GB2436681A

GB2436681A - Fluorescent assay

Info

Publication number: GB2436681A
Application number: GB0606450A
Authority: GB
Inventors: John Phillip Vessey; Adrian Richard Gray
Original assignee: Quotient Diagnostics Ltd
Current assignee: Quotient Diagnostics Ltd
Priority date: 2006-03-31
Filing date: 2006-03-31
Publication date: 2007-10-03
Anticipated expiration: 2026-03-31
Also published as: EP2005164B1; US7998742B2; US20090176309A1; GB2436681B; ATE541216T1; PL2005164T3; WO2007113590A2; ES2377232T3; EP2005164A2; JP2009531700A; JP5118126B2; WO2007113590A3; CN101416054A; GB0606450D0

Abstract

A method and apparatus to estimate the concentration of a nonfluorescent substance (e.g. haemoglobin or glycated haemoglobin) in a fluorescent assay by separately estimating the non time dependent alteration attributed to inherent filter effects from the time dependent alteration caused by the assay chemistry. Such a method obviates the requirement for a separate photometric or other measurement thereby simplifying the methodology and associated instrumentation. In another method the concentration of a non-fluorescent analyte is measured by firstly choosing a fluorescent marker with an excitation and emission spectrum which overlaps the absorption Spectum of the analyte, measuring the marker's fluorescence spectrum. Then adding the analyte and measuring the fluorescent emission. The concentration of the analyte is calculated from the difference between the 2 fluorescent measurements.

Description

FLUORESCENT ASSAY

The present invention relates to methods of carrying out an assay for a non-fluorescent substance in a sample, and further relates to apparatus for conducting assays according to such methods.

European patent number EP 0,772,779 describes the assay of glycated proteins by fluorescence quenching. The prior art as exemplified by this document describes the assay of glycated haemoglobin using the quenching of fluorescence produced when said glycated haemoglobin binds to a conjugate produced by the chemical linking of specific fluorophors to boronic acid groups. This concentration dependent quenching is then compared to a measure of total haemoglobin made by conventional photometric means,

which is established prior art.

However during this prior art method there is an unavoidable non-specific level of fluorescence quenching that is not caused by the binding of said conjugate to glycated haemoglobin but instead by an inherent filter effect.

This inherent filter effect is caused by the haemoglobin molecule per se absorbing both the exciting radiation and the emitted fluorescent light caused by the spectral overlap of the haemoglobin absorption spectrum with the fluorescence excitation and emission spectra. This background fluorescence quenching must be compensated for in calculation in order to allow quantification of the specific quenching that is attributed solely to the concentration of glycated haemoglobin and hence its quantum. The inventors of EP 0,772,779 achieved this by determining the optical density of the reaction solution at a suitable wavelength (e.g. 405nm or 415nm) and P14011-SP-02doc 31/03/06 subtracting a pro rata element of the total fluorescence quenching dependent on this optical density measurement.

In the method disclosed in EP 0,772,779 the total haemoglobin concentration is measured using conventional photometric methods such as absorbance at 405nm or 415nm. For that method to function as described therein the user must make two independent measurements on separate instruments: a photometer to measure the total optical density and a fluorimeter to measure the fluorescence quenching. For the test to have any commercial utility, for example in the management of diabetes, any instrument designed and manufactured specifically for the test must consequently have the dual functionality of both a fluorimeter and a photometer. This is costly, more complex and undesirable.

The quenching of fluorescence by haemoglobin as described in EP 0,772,779 has two constituents. The first, an initial instantaneous drop in fluorescence associated with the haemoglobin specimen acting like a neutral density filter at the fluorescence excitation and emission wavelengths; and the second a time dependent quenching of the fluorescence signal associated with the time course of the chemical binding of glycated haemoglobin to the fluorescent conjugate reagent.

The present invention aims to overcome the problems of the prior art method, as described above and further discussed below, in the hope of developing a significantly improved assay method, as well as to design instruments automatically to achieve the desired results. As a result of this careful study, the present inventors have ascertained that by following the fluorescence alteration time course as the assay reaction progresses, the P14O1-SP-O2doc 31/03106 background fluorescence alteration, i.e. an instantaneous decrease or increase not dependent upon the reaction chemistry, can be determined separately from the time dependent fluorescence quenching attributed to the chemical binding of the target compound to the fluorescent reagent. Leading on from this they have also found that an alteration in detected fluorescence, in particular a quenching effect, when a substance is added to a fluorescent reagent can be used to calculate the concentration of that substance which is not itself fluorescent.

According to a first aspect of the present invention there is provided a method of carrying out an assay for a non-fluorescent substance in a sample, which method comprises: (a) carrying out a reaction in solution between an assay sample and a fluorescent marker compound, the sample and marker compound being combined at time t0; (b) exciting the fluorescence in the marker compound, wherein the nature of the marker and the nature of the excitation are such that the said fluorescence occurs at a wavelength at which said fluorescence is altered by the reaction of the marker compound with the non-fluorescent substance; (c) detecting the resulting fluorescence as the reaction progresses; (d) calculating from the detected fluorescence the values of F0 being the fluorescence at time to and F0. being the fluorescence at time L, which is the point at which all of the non-fluorescent substance has reacted with the marker compound, or the reaction has attained equilibrium; (e) calculating from the values of F0 and F0. the background change in fluorescence attributable to the reaction in step (a); and P14011-SP-02doc 31/03/06 (f) determining from the calculated values and a suitable calibration algorithm the concentration of the non-fluorescent substance present in the sample at to prior to reacting with the fluorescent marker compound.

As used herein, when referring to the substance to be assayed (the analyte) the term non-fluorescent does not mean that the substance is totally non-fluorescent. Instead it means that it is has a different excitation and or emission spectra to the fluorescent marker compound at the relevant excitation and emission wavelengths.

A major currently intended use of the present invention is in the assaying of glycated haemoglobin in blood. Indeed, the genesis of the present invention lies in improving a process for this assay, and in particular assaying the amount or proportion of glycated haemoglobin (i.e. haemoglobin to which glucose has become non-enzymatically bound) within a sample. For this reason the following description will often describe the invention in respect of this, though the present invention is equally applicable to the assaying of many different substances -in particular such substances where the absorption characteristics of the contents of the sample are prone to interfere with the fluorescent characteristics of an appropriate fluorophor.

