analyser (MIRA, Roche) according to the following method: -
• Reagent B12 as the 2nd reagent to be added. Assay parameters were set so that
• Additions should be 1st reagent, add sample, add reagent B12 and read at cycle 1-15 (25 sec apart) at 600nm.
• the change in absorbance at 600nm between cycle 2 and cycle 15 (350 sec) is the reading ( ⁇ 60 ⁇ )
• the reagents are usable in a dry chemistry format. In order to minimise the interaction of the reagents before dry they were added in order and after the final reagent were immediately frozen prior to drying under a vacuum (lyophilisation) .
• Reagent A (0.025 ml) was dispensed as spots on to filter paper (Biorad) and allowed to air dry and placed in a plastic bag on dry ice for 60 min. Ice cold Reagent B (0.025ml) was dispensed as spots on top of the previously dispensed Reagent A, replaced in the plastic bag on dry ice for 30 min. The filter paper was placed in a freeze drier overnight.
• Bovine serum (0.01 ml) with haptoglobin at 0.74and 0.35 g/1 (1:2 and 1:4 dilution of the 1.4 g/1 standard used in example 4) and BSA at 5 g/1 and 2.5 g/1 placed on the dried spots and incubated for about 5 min with colour development recorded by scanning.
• the new reagents when dried onto filter paper gave a deep blue/purple reaction with 5 min of application of a sample containing Haptoglobin but a negative reaction with BSA.
• Reagent A Haemoglobin/glucose oxidase/aminoantipyrine
• Glucose oxidase (Sigma) 0.6 mg/ml (105 U/ml)
• Equine haemoglobin 0.26 mg/ml
• Triclosan (Fluka) 0.0001% (v/v)
• Triclosan (Fluka) 0.0001 % (v/v)
• Reagent 1 A2 : 200 ⁇
• Reagent 2 B13: 90 ⁇
• Samples 1-5 are from a calf during an acute phase reaction.
• Example 7 Optimised Wet Chemistry Assay Canine samples
• Reagents as in Example 6 used on a Pentra 400 (Horiba Abx Ltd) analyser to determine haptoglobin in canine serum samples. Samples were diluted 1:10 prior to analysis in 0.9% (w/v) NaCl and analysed in duplicate. Results given are after calculation to adjust for the dilution and are the mean of the duplicates.
• Reagent 1 A2 : 150ul
• Reagent 2 B13: 60ul Blank and Calibrator Diluent: 2% (w/v) bovine serum albumin (BSA)
• Triclosan (Fluka) 0.0001% (v/v)
• Calibrators haptoglobin at 1.48 g/L, 0.74 g/L, 0.37 g/L, 0.19 g/L diluted in calibrator diluent,
• Samples 6-7 are from dogs with haptoglobin in the range found in healthy dogs and samples 8-9 are from dogs with inflammatory or infectious conditions.

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  • 2010-12-20 GB GBGB1021509.3A patent/GB201021509D0/en not_active Ceased
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