WO2007028983A1

WO2007028983A1 - Protein assay

Info

Publication number: WO2007028983A1
Application number: PCT/GB2006/003293
Authority: WO
Inventors: Samir Chahine; Callum Livingstone; James Davis
Original assignee: The Nottingham Trent University
Priority date: 2005-09-09
Filing date: 2006-09-06
Publication date: 2007-03-15
Also published as: GB0518460D0; WO2007028983A8

Abstract

The invention provides a method for determining the relative amount of a sulphydryl-containing protein, such as albumin, compared with lower molecular weight thiol-containing compounds in a sample containing both sulphydryl containing protein and lower molecular weight thiol-containing compounds, comprising the step of contacting the sample with substituted or non-substituted 2.2'-dithio(bis)benzothiazole reagent (the first reagent) or a derivative thereof and determining directly or indirectly an amount of reacted thiol compounds that have reacted with the first reagent (reacted thiols) and/or thiol-containing compounds that have not reacted (unreacted thiol) with the first reagent. The method allows the discrimination between, for example, albumin and low molecular weight species such as glutathione, cysteine and homocysteine. The first reagent may be provided in a column and the sample passed through the column.

Description

Protein Assay

The invention provides assays and kits for the identification and differentiation between proteins, such as sulphydryl albumin, and low mono-molecular weight thiols, such as glutathione (GSH), Cysteine (CSH) and homocysteine (HCSH) in, for example, blood plasma, saliva and urine.

The concentration of anti-oxidant thiol (RSH) within plasma has been shown to be a potentially valuable semi-quantitative indicator through which the severity of various injuries or diseases can be assessed/^1"4' The analysis of the latter within mainstream practice, however, invariably requires the referral of the sample to specialist laboratories with the time delays often negating the diagnostic advantage. While Ellmans Reagent (5,5'-dithio-(2-nitrobenzoic acid))⁵ has long been the mainstay of routine thiol analysis, it is unable to speciate between sulphydryl albumin and the low mono-molecular species, such as glutathione (GSH), cysteine (CSH) and homocysteine (HCSH) and, as a consequence, can only deliver total plasma thiol (PSH) concentration.⁴ The latter is widely recognised as having clinical merit but there is considerable debate as to whether mono, macro or total thiol respond in equal measure to the onset of physiological stress.⁴

There is therefore a need to identify a method of being able to distinguish between albumin and low molecular weight thiols in samples of, for example, plasma. Ideally the assay should be easy to use to allow the testing of samples to be carried out within, for example, hospital laboratories.

The inventors have unexpectedly found that this selection may be carried out using 2,2'-dithio(bis)benzothiazole. This compound has hitherto not been used for this purpose. This is also known as WestcoMBTS and has previously been used as a general purpose accelerator for sulphur cures in natural and synthetic rubber processing.

In principle this compound was expected to react with free sulphydyl thiols in a similar manner to the mechanism observed for Ellman's Reagent, resulting in a mixed disulphide complex and the release of a mercaptobenzothiazole (MBT) anion. The 2,2'-dithio(bis)benzothiazole reagent is insoluble in aqueous solutions. Passing, for example, cysteine, through a column of the reagent produced a filtrate containing mixed disulphide complex. However free MBT was not observed in the filtrate. The MBT is thought to be insoluble within the filtrate and retained within the packing material in the column. This may be released by the addition of an amino acid moiety to the complementary thiazole component to aid solubilisation.

Unexpectedly, it was found that passing albumin through a column of the reagent did not result in any detectable reaction of the reagent with the protein. This unexpectedly allows low molecular weight thiols and high molecular weight albumins to be distinguished.

The invention provides a method for determining the relative amount of a sulphydryl-containing protein, such as albumin, compared with low molecular weight thiol-containing compounds in a sample containing both sulphydryl-containing protein and low molecular weight thiol-containing compounds, comprising the step of contacting the sample with substituted or non-substituted 2,2'-dithio(bis)benzothiazol reagent ("the first reagent") or a derivative thereof and determining directly or indirectly an amount of thiol compounds ("reacted thiol"), that have reacted with the first reagent and/or thiol-containing compounds that have not reacted with the first reagent ("unreacted thiol").