The first aspect of the present invention requires a marker compound that is specific for the substance to be assayed and the fluorescence of which is appropriately altered (possibly increased, but more usually decreased) by the binding of the marker to the substance.

The marker compound will usually comprise a fluorophor and a linking group adapted to bind selectively to the substance. The selection of an appropriate fluorophor and linking group combination for a particular target P14011-SP-02.doc 31/03/06 substance may be made based on experimental testing or on the known characteristics of existing fluorophors and linking groups relative to the substance to be assayed. A range of known fluorophors is mentioned in EP 772,779, the content of which is incorporated herein by reference. For a glycated protein such as glycated haemoglobin a suitable marker could include a boronic acid group bound by a connecting group to a fluorescein derivative. The boronic acid binds to the cis-diol group of a glycated protein but is not protein specific. It is preferred that the method is adapted specifically for the detection of glycated haemoglobin and a suitable marker for this might have the formula: HO. 0 0

OH H"r

H

The calibration algorithm used in section (f) may be of the form y = mx + c, where m and c are the slope and intercept calibration constants and x = (Fo -F)I F0. The target non-fluorescent substance may be a subspecies (e.g. glycated haemoglobin) of a main substance (e.g. total haemoglobin in all forms), and the subspecies will be selectively reactive with the marker compound as compared to the main substance. In such a case it may be desirable to known the overall concentration of the main substance P14011-SP-02.doc 31103/06 (including the subspecies) present in the sample at to, prior to reacting with the fluorescent marker compound. This may be determined from the calculated values and a suitable calibration algorithm of the form y = m'x + where m' and c' are the slope and intercept calibration constants and x = Log (Faflç/ Fo).

The calculation of the values of F0 and Ft,,. in step (d) preferably includes plotting the detected fluorescence data against time, and applying a best fit curve to the plotted points. The plotting may be physical or even manual, but is often achieved automatically and mathematically by fitting a mathematical function of a curve of best fit to the data and extrapolating that function to time to and L, without the generation of a graph as such. The curve fitting may be achieved by any suitable mathematical method, and two particularly appropriate ones are described later in this specification. Using these regimes the recorded data is used to minimise variance and then the curve is extrapolated to provide values for F0 and F. In a fully automated instrument system, where blood is added to the reagent and mixed by the instrument, the initial quenching might possibly be determined within the first 5 seconds or so after blood addition and mixing.

However, in a non-automated system, the monitoring of the initial binding reaction is further delayed due to the finite time required by an operator physically to add the blood specimen to the reaction cuvette containing the reagent, mix and return the reaction cuvette to the fluorimeter. Therefore, the actual fluorescence level at to, immediately after the addition of the blood specimen, cannot directly be measured in either the manual or automatic system. To overcome this, the fluorescence level F0 at time to i.e. immediately P14011-SP-02doc 31/03/06 after sample addition and mixing but before reaction has occurred, is determined by back extrapolation of a curve fitted to the time course fluorescence data, based upon the rate equation of the chemical binding reaction. Further, by forward extrapolation of this curve fit beyond the data collection period, the fluorescence level F. at time L, the reaction end-point, is also determined. Determining the fluorescence levels at to and L in this manner has been shown to produce reliable and accurate results.

The time period over which the fluorescence of the reaction mixture is measured may be anything suitable for the use to which the assay is put. A long period gives greater accuracy, but a slower assay process, whereas a short period is convenient, but may leads to less precise results as the amount of data from which to extrapolate becomes sparse. The suitable length of the measurement period will depend on the substance being assayed and the time profile of its reaction with the fluorescent marker. In is respect of haemoglobin boronate binding to cis diol groups, a measurement period of about 3 minutes is usually appropriate.

It is preferred that before the sample is combined with the marker compound in step (a), the marker compound alone (which is usually in the form of a reagent solution) is excited by incident electromagnetic radiation of a suitable wavelength A (i.e. the same as that at which excitation occurs during reaction with the sample) and the resultant initial fluorescence (FBIank) at the emission frequency is detected. This in combination with the value of F0 can be used to calculate initial alteration in the fluorescence, which in turn may be used to calculate the fluorescence optical density (FOD) using the equation: P4O11-SP-O2doc 31/03/06 FOD = Log [FBIank/FO] (Equation 1) This log function of the initial background alteration (usually a quenching effect) is designated as FOD. FOD has been found to be directly proportional to the optical density of the reaction solution at a given wavelength. In the instance where the assay concerns haemoglobin, it is proportional to the optical density measured at 415nm (See Fig 3). As the optical density, at 415nm, is in turn proportional to total haemoglobin concentration, the measurement of background fluorescence quenching can therefore also be used to determine total haemoglobin concentration. This background fluorescence quenching determination of total haemoglobin can therefore be used to replace the optical density determination, otherwise made by a separate photometric apparatus.

It may not be necessary to determine FBIaflk before every assay, as this value may be a standard or constant value depending on the reaction cuvette and the marker compound reagent used.

According to a second aspect of the present invention there is also provided a method of determining the concentration of a non-fluorescent substance, comprising exciting a matching fluorescent reagent the excitation and emission spectrum of which overlaps the absorption spectrum of the non- fluorescent substance; detecting the resultant fluorescence; adding the non-fluorescent substance to the matching fluorescent reagent; detecting the fluorescence after the addition; and calculating the concentration of the non-fluorescent substance from the difference between the fluorescence detected before and after addition of the non-fluorescent substance.

P14011-SP-02 doc 31/03/06 It is well known to calculate the concentration of a substance in solution using optical density measurements in comparison to established standards.

However the second aspect of the present invention allows a concentration to be experimentally derived without the use of a photometer.

The method of the second aspect of the invention can be used where there is no reaction between the non-fluorescent substance and the reagent, which may be similar to the marker compound described above. However there may be a time dependent reaction that alters (either to increase or decrease) the detected fluorescence. By monitoring this and plotting a graph (either actually or notionally) as described with respect to the first aspect, the value of the initial alteration effect may be accurately estimated by extrapolation from the measured data.

