Method and reagent for the determination of fructosamine

Abstract: A system for determining the concentration of fructosamine in sera which consists of a first reagent in which a tetrazolium salt which reduces all reactive substances in sera including fructosamine and a second reagent which is responsive to all reactive substance in sera other than fructosamine. The difference in color change as between the two allows the determination of concentration of fructosamine.

Description: Field of the Invention Present invention relates to a stable single reagent system for rapid determination of fructosamine concentration in sera. Background of Invention Fructosamine is an index of the existence of diabetes mellitus in humans. Its measure can be effectively used to reflect patient compliance with treatment, quality of care and efficacy of insulin therapy. Fructosamine is a product of the interaction of serum glucose with serum proteins, wherein the glucose binds to the amino groups of proteins to form an aldimine, a Schiff base, which undergoes molecular rearrangement to form a stable ketoamine also known as fructosamine. The structure, analysis and clinical usefulness of fructosamine is described by Armbruster in Clinical Chemistry , Vol 33. No. 12, pg. 2153-63 (1987) incorporated herein by reference. A method and composition for direct determination fructosamine in blood samples are described in U.S. Patents 4,642,295 and 4,645,742 to Baker. The method for determining the level of fructosamine in a blood sample or a sample derived from blood generally involves maintaining the sample at a temperature below 50°C and at a pH between 10 and 11 and adding a coloring agent (nitro blue tetrazolium or NBT) to the sample. After a first time delay of 10 minutes a first color measurement is taken at a predetermined wavelength of 550 nm and after a second time delay of 2 additional minutes a second color measurement at the predetermined wavelength is taken. The fructosamine level in the sample is determined by comparing the change between the first and second color measurements. This system is employed by Roche and sold as the RoTAG ™ plus fructosamine assay. It consists of a buffer (pH 10.3), NBT tablets and a calibrator in solid form. The dye when dissolved is stated to be stable for up to 2 weeks at 2-8°C and the calibrator when reconstituted is stated to be stable for up to 4 weeks at 2-8°C. A principal shortcoming of the system is a long response time. Conventionally the art desires an assay to be complete in less than 10 minutes and automated analyzers are designated to operate on that cycle. Longer time spans require an undesirable modification to an analysis system and a need exists for an accurate assay which would enable completion within 10 minutes at the normal assay temperature of 37°C. A separate need is accuracy of the assay. It has been the ongoing quest of the assignee of this invention to develop time stable, single-liquid reagent assay compositions in which each unit is formulated at the source of manufacture and has a shelf life sufficient to remain stable from time of manufacture, storage at the point of manufacture, shipment to any destination in the world and storage at the destination until use. Products to obtain commercial acceptability must have a shelf life of 12 months or more at 2 to 10°C more typically 18 months at 2 to 10°C, which corresponds to a shelf life of at least about 3 days at 41°C. As indicated, to be commercially adaptive to all spectrophotometric instruments, the compositions must have response time within 10 minutes at 37°C. It is therefore, the object of this invention to provide a time stable liquid single reagent system for the determination of fructosamine in sera which has a commercially acceptable response time and a stability which is equivalent to at least 12 months at 2 to 10°C. Summary of the Invention The present invention is directed to a novel single reagent system for the rapid determination of fructosamine in sera and procedure therefore. The novel single reagent system consists of two separate factory formulated reagents preferably supplied with a standard and more preferably with controls for use in the accurate determination of fructosamine in human sera in less than 10 minutes at 37°C. The first reagent ("Reagent A" herein) is responsive to all the reactive substances in sera. The second reagent, ("Reagent B" herein), contains a compound that binds to fructosamine and inhibits the ability of