Fructosmaine reagent and calibrator system

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 THE 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. Pat. Nos. 4,642,295 and 4,645,742 to Baker incorporated herein by reference. 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.degree. 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.TM. 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.degree.-8.degree. C. and the calibrator when reconstituted is stated to be stable for up to 4 weeks at 2.degree.-8.degree. 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.degree. 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 commercially acceptability must have a shelf life of 12 months or more at 2.degree. to 10.degree. C. more typically 18 months at 2.degree. to 10.degree. C., which corresponds to a shelf life of at least about 3 days at 41.degree. C. As indicated, to be commercially adaptive to all spectrophotometric instruments, the compositions must have response time within 10 minutes at 37.degree. 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.degree. to 10.degree. 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.degree. C.

There is provided a two reagent system for the determination of fructosamine in sera. The first is an alkaline aqueous reagent containing a tetrazolium salt which reacts with all reactive substances in sera including frutosamine an interfering substance to exhibit a measurable first change of color within a predetermined time span of less than about 10 minutes. The second reagent is identical to the first alkaline aqueous reagent, but includes a substance which inhibits reactions of fructosamine with the tetrazolium salt to produce, by reduction of the tetrazolium salt with the reactive substances including interfering substance other than fructosamine, a measurable second change of color in the same predetermined time span as the first reagent. This enables 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 is produced on the same proportional amount of sera and reagents and after identical predetermined time spans.

The first reagent ("Reagent A" herein) is responsive to all the reactive substances in sera. The second reagent, ("Reagent B" herein), contains a boric acid 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 0.5 mMor 252.85 mg/l p-Iodonitrotetrazolium Violet (INT), 0.1M or 12.4 g/l Sodium Carbonate Monohydrate (Na.sub.2 CO.sub.3) 0.1M or 8.4 g/l Sodium Bicarbonate (NaHCO.sub.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 NP40 contained in the deionized water. Solution pH is about 11.0.

Reagent B is identical to Reagent A, but contains 0.12M or 7.416 g/l Boric Acid (H.sub.3 BO.sub.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.degree. C. The difference in absorbance (ABS) for Reagent A and Reagent B are determined and a difference 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 manufacture and sold by Roche.

DETAILED DESCRIPTION

Present invention is directed to the determination of fructosamine concentration in sera. It is based on the use of one reagent to 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 boric acid which prevents fructosamine from reducing the tetrazolium salt. By the ten 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 ##EQU1## with fructosamine concentration of the same being determined by the equation:

F.times.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 polyethoxy ethanol 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 0.4M, tetrazolium salt (INT) in a concentration of about 0.1 to 5 mM, urea in a concentration of 0.1 to about 1M, 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 1M. Functional pH range is about 8.5 to about 1.5, 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.degree. C. which correlates to a life time of at least 15 to 18 months at 2.degree.-10.degree. 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.degree. 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.

TABLE 1______________________________________Criteria This Invention RoTAG______________________________________Reagent Type Single Liquid Two part Liquid & TabletBlank Yes NoCalibrator Single Point Single Point Liquid LyophilizedControls Two Levels Two Levels Liquid LyophilizedStability 15 mos. @ 4.degree. C. Reagent: 2 Wks. after reconst. Cal/Control: 4 wks after reconst.Kinetics Spline Fit Spline FitLinearity 10-1600 umol To 1000 umolTemperature 37.degree. C. 37.degree. C.of ReactionResponse Time <5 Minutes 10-12 MinutesSpecificity Excellent GoodPrecision: 280 umol% CV Run to Run 2.8 2.4With-In Run 2.9 1.9500 umolRun to Run 2.8 1.7With-In Run 2.4 2.3Fructosamine 196-279 umol 200-272 umolNormal Range______________________________________

The preferred composition Reagent A, which Table 1 is based on, is one containing 0.5 mM p-Iodonitrotetrazolium Violet (INT), 0.1M Sodium Carbonate Monohydrate (Na.sub.2 CO.sub.3), 0.1M Sodium Bicarbonate (NaHCO.sub.3), 0.1M Urea, 1.0 v/v Triton X-100 and 1% Tergitol NP40 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 0.12M Boric Acid (H.sub.3 BO.sub.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 a 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 (umol). 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, 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.sub.2 O252.85 mg p-Iodonitrotetrazolium Violet12.4 g Na.sub.2 CO.sub.3 Monohydrate8.4 g NaHCO.sub.36. g Urea10. ml Triton X-10010 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.sub.2 O252.85 mg p-Iodonitrotetrazolium Violet12.4 g Na.sub.2 CO.sub.3 Monohydrate8.4 g NaHCo.sub.36. g Urea10. ml Triton X-1007.416 g H.sub.3 BO.sub.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 micron cellulose nitrate filter: 2. 0.50 grams Human Albumin Powder Fraction V (Armour Pharmaceutical Co.). 3. 7.20 grams of D-Glucose. 4. 0.01 g NaN.sub.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.degree. 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.degree. 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 0.5 g 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 micron cellulose nitrate filter (Whatman). Fructosamine value was in the range of 378-462 umol 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 micron cellulose nitrate filter (Whatman). Fructosamine value was in the range of 229-281 umol 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 Dilutent was employed. Fructosamine level was 457-559 umol.