When measuring the concentration of a substance that does not react with the marker to change the fluorescence, there is no need to extrapolate as the fluorescence measurement taken some time after addition of the sample will be the same as that immediately thereafter. But when the sample and reagent react, the back extrapolation to to may be used to overcome the inability to measure the F0 value directly.

Since the various aspects of the present invention describe means for making accurate determination of the amount or concentration of a substance present or the amount of a subspecies of that substance (such as the level of glycated haemoglobin within the total haemoglobin) that is present from the analysis of the fluorescence data alone, the design and construction of any associated instrument is greatly simplified, with costs substantially reduced as P1401 I-SP-02.dcc 31/03/06 -10 -a consequence. In particular, simpler apparatus not including a spectrophotometer may be used to give better results.

According to a third aspect of the present invention there is yet further provided apparatus for the assay of a substance in a sample and using a fluorescent marker compound, which apparatus comprises: (a) a reaction vessel adapted to hold an amount of the fluorescent marker compound and an assay sample, which marker compound together with the substance in the sample undergo a reaction beginning at time to and ending at time t,. that alters the fluorescent characteristics of the marker compound; (b) a source of electro-magnetic radiation having a wavelength A to excite the fluorescence in the marker compound; (c) detecting means to measure the resulting fluorescence as the reaction progresses; and (d) a processor adapted: (1) to calculate from the measured fluorescence the values of F0 being the fluorescence at time to and F. being the fluorescence at time L, when all of the substance has reacted with the marker compound; (2) to calculate any background change in fluorescence not attributable to and that attributable to the reaction between the substance and the marker compound; and (3) to determine from the calculated values the amount of the non-fluorescent substance and/or a sub-species thereof present in the sample at to.

For the aspects of present invention to have a benefit, at least one constituent element of the sample -be it the substance that is to be quantified, a related substance that needs quantification in contrast to the P14011-SP-02.doc 31103/06 -11 -desired substance, or a contaminant -must have an absorption spectrum that overlaps to some degree with the excitation and emission spectra of the fluorescent marker/reagent. Consequently the wavelength of the exciting radiation and the nature of the fluorescent marker must be matched to each other and selected based upon the nature of the assay to be performed, which in turn includes the characteristics of the substance to be quantified and other substances liable to be found in the sample.

Many modifications of the methods and apparatus of the present invention described herein will be apparent to the skilled man and these modifications fall within the scope of the present invention. However in order that its principles may be better understood, but by way of example only, the present invention will now be described in detail with reference to certain examples and where appropriate to the accompanying drawings in which: Figure 1 is a graph depicting the relationship between optical density (OD) at 415 nm and quantity of haemoglobin added; Figure 2 is a plot of the fluorescence signal time course during a glycated haemoglobin I eosin-boronic acid binding reaction; Figure 3 is a graph showing the relationship between OD at 415 nm and FOD derived from the background fluorescence quenching; Figure 4 is a graph showing the spectral differences between different haemoglobin types; Figure 5 is a graph showing the absorption spectrum of bilirubin; Figure 6 is a graph plot of fluorescence data against time for sample 1

in Example 1;

P14011-SP-02.doc 31/03/06 -12 -Figure 7 is a plot of Glycated haemoglobin concentration v Specific Quenching for Example 1; and Figure 8 is a plot of total haemoglobin concentration v Fluorescence Optical Density (FOD) for Example 1.

The present invention may be used to assay haemoglobin and in particular glycated haemoglobin in blood. In such a method the fluorescent marker compound, which may be fluorescein-boronic acid or as in this example an eosin-boronic acid compound, is introduced into a cuvette and prior to the introduction of a blood specimen, is excited in a fluorimeter by EM radiation at a suitable wavelength (5lOnm) and the fluorescence blank (FBIank) is measured by detecting emission at 580nm. These wavelengths were chosen because with a move off the Eosin Y excitation peak improved assay performance is found. The chosen excitation wavelength corresponding to a point where the absorption spectra of the many different haemoglobin species found in blood (i.e. carboxy-haemoglobin, oxy-, deoxy-, met-etc.) has the smallest variation. Likewise the emission wavelength monitored, 580nm (with a 4Onm bandpass), is chosen so as to minimise differences between the integrated absorption, over the emission bandpass, for the various haemoglobin species. The cuvette is removed from the fluorimeter or left in place and the blood sample is immediately added and mixed and the fluorescence over a time course is detected and recorded. This data is plotted and a curve is fitted to the data set. Figure 2 shows such a reaction time profile where the initial fluorescence FBIank labelled 10 is recorded prior to sample introduction at to (11). The actual experimental fluorescence data for the reaction is recorded over time (usually at I second intervals) from the P14011-SP-02.doc 31/03106 -13-reintroduction of the cuvette in a manual system or after mixing in an automatic system at point 13 until a suitable period has elapsed. By back extrapolation, the fluorescence level, F0 (see point marked 14) is determined att0 (11), i.e. the point when the sample was added but no reaction with the marker had occurred. By forward extrapolation (which is not shown because the end of the measure data is off the graph in Figure 2) the fluorescence level F (15) at the reaction end-pointL is similarly determined.

Two of many potential curve fit routines have been shown to be effective in the extrapolated estimation of F0 and F and the subsequent derivation of FOD and the specific quenching.

In the first an estimate Ft-estimate is made based on the fluorescence measured Ft-actuai at each of the one second measurement points throughout the 3 minute assay, using a curve fitting routine based upon the general rate equation below (Equ.2) F = F04-(F-Fo) x (1-e'8) Equ.2 where, = Fluorescence at time t seconds F0 = Fluorescence at time zero F = Fluorescence at time infinity (i.e. at reaction end) e = 2.7813 (natural log base) e = rate constant with F0, F and e being determined iteratively by minimising the sum of the squared variances between the fitted and measured value at each one P14011-SP-02doc 31103/06 -14 - second data point, i.e. (Ftactuai -Ft-estimated)2 is minimised by the fitting routine.

The second approach uses the rate equation for a second order chemical reaction in place of the general rate equation (Equ.2) used above.