fructosamine to reduce the tetrazolium salt. Reagent B therefore measures all reactive substances except fructosamine that are capable of reducing the tetrazolium salt. The absorbance change obtained with Reagent A less the absorbance change determined with Reagent B gives a net absorbance (Net Delta ABS) which is a measure fructosamine content of the sera. The concentration of fructosamine in the sample is determined by multiplying the Net Delta ABS by a factor (F) the ratio of fructosamine level of a standard to net absorbance of the standard determined using Reagent A and Reagent B. Presently preferred composition of Reagent A is about 0.5mM or 252.85 mg/l p-Iodonitrotetrazolium Violet (INT), 0.1M or 12.4 g/l Sodium Carbonate Monohydrate (Na 2 CO 3 ) 0.1M or 8.4 g/l Sodium Bicarbonate (NaHCO 3 ), 0.1M or 6.0 g/l Urea and 1.0% v/v or 10 ml/l Triton® X-100 and 1.0% or 10 g/l tergitol NP 40 contained in the deionized water. Solution pH is about 11.0. A preferred Reagent B is identical to said Reagent A, but contains 0.12 M or 7.416 g/l Boric Acid (H 3 BO 3 ) and also has a pH of about 11.0. The assay protocol is to measure the color change (absorbance) of Reagent A after 2 and 4 or 3 and 5 minutes of reaction and Reagent B at the same 2 and 4 or 3 and 5 minute intervals at a wavelength of about 500 nm at a standard temperature 37°C. The difference in absorbance (ABS) for Reagent A and Reagent B are determined and a differential between the two differentials is then determined. The differential is multiplied by the factor (F) to determine the concentration of fructosamine in micromoles (umol). In the system, the use of INT in Reagent A enables reduction of INT by fructosamine and all other interfering substance while Reagent B causes reduction of INT by the interfering substances other than fructosamine. By differential therefore, the amount of fructosamine is determined. This indirect system of determining fructosamine accounts for all of the reactive substances and in so doing effectively eliminates the influence of the reactive substances in a fructosamine determination procedure. Most significantly, the system enables the use of any absorbance spectrophotometer for the determination of fructosamine in sera since the analysis can be accomplished within in the commercially desired 10 minute time frame. Brief Description of the Drawings FIG. 1 shows the linearity of the two single reagent fructosamine assay system of the invention; FIG. 2 illustrates retention of linearity for diluted sera; and FIGS. 3 and 4 correlate the performance of the Fructosamine Assay System of the invention against RoTAG plus a fructosamine assay manufactured and sold by Roche. Detailed Description The present invention is directed to the determination of fructosamine concentration in sera. It is based on the use of one reagent which is reacted with (reduced by) fructosamine in addition to the most common reactive substances in sera and a second reagent which reacts with the same reactive substances but not fructosamine. By differential therefore, the fructosamine concentration in sera can be determined without the analysis being impacted to any significant degree by the reactive substances. Each reagent contains the same color producing substance preferably p-Iodonitrotetrazolium Violet (INT). The second reagent is identical to the first but contains a compound which prevents fructosamine from reducing the tetrazolium salt. By the term reactive substances as used herein there is meant as any substance that may be present in the sample which may be capable of reducing a tetrazolium salt. Such substances include glucose, hemoglobin, bilirubin, ascorbic acid, uric acid, triglycerides, and the like. Using standard commercial spectrophotometers, the absorbance change for the first reagent minus the absorption change for a second reagent gives a net absorbance change (Net Delta ABS) due to fructosamine. Concentration of fructosamine is then determined by multiplying the net absorbance change by a factor (F) which is derived by dividing the fructosamine concentration of a known standard by the net absorbance change of the standard as determined by measurements taken with Reagent A and Reagent B according to the formula F = Fructosamine Conc. of Standard (Net ABS Reagent A - Net ABS Reagent B) with fructosamine concentration of the same being determined by the equation: F x Net Delta ABS (Sample) = umol fructosamine. Each composition used in the system is a single liquid