Glycation of Polyiysine

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-1-lysine. 3. 4.75 grams of D-glucose. 4. 0.007 grams of NaN.sub.3 (sodium azide)

Each was separately dissolved and the container parafilmed and incubated at 37.degree. 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 micron 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 a assay temperature of 37.degree. C. Reagent volume was 1.0 ml, sample volume was 100 ul. 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.degree. 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 ul 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: ##EQU2##

A calculation factor (F) was determined by dividing the concentration of the standard by the Net Delta ABS of the standard. ##EQU3##

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.times.Net Delta ABS=umol Fructosamine

The assay system linearity was assayed for linearity using 300 umol calibrator and five levels of human serum pool spiked with various amounts of fructosamine. Table 2 and FIG. 1 summarize the results.

TABLE 2______________________________________REAGENT LINEARITYAssayed on the Cobas MiraProtocol: 500 nm, 37.degree. C., RV = 200 ul, SV = 20 ulAbsorbance Measured at 3 and 5 minutes Actual Net Actual TheoreticalSample Delta ABS [umol] [umol]______________________________________300 Std .0410 300 -- Mix of 1 & 51 .0348 255 255 12 .0614 449 439 [(3 .times. 1) + 5]/43 .0863 632 623 (1 + 5)/24 .1115 816 806 [1 + (3 .times. 5)/55 .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.

TABLE 3______________________________________Glycated Polylysine Dilution Protocol Dilution DilutionFactor Level = with NaCl______________________________________1 1X Glyc. Poly. + 19X NaCl .052 1x Glyc. Poly. + 9X NaCl .103 1X Glyc. Poly. + 3X NaCl .254 1X Glyc. Poly. + 1X Nacl .505 1X Glyc. Poly + 1X NaCl .756 Undiluted Glyc. Poly. (Neat) 1.00______________________________________ Level 4 (Dilution Factor .50) = 778 umol per Roche RoTAG

Table 4 and FIG. 2 summarize the linearity results which establishes that the system is functional for diluted sera.

TABLE 4______________________________________LINEARITY/DYNAMIC RANGEAssayed on the Cobas MiraMAS protocol: 500 nm, 37.degree. C., RV = 200 ul, SV = 20 ulAbsorbance Measured at 3 and 5 Minutes Dilution NetSample Factor Delta ABS Umol Fructosamine______________________________________300 Std / .0399 3001 .05 .0093 702 .10 .0217 1633 .25 .0590 4444 .50 .1163 8745 .75 .1784 13416 1.00 .2310 1737______________________________________

Precision

Biocell Blood Bank Segments Pool was spiked with glycated human serum to levels of 280 umol and 500 umol fructosamine for use in precision (Run to run) and within-run studies. Table 4 lists the results of Run to Run Performance.

TABLE 4______________________________________RUN TO RUN PERFORMANCEAssayed on the Cobas MiraProtocol: 500 nm, 37.degree. C., RV = 200 ul, SV = 20 ulAbsorbance Measured at 3 and 5 MinutesStandard: In-House 300 umolBased on Single Calibration Day 1Daily Value is Average of Triplicate AnalysesDay 280 umol 500 umol______________________________________1 272 4922 277 5133 288 5054 284 4785 270 488N 5 5Minimum 270 478Maximum 288 513Mean 278 495Std 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.

TABLE 5______________________________________WITH-IN RUN REFERENCEAssayed on the Cobas MiraProtocol: 500 nm, 37.degree. C., RV = 200 ul, SV = 20 ulReaction Measured from at 3 and 5 minutesStandard: 300 umolSample Repititions______________________________________280 umol 294 293 289 297 285 282 277 272 291 272 292 276 285 304 289 294 290 290 288 281500 umol 501 491 494 504 514 511 497 504 515 523 495 494 503 507 510 511 475 515 525 498______________________________________ 280 umol 500 umol______________________________________N 20 20Minimum 272 475Maximum 304 525Mean 287 504Std Dev 8.4 11.9% CV 2.9 2.4______________________________________ The WithIn 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.degree. C. and Roche RoTAG Plus. The results are tabulated and presented in Table 6.