Whereby: A+B-C V0= k[A][B] where, V0 is the rate of reaction, k = rate constant The rate constant, k, is defined by k = A. e -E/(RT) where, E = constant, A = activation energy, R = gas constant and T = Absolute temperature V0 is used to calculate the fluorescent intensity for each second, the concentrations of the reactants modified and V0 then re-calculated for the next time point. An iterative procedure minimises the sum of the squared variances between the estimated value and the measured value by adjusting the values for E, A and R as in the previous example.

However, it should be noted that at least in respect of haemoglobin quantification the present invention relates to the principles of estimating the initial fluorescence drop caused by the quenching of fluorescence due to the P14011-SP-02.doc 31/03/06 -15-inherent filter effect of haemoglobin, and then possibly using this to estimate the concentration of the substance or indeed any quenching material in the sample. The mathematical modelling of the data can be achieved by other methods of curve fitting which may be equally acceptable in practice.

The FOD parameter, fluorescence optical density, is determined via the following expression: FOD = Log [FBIank/Fo] Equ.1 FOD has been found to be linearly related to the total haemoglobin concentration. Haemoglobin concentration has been shown to be proportional to the optical density of a blood sample and 415nm and Figure 3 shows that FOD is directly proportional to the OD of the sample. The initial alteration, which when assaying blood samples for haemoglobin is a drop (or quenching), when calculated as a log function (FOD), is correlated to the measurement of haemoglobin by absorption photometry at other wavelengths.

In addition to the benefits discussed above, the present invention also has other important beneficial consequences particularly in the assay of blood for the presence or quanta of glycated haemoglobin. Haemoglobin in red blood cells exists in a variety of states depending on the oxygenation status of the donor's blood and his/her physiological condition. Deoxyhaemoglobin, oxyhaemoglobin, carboxyhaemoglobin, sulphydryl haemoglobin and met-haemoglobin are some of the types, or chemical modifications of, haemoglobin that may be present in blood samples in varying amounts depending on their age and source. Each type of haemoglobin is characterised by a unique absorption spectrum when subjected to light of varying wavelengths (see Figure 4 for a comparison of oxyhaemoglobin and P14011-SP-02doc 31/03f06 -16 -deoxyhaemoglobin). Stored and processed blood samples in particular accumulate a significant proportion of met-haemoglobin, where the ferric ion is oxidised, changing its absorption spectrum and accounting for its deep red/brown colouration.

The prior art as exemplified by EP 0,772,779 does not appreciate or counter the influence of the state of the haemoglobin molecule with its associated absorption spectrum, on its ability to quench fluorescence both by virtue of its intrinsic absorption and also by virtue of its spectral overlap with the fluorescent conjugate. Instead it expects that a measurement of haemoglobin at either 405 or 415nm accurately predicts the initial quenching drop caused by the presence of the haemoglobin in the sample. This is only true for haemoglobin samples that have identical spectra since the initial quenching drop is caused by secondary haemoglobin absorption peaks which correspond to the excitation and emission wavelengths A of the fluorophor used. It is the secondary absorption peaks that show most variability between haemoglobin states.

These differences in haemoglobin types in the assay are so significant that in order to calibrate the assay, standards having the same composition of haemoglobin types as the unknown samples under investigation must be used. Of course, in practice, the status of an individual's haemoglobin profile is unknown at the time of measurement even if that sample is freshly drawn either from a finger stick injury or from venepuncture and, therefore, the assay's utility as a point of care method is compromised, as unacceptable inaccuracies could occur.

P14011-SP-02.doc 31/03106 -17-Moreover, clinical assays for HbAlc and other glycated haemoglobins are often subjected to a process of verification involving the distribution of stored blood samples by a central laboratory and their simultaneous analysis by different systems. If the assay does not perform identically between fresh patient samples and stored or laboratory manipulated blood samples, unacceptable bias will occur and the assay will fail to achieve the correct values.

Therefore, in addition to the simplification of the instrumentation and measurement requirements, an additional benefit of the present invention is the fact that an optical density measurement based on the intrinsic fluorescence quenching of the sample (FOD) reduces errors that may be introduced into the test methodology by variations in the composite spectrum of a given individual's haemoglobin profile because the spectra of the various haemoglobin types at these wavelengths are more closely matched.

A'final benefit from the present invention is the reduction of errors caused by the presence of many interfering factors that might influence absorption measurements at 405 or 415nm. For example, patients who suffer from liver disease that manifests itself in jaundice (i.e. yellowing of the skin and eyeballs) have high circulating levels of bilirubin. Bilirubin is a circulating by-product of haemoglobin metabolism and has significant overlapping absorption with the major peaks of haemoglobin (Figure 5). The method of the prior art, which estimated the background fluorescence quenching from an optical density measurement of haemoglobin at its major absorption peaks, would be influenced by elevated circulating levels of bilirubin. The present invention, by estimating total haemoglobin by background fluorescence P14011-SP-02.doc 31/03/06 -18-quenching at wavelengths greater than 500nm, eliminates this potential source of error.

Example I

A known volume (2.5mL) of reagent comprising a buffer (principally HEPES) and a fluorescent market compound (Eosin Boronate) was introduced into a reaction cuvette. This was introduced into an apparatus accordingto the present invention and was exited at 5lOnm. Blank fluorescence readings were taken at I second intervals.

Then a volume of blood, whose total haemoglobin content/concentration and percentage of glycated haemoglobin is known, was introduced into the cuvette at time to and mixed. The fluorescence at 510 nm was then measured every second up to 3 minutes reaction time. The time taken to effect mixing results in the fluorescence only being recorded from about the 7th second onwards.

The blank and blood readings of fluorescence signal and reference are laid out in Table I below.

Table I

Reading Raw Fluorescence Data Fluorescence Signal Fluorescence Fit Number Minus Fluorescence Signal!