reagent. The reagents (Reagent A and B ) require no dilution. Quality control is at the point of manufacture. They are aqueous solutions of a carbonate buffer system to maintain reagent pH in the range of about 8.5 to about 11.5 and preferably about 11. Use of other buffer systems such as AMP or CAPS, results in the loss of sensitivity and an increase in average increase in interference. The tetrazolium salt used in this invention is a compound which will be reduced by fructosamine and interfering substances in the absence of boric acid to a colored formazan product but will only be reduced by the interfering substance other than fructosamine in the presence of boric acid. The preferred tetrazolium salt is p-Iodonitrotetrazolium Violet (INT). Urea is provided in an amount sufficient to minimize interference due to ascorbic acid. Triton® X-100 (Octylphenoxy polyethoxyethanol a non-ionic surfactant having an HLB value of 13.5) solubilizes the products of reduction and increases reagent sensitivity by at least about 30% as compared to a solution without Triton® X-100. There may be optionally present another non-ionic surfactant Tergitol® NP-40 (an ethoxylated nonylphenol having an HLB value of 17.8) to clarify the sample and minimize interference due to elevated serum triglyceride levels, and which also increases sensitivity. Boric acid is provided to complex with fructosamine and inhibit the reduction of INT by fructosamine. Any other compound which inhibits reduction of INT by fructosamine but not the other reactive substances may be used in place of boric acid. The carbonate buffer can be used in a concentration of from about 0.01 to about 0.4 M, tetrazolium salt (INT) in a concentration of about 0.1 to 5 mM, urea in a concentration of 0.1 to about 1 M, Triton® X-100 in a concentration of 0.1 to about 4% by v/v, Tergitol NP-40 in a concentration of about 0 to about 3% v/v of solution. For the non-fructosamine responsive reagent (Reagent B) boric acid is employed in a concentration of about 0.05 to about 1 M. Functional pH range is about 8.5 to about 11.5, preferably 11. At a pH less than about 8.5 sensitivity decreases and the ascorbic acid interference increases. At pH above 11.5 glucose interferes substantially. The fructosamine analytical systems of this invention can be sold as a two component kit with or without a standard which can be separately sold and with or without upper level and lower level controls for calibrating a system which can be sold with the kit or separately. The reagents, standards and control employed in the instant invention have been established to withstand a stress of three days at 47°C which correlates to a life time of at least 15 to 18 months at 2-10°C, the normal temperature at which they stored. Being in liquid form there is eliminated the error of reconstituting a lyophilized product and all quality control is exercised at the site of manufacture for uniform performance. More significantly, an assay can be completed in 5 minutes at 37°C which makes the system adaptive to continuous analyzers which is not the case for the system sold by Roche under the trademark RoTAG. A comparison of the two systems is shown in Table 1 below. Criteria This Invention RoTAG Reagent Type Liquid Single Liquid Two part & Tablet Blank Yes No Calibrator Single Point Liquid Single Point Lyophilized Controls Two Levels Liquid Two Levels Lyophilized Stability 15 months at 4°C Reagent: 2 weeks after reconst. Cal/Control:4 weeks after reconst. Kinetics Spline Fit Spline Fit Linearity 10-1600 µmol To 1000 µmol Temperature of Reaction 37°C 37°C Response Time < 5 Minutes 10-12 Minutes Specificity Excellent Good Precision: 280 µmol % CV Run to Run 2.8 2.4 With-In Run 2.9 1.9 500 µmol Run to Run 2.8 1.7 With-In Run 2.4 2.3 Fructosamine Normal Range 196-279 umol 200-272 µmol The preferred composition Reagent A, which Table 1 is based on, is one containing 0.5 mM p-Iodonitrotetrazolium Violet (INT), 0.1 M Sodium Carbonate Monohydrate (Na 2 CO 3 ), 0.1 M Sodium Bicarbonate (NaHCO 3 ), 0.1 M Urea, 1.0 v/v Triton® X-100 and 1% Tergitol NP 40 dissolved in deionized water and adjusted to a pH of 11.0. The preferred composition of Reagent B is identical to the Reagent A except that it contains about 0.12 M boric acid (H 3 BO 3 ) also a pH at 11.0. Reagent A in addition to being reduced by fructosamine will also be reduced by glucose, hemoglobin, bilirubin, ascorbic acid, uric acid and triglycerides within a time frame of 5 minutes or less enabling