TABLE 6______________________________________PATIENT SAMPLE CORRELATION FRUCTOSAMINESYSTEM (FS) vs ROCHEAssayed on the Cobas Mira 37.degree. C.FS: RV = 200 ul, SV = 20 ulWavelength 500 nmStandard: 300 umolAbsorbance Measured at 3 and 5 minutesROCHE: RV = 200 ul, Dil. Vol. = 30 ul, SV = 10 ulWavelength 550 nmStandard: Roche 442 umolAbsorbance Measured at 10 and 12 minutes Roche FS umol umol______________________________________Normal Samples 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 24510 232 24111 224 23412 231 23213 240 25014 174 16515 240 24716 240 19717 229 20618 202 19319 206 20220 222 19021 282 26322 262 24623 251 21524 252 21425 245 19426 276 22027 196 19828 238 21829 253 25930 212 23131 219 21132 219 22633 235 23734 192 20135 210 23036 212 22137 240 22938 253 25339 244 26640 242 24441 171 18042 233 25043 228 23944 196 25045 208 24246 202 22047 209 20948 252 25849 245 22750 191 20351 222 24152 246 24053 473 49554 214 21755 225 23356 226 21157 244 22658 243 26359 226 26060 208 22961 243 23662 200 19763 171 16864 215 21465 284 21566 248 27567 269 28968 203 20469 234 23470 222 22371 209 24272 227 21973 203 21574 227 24575 225 23876 209 20277 229 20678 224 21679 230 25580 247 20381 189 15282 251 19883 344 35784 171 17485 616 671Diabetic Samples86 375 36587 295 27888 374 37989 254 25090 397 45091 291 27792 377 38393 377 38394 367 35995 250 24396 395 43297 283 27898 378 40099 282 283100 376 390101 244 238102 386 447103 278 266Glyc. LRRMC104 291 256105 473 477106 310 343107 327 360108 451 476109 513 541110 371 335111 489 485112 286 266113 345 342Glyc. Biocell114 286 262115 403 389116 534 517117 668 673118 799 835119 917 938120 1102 1100121 1180 1196122 1304 1357N 122 122Minimum 165 149Maximum 1304 1357Mean 301 302Std Dev 185 192Linear 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.degree. 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.degree. C. and 47.degree. 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.

TABLE 7______________________________________FRUCTOSAMINE STRESS STUDY - Ai ANDENDOGENOUS RATEReagent A and B stresses 3 days at 4.degree. C. and 47.degree. C.assayedon the Gilford Stasar III - 500 nm, 37.degree. C. Reactionmeasured at 0 and 5 minutesReagent/Temperature ABS, 0 Min ABS, 5 Min______________________________________Reagent A 4.degree. C. .060 .062Reagent A 47.degree. C. .373 .375Reagent B 4.degree. C. .068 .069Reagent B 47.degree. C. .383 .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.

TABLE 8______________________________________FRUCTOSAMINE STRESS STUDYReagent A and B Stressed 3 days at 4.degree. C. and 47.degree. C.Standard; 320 umol stressed 3 days at 4.degree. C. and 47.degree. C.Assayed on the Cobas Mira 500 nm, 37.degree. C. ReagentVolume 200 ul, Sample Volume 20 ul absorbancemeasured at 3 and 5 minutes.Patient 4.degree. C. 47.degree. C. 47.degree. C.Sample umol umol % 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 9 170 182 11310 650 616 9511 282 280 9912 501 462 9213 331 325 9314 366 371 10115 487 471 9716 555 537 9717 365 368 10118 486 483 9919 274 291 10620 359 366 102______________________________________

In a couple of instances the 47.degree. 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.degree. C.

Claims

1. A reagent system for the determination of fructosamine in sera which comprises:

(a) a first liquid 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 alkaline aqueous reagent identical to the first alkaline aqueous reagent but including a substance which inhibits reaction of fructosamine with the tetrazolium salt by binding or complexing to the fructosamine 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 produced on the same proportional amount of sera and after identical predetermined time spans.

2. The 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 enable the determination of a factor used as multiplier used to compute from the difference in color change the fructosamine concentration in a sera sample.

3. An assay system as claimed in claim 2 further including a liquid normal level fructosamine control and a liquid high level fructosamine control.

4. An assay system as claimed in claim 2 further including a liquid normal level fructosamine control having a fructosamine range of 229-281 .mu.mol, when assayed, and a liquid high fructosamine level control having a fructosamine range, when assayed, of 457-559 .mu.mol.

5. An assay system as claimed in claim 3 further including a liquid normal fructosamine level control having a fructosamine range of 229-281 .mu.mol, when assayed, and a liquid high fructosamine level control having a fructosamine range, when assayed, of 457-559 .mu.mol.

6. A method for determining fructosamine in sera which comprises:

(a) determining by absorbance a first color produced by reaction of a first amount of sera, with a first amount of a liquid alkaline aqueous reagent to produce 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.degree. C.;

(b) determining absorbance of a second color produced by reaction of the same amount of reagent with the same amount of said sera using a second liquid alkaline aqueous reagent which produces a second color change by reaction of the same amount of said tetrazolium salt with 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, by binding or complexing to the fructosamine the change in absorbance being determined for the same predetermined period of time; and

(c) determining the fructosamine concentration in the sera as function of the difference in the first and second color change produce using the first liquid alkaline aqueous reagent which reacts with fructosamine and the second liquid alkaline aqueous reagent which is inhibited from reacting with fructosamine.