(Seconds) Background Fluorescence

Signal Reference Reference Blanks _______________________________ ________ 1 3564362 51585883 3563988 0.069088436 ________ 2 3564298 51585628 3563924 0.069087537 ________ 3 3564874 51585846 _______ 3564500 0.069098411 4 3566215 51586743 3565841 0.069123205 ________ 3565496 51586091 3565122 0.069110141 ________ P14011-SP-02.doc 31/03/06 -19-Reading Raw Fluorescence Data Fluorescence Signal Fluorescence Fit Number Minus Fluorescence Signal I

(Seconds) Background Fluorescence

Signal Reference Reference Bloods _________ ____________________ _______________ _________ 1 0 --374 _______________ 0.0575 _____ 2 0 -374 _______________ 0.0575 _____ -0 -374 ______ 0.0575 __________ 4 0 -374 _______________ 0.0574 ________ 5 0 -374 _____________ 0.0574 __________ 0 -374 _______________ 0.0574 _________ 2955821 51583592 2955447 0. 057294323 0.0573 _________ 2955062 51582679 2954688 0.057280623 0.0573 ________ 2953008 51583518 2952634 0.057239873 0.0573 2949933 51582543 2949559 0.057181341 0.0572 11 2949493 51583139 2949119 0.057172151 0.0572 _______ 12 2946591 51582599 2946217 0.05711649 0.0572 13 2945289 51583468 2944915 0.057090287 0.0571 14 2944638 51583291 2944264 0.057077863 0.0571 -15 2943483 51583517 2943109 0.057055222 0.0571 16 2941483 51582296 2941109 0.057017799 0.0571 _____ 17 2939910 51584175 2939536 0.056985229 0.0570 18 2938487 51583010 2938113 0.056958929 0.0570 19 -2937585 51582202 2937211 0.056942334 0.0570 2935457 51583227 2935083 0.056899949 0.0569 21 2934545 51584002 2934171 0.056881415 0.0569 22 2933246 51582555 2932872 0.056857827 0.0569 --23 -2932307 51583308 2931933 0.056838794 -0.0569 24 2930244 51583334 2929870 0.056798771 0.0568 2928809 51583527 2928435 0.05677074 0.0568 26 2928925 51582154 2928551 0.0567745 0.0568 27 2927934 51582712 2927560 0.056754674 0.0568 28 2925072 51583693 2924698 0.056698112 0.0567 29 -2924539 51582904 2924165 0.056688646 0.0567 ______ 30 2924052 51583196 2923678 0.056678884 0.0567 31 2921778 5158?587 2921404 0.056635469 0.0567 32 2921581 51583313 2921207 0.056630853 0.0567 33 2918178 51581710 __________ 2917804 0.05656664 0.0566 34 2918348 51581902 2917974 0.056569725 0.0566 2916054 51582192 2915680 0.056524934 0.0566 36 2915194 51582289 2914820 0.056508155 0.0566 37 2914844 51582120 2914470 0.056501555 0.0565 38 2914629 51581901 2914255 0.056497627 0.0565 39 2912071 51581619 2911697 0.056448345 0.0565 2911685 51581776 2911311 0.056440689 0.0565 41 2909430 51580144 2909056 0.056398757 0.0565 42 2909121 51581358 2908747 0.056391 439 0.0564 43 2908666 51581300 2908292 0.056382681 _J10564 44 2906914 51581161 2906540 0.056348867 0.0564 --2905229 51581336 --2904855 0.056316009 0.0564 -46 --2904469 51581125 2904095 0.056301506 0.0564 47 2903867 51580515 2903493 0.0562905 0.0564 48 2903108 51580361 2902734 0.056275954 0.0563 49 2902382 51580998 2902008 0.056261184 0.0563 2900836 51580764 2900462 0.056231466 0.0563 51 2899915 51580441 2899541 0.056213963 0.0563 52 2899699 51580109 2899325 0.056210137 0.0563 53 2899117 51579994 2898743 0.056198979 0.0563 54 2898402 51579715 2898028 0.056185421 0.0562 2896664 51580326 2896290 0.05615106 0.0562 56 2897052 51580613 2896678 0.05615827 0.0562 57 2896426 51580073 2896052 0.056146722 0.0562 58 2895070 51580024 --2894696 0.0561 20486 0.0562 59 2894867 51579530 2894493 0.056117088 0.0562 2893832 51579129 2893458 0.056097458 -0.0562 P14011-SP-02.dcc 31103/06 -20 -Reading Raw Fluorescence Data Fluorescence Signal Fluorescence Fit Number Minus Fluorescence Signal I