absorbance color change determinations to be made at time spans of 2 and 4 minutes, preferably 3 and 5 minutes to enable a differential absorbance (Net Delta ABS) for the reduction products to be obtained. Using Reagent B the same substances are reacted with and produce an absorbance color change in the tetrazolium salt but the reaction does not involve fructosamine which is prevented from reacting with the tetrazolium salt by boric acid. The preferred assay procedure of the instant invention involves adding to a sample of sera a Reagent A in which INT is reduced by fructosamine and the interfering substances to provide a change in the absorbance as a function of time of, determining the change in absorbance after a given time 2 and 4 or 3 and 5 minute intervals, simultaneously or sequentially reacting another sample of the same sera containing a composition identical to Reagent A but also containing boric acid which prevents the reduction of the tetrazolium salt with fructosamine, and measuring the absorbance for the same time interval as the first sample. The difference in absorbance for the first sample which is responsive to fructosamine (First Delta ABS) less the difference in absorbance to the second same which is non-responsive to the presence of fructosamine (Second Delta ABS) gives a Net Delta ABS which is multiplied by the Factor (F) to give fructosamine concentration in micromoles (µmol). Alternately the micromoles concentration can be determined for each sample with the difference being the fructosamine concentration of the sample. The fructosamine Assay system of the invention preferably consists of two reagents Reagent A and B which are normally provided with a standard calibrator, a normal level control and a high level control. The preparation and use of which are described below. Preparation of Reagent A With constant stirring there was sequentially combined at room temperature the following: 800 ml Deionized H 2 O 252.85 mg p-Iodonitrotetrazolium Violet 12.4 g Na 2 CO 3 Monohydrate 8.4 g NaHCO 3 6.0 g Urea 10.0 ml Triton® X-100 10 g Tergitol NP 40 Solution pH was adjusted to 11.0 with NaOH with constant stirring. Preparation of Reagent B With constant stirring at room temperature the following were combined: 800 ml Deionized H 2 O 252.85 mg p-Iodonitrotetrazolium Violet 12.4 g Na 2 CO 3 Monohydrate 8.4 g NaHCo 3 6.0 g Urea 10.0 ml Triton® X-100 7.416 g H 3 BO 3 (Boric Acid) 10 g Tergitol NP 40 As with Reagent A pH was adjusted to 11.0 with NaOH with constant stirring. Preparation of Calibrator/Control Highly Glycated Fructosamine Intermediate With constant stirring at room temperature the following were combined: 1. Filtered 50 milliliters of human serum through a 0.2 µm cellulose nitrate filter: 2. 0.50 gram Human Albumin Powder Fraction V (Armour Pharmaceutical Co.). 3. 7.20 grams of D-Glucose. 4. 0.01 g NaN 3 (Sodium Azide). Each constituent was added separately with stirring until it went into solution. The solution was sealed in its container with parafilm and incubated for 4 days (96 hours) at 37° C. The solution was dialyzed against 1 liter of deionized water at room temperature for 4 hours, changing the water every hour using a Spectrapor® semi-permeable membranes, (M.W. Cutoff 12-14,000), where M.W. = molecular weight. After 4 hours, the water was changed one more time and dialysis continued at 4°C for an additional 20 hours. At the end of 20 hours the solution was warmed to room temperature, filtered through a 0.2 micron cellulose nitrate filter (Whatman). Human Serum Diluent Intermediate A highly glycated fructosamine intermediate for use in formulating of controls and calibrators was prepared using the procedure described for the Highly glycated fructosamine except that using 100 ml of human serum was employed and dialysis was with 2 liters of deionized water. Working Fructosamine Standard Formulation The highly glycated fructosamine intermediate was diluted with the human serum diluent intermediate in proportion of 1:40 v/v and stabilized by adding 0.5g of human albumin powder fraction V, 25 mg of D-glucose and 0.5 ml of Additive AB for each 50 ml of solution. The resultant stabilized standard was filtered through a 0.2 µm cellulose nitrate filter (Whatman). Fructosamine value was in the range of 378-462 µmol when assayed. Normal Level Control Formulation The highly glycated fructosamine intermediate was diluted with the human serum diluent intermediate by adding 2.5 ml of glycated fructosamine to 47.5 ml of Diluent. The mixture was stabilized by adding 0.5 g of human albumin powder fraction V, 25 mg of D-glucose and 0.5 ml of additive AB and filtered through a 0.2 µm cellulose nitrate filter (Whatman). Fructosamine value was in the range of 229-281 µmol when assayed. Abnormal High Level Control The procedure for formulating the normal level control was followed except that 14 ml glycated fructosamine, 36 ml of diluent was employed. Fructosamine level was 457-559 µmol. Glycation of Polylysine With constant stirring at room temperature there was combined: 1. 