(Seconds) Background Fluorescence

Signal Reference Reference 61 2893835 51578832 ___________ 2893461 0.056097839 0.0561 62 2893142 51580065 2892768 0.056083062 0.0561 -63 2891979 51579300 2891605 0.056061346 0.0561 64 2891624 51580313 2891250 0.056053363 0.0561 -65 2889838 51579562 2889464 0.056019553 0. 0561 66 2890166 51579307 2889792 0.056026189 0.0561 67 2888871 51579263 2888497 0.05600113 0.0561 68 2888266 51579055 2887892 0.055989626 0.0560 69 2887788 51578116 2887414 0.055981378 0.0560 2887371 51578675 2886997 0.055972686 0.0560 71 2887694 51578175 2887320 0.055979491 0.0560 72 2887040 51578384 2886666 0.055966585 0.0560 73 2886642 51577925 2886268 0.055959366 0.0560 74 2885106 51577596 2884732 0.055929943 0.0560 2884262 51576184 -. 2883888 0.05591511 0.0560 76 2884732 51577237 2884358 0.055923081 0.0560 77 2884096 51577591 2883722 0.055910366 0.0559 78 2883331 51576616 2882957 0.055896591 0.0559 79 2882382 51577518. 2882008 0.055877214 0.0559 2883826 51577070 2883452 0.055905696 0.0559 81 2882045 51577415 2881671 0.055870792 0.0559 82 2881718 51577408 2881344 0.055864459 0.0559 83 2881424 51577078 2881050 0.055859116 0.0559 84 2881564 51576485 2881190 0.055862473 0.0559 -2882487 51575751 2882113 0.055881164 0.0559 86 2879464 51575861 2879090 0.055822432 0.0559 87 2879806 51575495 2879432 0.055829459 0.0558 88 2878031 51575609 2877657 0.05579492 0.0558 89 2877710 51576230 2877336 0.055788025 0.0558 2876336 51576600 2875962 0.055760985 0.0558 91 2877713 51576006 _____ 2877339 0.055788325 0.0558 92 2877616 51576122 ---2877242 0.055786319 0.0558 93 2877104 51578012 2876730 0.055774348 0.0558 94 2874740 51577685 2874366 0.055728868 0.0558 2875728 51576443 2875354 0.055749366 0.0558 _______ 96 2874720 51576213 2874346 0.055730071 0.0558 97 2875418 51576641 2875044 0.055743142 0.0558 98 2875735 51576325 2875361 0.055749629 0.0558 99 2875128 51576472 2874754 0.055737701 0.0557 2874927 51576492 2874553 0.055733783 0.0557 101 2874846 51576435 2874472 0.055732274 0.0557 102 2873640 51576098 2873266 0.055709255 0.0557 103 2873382 51575645 2873008 0.055704742 0.0557 104 2872954 51576065 2872580 0.05569599 0.0557 2872727 51575160 -2872353 0.055692566 0.0557 106 2871226 51575773 2870852 0.055662801 0.0557 107 2872402 51575375 2872028 0.055686032 0.0557 108 2872774 51576582 2872400 0.055691942 0.0557 109 2870911 51575349 __________ 2870537 0.055657151 0.0557 2871892 51575295 2871518 0.05567623 0.0557 __1 2870390 51575094 2870016 0.055647325 0.0557 112 2870840 51576083 2870466 0.055654983 0.0557 113 2870388 51575798 2870014 0.055646526 0.0556 114 2869947 51574332 2869573 0.055639557 -0.0556 -. --115 2870353 51573855 --2869979 0.055647944 0.0556 116 2869297 51574060 2868923 0.055627247 0.0556 117 2868911 51573600 2868537 0.055620259 0. 0556 _______ 118 2868895 51573772 -2868521 0.055619763 0.0556 119 2868030 51573821 2867656 0.055602939 0.0556 2868701 51574367 2868327 0.05561536 0.0556 121 2866233 51574457 2865859 -0.05556741 0.0556 P14011.SP-02.doc 31/03/06 -21 -Reading Raw Fluorescence Data Fluorescence Signal Fluorescence Fit Number Minus Fluorescence Signal!

(Seconds) Background Fluorescence

Signal Reference Reference 122 2867978 51573835 2867604 0.055601915 0.0556 123 2867313 51573376 2866939 -0.055589516 0.0556 124 2867172 51573576 2866798 0.055586566 0.0556 2866858 51573178 2866484 0.055580907 0.0556 126 2867058 51573583 2866684 0.055584348 0.0556 127 2867722 51573210 2867348 0.055597625 0.0556 128 2867285 51574328 2866911 0.055587947 0.0556 129 2858225 51574505 ______ 2857851 _.055412088 0.0556 2867356 51573382 -2866982 0.055590343 0.0556 -131 2863993 51573955 2863619 0.055524518 0.0555 132 2865453 51573157 2865079 0.055553687 0.0555 -133 2865181 51574269 2864807 0.055547215 -0.0555 _______ 134 2864123 51573728 2863749 0.055527283 0.0555 2864932 51572876 2864558 0.055543887 0.0555 136 2863379 51572485 2863005 0.055514195 0.0555 137 2863690 51573048 2863316 0.055519619 0.0555 138 2864278 51572945 2863904 0.055531132 0.0555 139 2864629 51572443 2864255 0.055538478 0.0555 -140 2862974 51573092 _______ 2862600 0.055505689 -0.055_ -141 2863867 51572498 -2863493 0.055523644 0.0555 142 2863679 51573115 2863305 0.055519334 0.0555 143 2862000 51572406 2861626 0.055487541 0.0555 -144 2862424 51573097 2862050 0.055495019 0.0555 2861464 51572506 2861090 0.05547704 0.0555 ______ 146 2862311 51572555 2861937 0.055493411 0.0555 147 2862304 51571108 2861930 0.055494832 0.0555 148 2862010 51571937 2861636 0.05548824 0.0555 149 2859734 51572024 2859360 0.055444014 0.0555 2860693 51571533 2860319 0.055463137 0.0555 151 2860913 51571985 2860539 0.055466917 0.0555 152 2862295 51572314 2861921 0.05549336 0.0555 153 2861267 51571491 2860893 0.055474312 0.0555 154 2861138 51572499 2860764 0.055470727 00555 2860113 51570936 2859739 0.055452532 0.0555 156 2859600 51571386 2859226 0.055442101 0.0555 157 2860578 -51571930 2860204 0.05546048 0.0555 158 2858371 51571186 2857997 0.055418485 0.0554 159 2859688 51570801 2859314 0.055444436 0.0554 2860773 51571318 2860399 0.055464919 0.0554 161 2857694 51570305 2857320 0.055406304 0.0554 162 2859769 51570656 2859395 0.055446163 0.0554 163 2859690 51571693 2859316 0.055443516 0.0554 164 2859449 51570510 2859075 0.055440115 0.0554 2860192 51570519 ______ 2859818 _055454513 0.0554 -166 --2858530 51569196 2858156 0055423707 -00554 167 2858942 51570525 2858568 0.055430268 0.0554 -168 2858447 51570477 2858073 0.055420721 0.0554 169 2857559 51570836 2857185 0.055403116 0.0554 2857736 51570526 2857362 0.055406881 0.0554 171 2857315 51569676 2856941 0.055399631 0.0554 172 2857600 51569981 2857226 0.055404829 0.0554 173 2858461 51570608 2858087 0.055420851 0.0554 174 2856840 51570008 2856466 0.055390063 0.0554 2857004 51569874 ___________30 0.055393387 0.0554 176 2856544 51569621 2856170 0.055384739 0.0554 177 2856786 51569880 2856412 0.055389154 0.0554 178 2856348 51570223 2855974 0.055380292 0.0554 179 2857336 51569511 2856962 0.055400215 0.0554 2857080 51570092 2856706 0.055394627 0.0554 P14011-SP-02.dcc 31/03f06 -22 -In addition to the raw fluorescence data, Table 1 also shows various mathematical operations carried out on that data. The first mathematical operation is to subtract this fluorescence background (FBgd) from the raw fluorescence data, for both blank and the blood readings. The fluorescence background signal (FB9d) is derived from a reading of the buffer solution, with no fluorescent marker reagent present and has previously been measured. It is predominantly a reader constant, this background is principally the electrical zero of the fluorimeter; as the fluorimeter optical filters have been optimised to reduce filter breakthrough' from the buffer effectively to zero'.