33 milliliters of 0.1M phosphate buffered saline (pH 7.4). 2. 1.0 grams poly-l-lysine. 3. 4.75 grams of D-glucose. 4. 0.007 gram of NaN 3 (sodium azide) Each was separately dissolved and the container parafilmed and incubated at 37°C for 4 days (96 hours). After 4 days the solution was dialyzed against 1 liter of deionized water at room temperature for 4 hours, changing the water every hour using Spectrapor® semi-permeable membranes, (M. W. Cutoff 12-14,000). At the end of 20 hours the solution was warmed to room temperature and filtered through a 0.2 µm cellulose nitrate filter (Whatman). The procedure makes a highly glycated polylysine compound that can be diluted with 0.9% NaCl to obtain a linearity set. Value Assignment Protocol Using the Roche RoTAG plus as the reference reagent for value assignment, the fructosamine standard, normal and abnormal controls and linearity set were evaluated according to instructions as detailed by Roche at 500 nm. The instrument employed was Gilford® Stasar III using mode ABS at a wavelength of 500 nm and an assay temperature of 37°C. Reagent volume was 1.0 ml, sample volume was 100 µl. The first ABS reading was taken at 3 minutes and a final ABS reading taken at 5 minutes. The procedure was to allow all reagents and standards to come to room temperature; equilibrate the spectrophotometer to 37°C; zero the spectrophotometer at 500 nm with deionized water. For each sample 1.0 ml of Reagent A (or Reagent B) was dispensed into a test tube. 100 µl of sample, standard or control was then added to each test tube. The reaction mixture was mixed and immediately aspirated into a flow cell. Absorbance values at 3 minutes and 5 minutes after aspiration were recorded. The Absorbance (ABS) at 3 minutes and 5 minutes for all tests was determined by the formula: (5 Min. ABS) - (3 Min. ABS) = Delta ABS (Delta ABS for Reagent A) - (Delta ABS for Reagent B) = Net Delta ABS. A calculation factor (F) was determined by dividing the concentration of the standard by the Net Delta ABS of the standard. EXAMPLE: F = 350 Net Delta ABS By multiplying the Net Delta ABS of each sample and control by F determines the fructosamine concentration of each sample and control according to the formula. F x Net Delta ABS = umol fructosamine The assay system linearity was assayed for linearity using 300 µmol calibrator and five levels of human serum pool spiked with various amounts of fructosamine. Table 2 and FIG. 1 summarize the results. REAGENT LINEARITY Assayed on the Cobas Mira Protocol: 500 nm, 37°C, RV=200 µl, SV=20 µl Absorbance Measured at 3 and 5 minutes Sample Actual Delta ABS Net [µmol] ActualTheoretical [µmol] 300 Std 0.0410 300 --- Mix of 1&5 1 0.0348 255 255 1 2 0.0614 449 439 [(3x1) + 5] /4 3 0.0863 632 623 (1 + 5)/2 4 0.1115 816 806 [ 1 + (3x5)]/5 5 0.1353 990 990 5 Linearity/Dynamic Range Protein glycation occurs at the epsilon amino groups of lysines. To achieve a fructosamine level to challenge linearity glucose was incubated with polylysine. Each lysine residue is a potential site of glycation. This concentrated glycated polylysine was diluted in 0.9% NaCl to obtain six levels of glycated polylysine as reported in Table 3. Glycated Polylysine Dilution Protocol Factor Dilution Level = Dilution with NaCl 1 1X Glyc. Poly. + 19X NaCl 0.05 2 1X Glyc. Poly. + 9X NaCl 0.10 3 1X Glyc. Poly. + 3X NaCl 0.25 4 1X Glyc. Poly. + 1X Nacl 0.50 5 1X Glyc. Poly + 1X NaCl 0.75 6 Undiluted Glyc. Poly. (Neat) 1.00 Level 4 (Dilution Factor 0.50) = 778 µmol per Roche RoTAG Table 4 and FIG. 2 summarize the linearity results which establishes that the system is functional for diluted sera. LINEARITY/DYNAMIC RANGE Assayed on the Cobas Mira MAS protocol: 500 nm, 37°C, RV=200 µl, SV=20 µl Absorbance Measured at 3 and 5 Minutes Sample Dilution Factor Net Delta ABS µmol fructosamine 300 Std / 0.0399 300 1 0.05 0.0093 70 2 0.10 0.0217 163 3 0.25 0.0590 444 4 0.50 0.1163 874 5 0.75 0.1784 1341 6 1.00 