The last three of the blank readings are averaged in order to establish FBIank.

The corrected fluorescence data is then divided by the reference. If this is plotted against time it produces a curve as shown in Figure 6. A curve is fitted to these values using a suitable algorithm as previously discussed and the values (as shown the right hand column) are also plotted on the graph in Figure 6. The values F0 and F,. can be derived for this sample by back and forward extrapolation of the data fit to to and t, respectively.

Table 2 shows the results of an equivalent assay run on 20 samples.

The sample (sample 1) assay shown in Table I is accompanied by 19 others who also had known reference amounts of total Haemoglobin (Total Hb), the concentration of glycated haemoglobin ([Aic]) and percentage glycated haemoglobin (%Alc).

Fblank (corrected) is the average of the "Fluorescence Signal! Fluorescence Reference" values for the blanks in Table 1.

P14011-SP-02.doc 31/03/06 -23 -For each sample, the Fluorescence Optical Density (FOD) and Specific Quenching (SQ) are calculated from FBIank, F0 and F using the formulae below; FOD = Log [(FBIaflk -FB9d) I (FO -FB9d)} [Equation 1 B] SQ = (Fo -F0)I (Fo -FBgd) [Equation 3] From FOD and SQ the concentrations of total haemoglobin and glycated haemoglobin (Aic) concentrations are calculated by respectively.

[Total Hb] = m. FOD + c [Equation 4] [Aic] m'.SQ + c' [Equation 5] The calibration constants m, m', c & c' are derived from standard curve (straight line) assay data using samples of known [Aic] and [Total Hb] concentrations. For the sample data provided c & c' are zero as the data has been constrained in both plots to go through zero.

The percentage glycated haemoglobin in the sample is then calculated thus; %Alc = ([Aic] / [Hb])*100% [Equation 6] %Alc = ((m' . SQ + C')! (m. FOD + c))*100 [Equation 7] P14011-SP-02 doc 31/03/06 -24 -If the value of the Total Haemoglobin concentration is plotted against FOD they have been found to be directly proportional and the graph may be summarised according to the equation y = mx + c in which y is the total Haemoglobin concentration and x is FOD. m and c can then be calculated for a particular set of specific reaction parameters to give a standard calibration curve. Figure 8 shows [Total Hb] v FOD for samples 1 to 20 with the line fitted thereto being summarised by the equation y = mx + c.

Likewise Specific quenching (SQ) has been found to directly proportional to the concentration of Aic, and the graph may be summarised according to the equation y = m'x + c' in which y is the glycated Haemoglobin concentration and x is specific quenching. m' and c' can then be calculated for a particular set of specific reaction conditions to give further standards.

Figure 7 shows [Aic] v SQ for samples 1 to 20 with the line fitted thereto being summarised by the equation y = m'x + c'.

For this example the standards derived from these are shown below in

table 3.

Table 3

[AlcJ -vs.-SQ Slope = m 0.1302 [Aic] -vs. -SQ Intercept = c' 0 [Total Hb] -vs. -FOD Slope = m 1.318 [Total Hb]-vs.-FOD lntercept=c 0 These calibration constants are derived for a particular set of reaction criteria, and these may be used for subsequent assays using the same criteria. Different reaction conditions may require different calibration constants to be derived.

P14011-SP-02 doc 31/03/06 -25 -Table 2 shows the calculated values of % Glycated Haemoglobin and a comparison to the known value indicates the accurate nature of the assay of the present invention. Indeed the standard deviation of the error in the sample data set is just O.4%A1 c, demonstrating the accuracy of the present invention.

P14011-SP-02.doc 31103/06

Table 2

Sample Reference Reference Reference Fblaflk Derived F, Derived F.. Inherent Specific] FOD Measured Number %Alc [Aid [Total Hb] (corrected) Fluorescence Fluorescence %Alc Quenching Quenching 1 5.65% 0.0059 0.1052 0.06863 0.05757 0.05525 16.12% 4.01% 0.07636 5.19% 2 5.05% 0.0045 0.0881 0.06487 0.05545 0.05354 14.52% 3.44% 0.06812 5.00% 3 11.05% 0.0125 0.1135 0.07210 0.06003 0.05448 16.74% 9.25% 0.07955 11.49% 4 10. 25% 0.0110 0.1077 0.06689 0.05540 0.05078 17.18% 8.33% 0.08188 10.05% 5.95% 0.0054 0.0913 0.06983 0.05987 0.05741 14.26% 4.10% 0.06683 6.06% 6 6.35% 0.0063 0.0987 0.06716 0.05661 0.05404 15.72% 4.54% 0.07426 6.03% 7 8.95% 0.0077 0.0864 0.06802 0.05867 0.05509 13.75% 6.11% 0.06426 9.39% 8 8.75% 0.0065 0.0744 0.06656 0.05796 0.05490 12.92% 5.28% 0.06009 8.67% 9 6.85% 0.0064 0.0928 0.06544 0.05560 0.05283 15.03% 4.99% 0.07075 6.96% 6.55% 0.0044 0.0675 0.07662 0.06797 0.06537 11.30% 3.81% 0.05205 7.24% 11 6.75% 0.0064 0.0952 0.06598 0.05575 0.05289 15.51% 5.13% 0.07318 6.93% 12 11. 55% 0.0109 0.0944 0.08622 0.07268 0.06664 15.70% 8.31% 0.07415 11.07% 13 7. 60% 0.0075 0.0993 0.06407 0.05362 0.05074 16.31% 5.38% 0.07734 6.87% 14 6.80% 0.0064 0.0944 0.06394 0.05443 0.05165 14.87% 5.11% 0.06993 7.22% 7.05% 0.0069 0.0983 0.06689 0.05628 0.05326 15.87% 5.36% 0.07503 7.06% 16 6.85% 0.0054 0.0795 0.06779 0.05883 0.05606 13.22% 4.70% 0.06158 7.54% 17 12. 95% 0.0135 0.1045 0.07168 0.05956 0.05325 16.92% 10.59% 0.08048 13.00% 18 6.10% 0.0060 0.0977 0.06451 0.05436 0.05194 15.73% 4.44% 0.07435 5.90% 19 5.90% 0.0062 0.1046 0.06804 0.05667 0.05419 16.71% 4.37% 0.07942 5.44% 9.00% 0.0084 0.0934 0.06881 0.05813 0.05421 15.52% 6.74% 0.07324 9.09% P1401 1-SP-02.doc 31/03/06

Claims

-27 -Claims 1. A method of carrying out an assay for a non-fluorescent

substance in an assay sample, which method comprises: (a) carrying out a reaction in solution between an assay sample and a fluorescent marker compound, the sample and marker compound being combined at time t0; (b) exciting the fluorescence in the marker compound, wherein the nature of the marker and the nature of the excitation are such that the said fluorescence occurs at a wavelength at which said fluorescence is altered by the reaction of the marker compound with the non-fluorescent substance; (c) detecting the resulting fluorescence as the reaction progresses; (d) calculating from the detected fluorescence the values of F0 being the fluorescence at time to and F. being the fluorescence at time L, when all of the non-fluorescent substance has reacted with the marker compound; (e) calculating from the values of F0 and F0. the background change in fluorescence attributable to the reaction in step (a); and (f) determining from the calculated values and a suitable calibration algorithm the concentration of the non-fluorescent substance present in the sample at to prior to reacting with the fluorescent marker compound.
2. A method as claimed in claim 1, in which prior to step (a) the marker compound is excited and the resultant initial fluorescence (FBIaflk) is detected.

P14011-SP-02dcc 31/03/06 -28 -
3. A method as claimed in claim 2, in which the values of F0 and FBIank are used to calculate the fluorescence optical density (FOD) using the equation: FOD = Log [FBIank/FO] (Equation 1)
4. A method as claimed in any of claims 1 to 3 in which the calculation of the values of F0 and F in step (d) includes fitting a mathematical function of a curve of best fit to the data and extrapolating that function to time to and L.
5. A method as claimed in any of the preceding claims where calibration algorithm used in section (f) is of the form y = mx + c, where m and c are the slope and intercept calibration constants and x = (F0 -F)I F0.

6. A method as claimed in any of the preceding claims where the non-fluorescent substance is a subspecies of a main substance, and the subspecies is selectively reactive with the marker compound, and wherein the overall concentration of the main substance and subspecies present in the sample at to, prior to reacting with the fluorescent marker compound, is determined from the calculated values and a suitable calibration algorithm of the form y = m'x + c'; where m' and c' are the slope and intercept calibration constants and x = Log(FBIaflk I Fe).

7. A method as claimed in any of the preceding claims, wherein the substance to be assayed is a glycated protein, and preferably is glycated haemoglobin.

8. A method as claimed in claim 7, wherein the marker compound contains a boronic acid group capable of selective binding with the cis-diol group of the glycated protein.

P1401 1-SP-02 dOe 31/03/06 -29 - 9. A method as claimed in any of the preceding claims, wherein the fluorescent marker compound has a principal excitation wavelength in the range of 200nm to 800nm, and preferably of approximately 5lOnm.

10. A method as claimed in any of the preceding claims, wherein the fluorescent marker compound contains a fluorescein residue.

11. A method as claimed in claim 10, wherein the fluorescent compound is a group of the formula F-NH-CS-NH-Ph-B(OH)2, wherein Ph is a phenyl group and F is a fluorescein residue.

12. A method as claimed in claim 10 or claim 11, wherein the fluorescent compound is a compound of the formula I: HO O o

OHI H/'N

H \B(OH)2 13. A method as claimed in any of the preceding claims, wherein the sample is a blood sample, a plasma sample, er-a serum sample, or a urine sample.

14. A method as claimed in claim I and substantially as herein described.

15. A method of determining the concentration of a non-fluorescent substance, comprising exciting a matching fluorescent reagent the excitation and emission spectrum of which overlaps the absorption spectrum of the non- fluorescent substance; detecting the resultant fluorescence; adding the non-fluorescent substance to the matching fluorescent reagent; detecting the P14011-SP-02 doc 31/03/06 -30 - fluorescence after the addition; and calculating the concentration of the non-fluorescent substance from the difference between the fluorescence detected before and after addition of the non-fluorescent substance.

16. A method as claimed in claim 15, in which the substance reacts with the fluorescent reagent to alter the fluorescence over time, and a range of fluorescence values are detected over a period of time starting at least 5 seconds after addition of the substance, and this range of measured values is used to extrapolate a value for the fluorescence directly after addition of the substance at time to.

17. A method as claimed in claim 15, and substantially as herein described.

18. Apparatus for the assay of a substance, in a sample, which comprises: (a) a supply of a fluorescent marker compound; (b) a reaction vessel adapted to hold an amount of the fluorescent marker compound and an assay sample, which together undergo a reaction beginning at time to and ending at time L that alters the fluorescent characteristics of the marker compound; (c) a source of electro-magnetic radiation having a wavelength A to excite the fluorescence in the marker compound; (d) detecting means to measure the resulting fluorescence as the reaction progresses; and (e) a processor adapted to calculate from the measured fluorescence the values of F0 being the fluorescence at time to, and F. being the fluorescence at time L when all of the substance has P14011-SP-02doc 31/03/06 -31 reacted with the marker compound; to calculate any background change in fluorescence not attributable to and that attributable to the reaction between the substance and the marker compound; and to determine from those calculated values the amount and/or concentration of the non-fluorescent substance and/or a sub-species thereof present in the sample at to.

19. Apparatus as claimed in claim 18, which is adapted to receive a blood derived sample and to perform and assay to determine the amount of haemoglobin, and/or glycated haemoglobin.

20. Apparatus as claimed in claim 18 or c'aim 19, in which A is approximately 5lOnm.

P14011-SP-02doc 31/03/06