0.2310 1737 Precision Biocell Blood Bank Segments Pool was spiked with glycated human serum to levels of 280 µmol and 500 µmol fructosamine for use in precision (Run to run) and within-run studies. Table 4 lists the results of Run to Run Performance. RUN TO RUN PERFORMANCE Assayed on the Cobas Mira Protocol: 500 nm, 37°C, RV=200 µl, SV=20 µl Absorbance Measured at 3 and 5 Minutes Standard: In-House 300 µmol Based on Single Calibration Day 1 Daily Value is Average of Triplicate Analyses Day 280 µmol 500 µmol 1 272 492 2 277 513 3 288 505 4 284 478 5 270 488 N 5 5 Minimum 270 478 Maximum 288 513 Mean 278 495 Std Dev 7.7 13.9 % CV 2.8 2.3 The results are excellent for a two reagent system. It also appears that the system can hold a calibration for at least 5 days. Within Run performance was also evaluated with the same two samples. Table 5 summarizes the results. WITH-IN RUN REFERENCE Assayed on the Cobas Mira Protocol: 500 nm, 37°C, RV=200 µl, SV=20 µl Reaction Measured from at 3 and 5 minutes Standard: 300 µmol Sample Repetitions 280 µmol 294 293 289 297 285 282 277 272 291 272 292 276 285 304 289 294 290 290 288 281 500 µmol 501 491 494 504 514 511 497 504 515 523 495 494 503 507 510 511 475 515 525 498 280 µmol 500 µmol N 20 20 Minimum 272 475 Maximum 304 525 Mean 287 504 Std Dev 8.4 11.9 % CV 2.9 2.4 The With-In Run precision is excellent. Correlation For correlation study 122 samples were assayed. 85 Normal Patients from Biocell Blood Bank Segment 18 Diabetic Patients 10 Randomly Glycated samples from LRRMC 9 Related Levels of Glycated Biocell Pool Each sample was assayed in triplicate. Each sample was assayed with fructosamine assay kept at 4°C and Roche RoTAG Plus. The results are tabulated and presented in Table 6. PATIENT SAMPLE CORRELATION FRUCTOSAMINE SYSTEM (FS) vs ROCHE Assayed on the Cobas Mira 37°C FS: RV = 200 µl, SV = 20 µl Wavelength 500 nm Standard: 300 µmol Absorbance Measured at 3 and 5 minutes ROCHE: RV = 200 µl, Dil. Vol. = 30 µl, SV = 10 µl Wavelength 550 nm Standard: Roche 442 µmol Absorbance Measured at 10 and 12 minutes Normal Samples Roche µmol FS µmol 1 195 176 2 188 184 3 216 245 4 165 149 5 220 178 6 216 239 7 219 209 8 234 265 9 215 245 10 232 241 11 224 234 12 231 232 13 240 250 14 174 165 15 240 247 16 240 197 17 229 206 18 202 193 19 206 202 20 222 190 21 282 263 22 262 246 23 251 215 24 252 214 25 245 194 26 276 220 27 196 198 28 238 218 29 253 259 30 212 231 31 219 211 32 219 226 33 235 237 34 192 201 35 210 230 36 212 221 37 240 229 38 253 253 39 244 266 40 242 244 41 171 180 42 233 250 43 228 239 44 196 250 45 208 242 46 202 220 47 209 209 48 252 258 49 245 227 50 191 203 51 222 241 52 246 240 53 473 495 54 214 217 55 225 233 56 226 211 57 244 226 58 243 263 59 226 260 60 208 229 61 243 236 62 200 197 63 171 168 64 215 214 65 284 215 66 248 275 67 269 289 68 203 204 69 234 234 70 222 223 71 209 242 72 227 219 73 203 215 74 227 245 75 225 238 76 209 202 77 229 206 78 224 216 79 230 255 80 247 203 81 189 152 82 251 198 83 344 357 84 171 174 85 616 671 Diabetic Samples 86 375 365 87 295 278 88 374 379 89 254 250 90 397 450 91 291 277 92 377 383 93 377 383 94 367 359 95 250 243 96 395 432 97 283 278 98 378 400 99 282 283 100 376 390 101 244 238 102 386 447 103 278 266 Glyc. LRRMC 104 291 256 105 473 477 106 310 343 107 327 360 108 451 476 109 513 541 110 371 335 111 489 485 112 286 266 113 345 342 Glyc. Biocell 114 286 262 115 403 389 116 534 517 117 668 673 118 799 835 119 917 938 120 1102 1100 121 1180 1196 122 1304 1357 N 122 122 Minimum 165 149 Maximum 1304 1357 Mean 301 302 Std Dev 185 192 Linear Regression Slope = 1.032883 Std Err = 0.011610 Y-Intercept = -8.67445 Std Err = 23.57628 Corr. Coef. 0.992503 FIGS. 3 and 4 illustrate the results of the correlation between fructosamine system of this invention 4°C and Roche RoTAG plus and establishes that excellent correlation of the two systems where the system of this invention is substantially faster recovery. Stress Test Reagents A and B and the standard were stressed at 4°C and 47°C for 3 days. Each of the reagents were then evaluated for Ai, endogenous rate and the reagent kit for recovery of 20 patient samples. Table 7 lists the results of the Ai analysis and endogenous rate. FRUCTOSAMINE STRESS STUDY - Ai AND ENDOGENOUS RATE Reagent A and B stresses 3 days at 4°C and 47°C assayed on the Gilford® Stasar III - 500 nm, 37°C Reaction measured at 0 and 5 minutes Reagent/Temperature ABS, 0 min ABS, 5 min Reagent A 4°C 0.060 0.062 Reagent A 47°C 0.373 0.375 Reagent B 4°C 0.068 0.069 Reagent B 47°C 0.383 0.384 The Ai increases upon stress, but remains acceptable. The endogenous rate from 0 - 5 minutes is less than 0.002 and is negligible. Table 8 shows the results of assaying the stresses reagents with the stressed standard and the 20 patient samples. FRUCTOSAMINE STRESS STUDY Reagent A and B stressed 3 days at 4°C and 47°C Standard; 320 µmol stressed 3 days at 4°C and 47°C Assayed on the Cobas Mira 500 nm, 37°C Reagent Volume 200 µl, Sample Volume 20 µl absorbance measured at 3 and 5 minutes Patient Sample 4°C µmol 47°C µmol 47°C % Rec 1 202 239 118 2 238 247 104 3 222 225 101 4 240 242 101 5 223 229 103 6 185 194 105 7 180 204 113 8 292 254 87 9 170 192 113 10 650 616 95 11 282 280 99 12 501 462 92 13 331 325 93 14 366 371 101 15 487 471 97 16 555 537 97 17 365 368 101 18 486 483 99 19 274 291 106 20 359 366 102 In a couple of instances the 47°C stressed over-recovers, but in most cases the recoveries are very good. Percent recoveries range between 87 and 118 percent. The stability is acceptable after 3 days at 47°C.

Claims: A reagent system for the determination of fructosamine in sera which comprises: (a) a first alkaline aqueous reagent containing a tetrazolium salt which reacts with all reactive substances in sera including fructosamine and interfering substances to exhibit a measurable first change of color within a predetermined time span of less than about 10 minutes in consequence of the reaction of the tetrazolium salt with fructosamine and the interfering substances; and (b) a second reagent identical to the first alkaline aqueous reagent but further including a substance which inhibits reaction of fructosamine with the tetrazolium salt to produce by reduction of the tetrazolium salt with the reactive substances including interfering substances other than fructosamine a measurable second change of color in the same predetermined time span as the first reagent to enable the determination of fructosamine concentration in sera by the difference in the first color change produced using the first alkaline aqueous reagent which reacts with fructosamine and the second color change produced using the second alkaline aqueous reagent each color change being produced on the same amount of sera and after identical preselected time spans. A reagent system as claimed in claim 1 in combination with a liquid standard containing a predetermined amount of fructosamine which when used with said first and second alkaline aqueous reagents enables the determination of a factor used as multiplier to compute from the difference in color change the fructosamine concentration in a sera sample. A reagent system for the determination of fructosamine in sera as claimed in claim 1 or 2 which comprises: (a) a first liquid reagent which is an aqueous solution of a carbonate buffer present in a concentration from about 0.1 to about 0.4 molar, a tetrazolium salt which is reduced by fructosamine and interfering substances in sera present in a concentration of about 0.1 to about 5 millimolar, urea in a concentration of about 0.1 to about 1 molar, octylphenoxy polyethoxy ethanol in a concentration of about 0.1 to about 4% volume by volume, and from 0 to 3% volume by volume ethoxylated nonylphenol, said solution having a pH of from about 8.5 to about 11.5; and (b) a second liquid reagent identical to the first reagent and further containing boric acid in a concentration of about 0.05 to about 1 molar. A reagent system as claimed in any one of claims 1 to 3, in which the tetrazolium salt is p-lodonitrotetrazolium Violet. A reagent system for the determination of fructosamine in sera as claimed in any one of the previous claims which comprises: (a) a first liquid reagent containing 0.5 millimolar p-lodonitrotetrazolium Violet, about 0.1 molar sodium carbonate monohydrate, about 0.1 molar sodium bicarbonate, about 0.1 molar urea, about 1% volume by volume of octylphenoxy polyethoxy ethanol, and 1% ethoxylated nonylphenol dissolved in the deionized water and having a pH of about 11; and (b) a second liquid reagent identical to the first reagent but further including about 0.12 molar boric acid and also having a pH of about 11, said reagent system being stable for at least 1 year at 2-10°C and cooperating to enable completion of a fructosamine assay within 10 minutes at 37°C. A reagent system as claimed in any one of the previous claims, further including a liquid standard containing a predetermined amount of fructosamine which when used with said first and second alkaline aqueous reagents enables the determination of a factor used as multiplier to compute from the difference in color change the fructosamine concentration in a sera sample. A reagent system as claimed in any one of the previous claims in which the standard provides a fructosamine value in the range of about 300 to 500 µmolars. A reagent system as claimed in any one of claims 1 to 7, further including a liquid normal level fructosamine control and a liquid high level fructosamine control. A reagent system as claimed in claim 8, further including a liquid normal level control having a fructosamine range of 229-281 µmol when assayed and a liquid high level control having a fructosamine range of 457-559 µmol when assayed. A method of determining fructosamine in sera which comprises: (a) determining absorbance of a first liquid reagent exhibiting a first change in color in consequence of reduction by fructosamine and other interfering substances in sera of a tetrazolium salt at two predetermined points in time, each within 10 minutes at 37°C; (b) determining absorbance of a second liquid reagent added in the same amount to an identical amount of said sera, said second liquid reagent producing a second color change by reaction of the same tetrazolium salt by interfering substances in the sera other than fructosamine in consequence of the reagent containing a substance which inhibits the reaction of fructosamine with the color producing tetrazolium salt, the change in absorbance being determined at same predetermined periods of time; and (c) determining the fructosamine concentration sera as function of the difference between the first color change and the second color change produced using the first liquid reagent which reacts with fructosamine and the second liquid reagent which is inhibited from reacting with fructosamine. A method as claimed in claim 10 which comprises: (a) adding to a first sample of sera a first liquid reagent which reacts with fructosamine and interfering substances and is an aqueous solution of a carbonate buffer present in a concentration from about 0.1 to about 0.4 molar, a tetrazolium salt which is reduced by fructosamine and interfering substances in sera present in a concentration of about 0.1 to about 1 molar, octylphenoxy polyethoxyethanol in a concentration of about 0.1 to about 4% volume by volume and from 0 to 3% volume by volume ethoxylated nonylphenol, said solution having a pH of from about 8.5 to about 11.5 and measuring the change in absorbance of the sera added reagent at 500 nm at 2 or 3 minutes of the addition of the reagent and at 2 minutes after the first measurement of absorbance; (b) repeating the procedure of step (a) using a second sample of the sera of the same volume as the first sample of sera by addition of a second liquid reagent identical in volume and content of the first reagent and further containing boric acid in a concentration of about 0.05 to about 1 molar; and (c) determining from the difference in change of absorbance in step (a) and the change of absorbance in step (b) the fructosamine concentration in the sera. A method as claimed in claims 10 or 11 in which the tetrazolium salt is p-lodonitrotetrazolium Violet. A method as claimed in any one of claims 10, 11 or 12 which comprises: (a) adding to a predetermined amount of sera a first liquid reagent containing 0.5 millimolar p-lodonitrotetrazolium Violet, about 0.1 molar sodium carbonate monohydrate, about 0.1 molar sodium bicarbonate, about 0.1 molar urea, about 1% volume by volume of octylphenoxy polyethoxy ethanol, and 1% volume by volume ethoxylated nonylphenol dissolved in the deionized water and having a pH of about 11 and determining absorbance at 37°C and 500 nm 2 and 4 minutes or 3 and 5 minutes after addition of the first liquid reagent to the sera; (b) repeating step (a) by adding to the same predetermined amount of another sample of the sera a second liquid reagent identical in volume and content of the first reagent but further containing about 0.12 molar boric acid and also having a pH of about 11; and (c) using the difference in absorbance determined in step (a) and the differential in absorbance in step (b) to determine the fructosamine content of the sera.

Inventors: BRAVO-LEERABHAND MARJORIE, KWAN SHING FAI, BRAVO-LEERABHAND, MARJORIE, KWAN, SHING FAI
Applicants: MODROVICH IVAN ENDRE, MODROVICH, IVAN E.
Application year: 1993
Main classification: G01N33
IPC codes: G01N33