Protein is intended to mean polypeptides having a molecular weight of at least 10,000 Da. Serum albumins typically have a molecular weight of at least 45,000 Da, and usually at least 60,000 Da.

Low molecular weight thiols typically have a molecular weight of below 1,000 Da. They are preferably mono-molecular species such as glutathione, cysteine and homocysteine. Such amino acids typically have a molecular weight of below 250 Da.

The structure of 2,2'-dithio(bis)benzothiazole is shown in Figure 1. This may be substituted by one or more side groups such as by adding one or more side chains to the benzothiazole ring to make chromogenic moieties. For example, the benzyl ring may be substituted by a nitro(NO2) group in a similar manner that Ellman's Reagent contains a nitro group. Furthermore, the solubility of the individual benzothiazole moieties making up the dimer may be adjusted by the addition of one or more side chains.

Preferably the sample is an aqueous sample such as blood, blood plasma, saliva, urine or serum.

The sample may be contacted with the first reagent by providing the reagent as a column through which the sample is passed. Alternatively the reagent may simply be mixed with the sample and the reagent allowed to precipitate. The first reagent selectively reacts with the low molecular weight thiol-containing compounds. The low molecular weight thiol-containing compounds selectively react with the first reagent to produce a mercaptobenzothiazole (MBT) conjugate. This conjugate may be detected directly or indirectly, for example, by reacting with further compounds. The sulphydryl-containing protein such as albumin has not been observed to substantially react with the first reagent. Hence, the protein will remain within the sample after it has been contacted with the first reagent and will contain free thiol. This free thiol left after contacting the sample with the first reagent may itself be determined either directly, or indirectly, by means of one or more additional reagents.

Preferably, the total amount of thiol-containing compounds in the sample is determined by taking an aliquot of the sample. This is then compared to the amount of low molecular weight thiol-containing compounds that have been determined to have reacted with the first reagent or alternatively to the amount of free thiol-containing compounds left, after contacting a different portion of the sample with the first reagent. If the total amount of free thiol in the sample is known and the amount of either low molecular weight thiol-containing compounds or the amount of unreacted thiol is known, then it is possible to determine the relative amount of sulphydryl-containing proteins, such an albumin, compared with low molecular weight thiol-containing compounds in the sample. This amount may be the relative ratio between the two or alternatively may be the actual concentrations of the two types of thiols. The total amount of thiol in the aliquot taken from the original sample may be determined by conventional assay techniques, such as using Ellman's Reagent. Ellman's Reagent is probably the most well known of compounds which are known to react with amino acids containing thiol groups. However, other compounds, such as iodoacetamides, maleimides, such as N-ethylmaleimide, or other thiol-detecting reagents known in the art may be used. Electrochemical methods of detecting thiols may be used, as discussed below.

Preferably, the first reagent is provided in a column and the sample is passed through the column to allow the low molecular weight thiol-containing compounds to react with the reagent. This column may be conveniently provided within a microfuge tube, such as those known as "Eppendorf tubes". However any convenient vessel or tube may provided to contain the column. The advantage of microfuge tubes is that they may be put within a microcentrifuge in order to increase the speed of passage of the sample through the column.

The reaction products from the reaction of low-molecular weight thiol-containing compounds may be detected using one or more electrodes. Hence the first reagent may be provided on the surface of one or more electrodes. The sample diffuses through the layer of the first reagent to the surface of the electrode. Alternatively the electrode may be used with samples previously contacted with the first reagent.

Preferably the low molecular weight thiols react with the first reagent to produce an MBT conjugate and the amount of MBT conjugate is detected.

Alternatively or additionally one or more detection reagents to convert the reacted thiol and/or unreacted thiol into detectable compounds may be provided. For example, the unreacted thiol may be then reacted with Ellman's Reagent or another thiol-reactive reagent known in the art (example as discussed above).

Ellman's Reagent forms a mixed dilsulphide with thiols, liberating the chromophore 5-mercapto-2-nitrobenzoic acid. This may be detected spectrophotometrically. The chromophore has an absorption maximum of approximately 410 nm, and e of approximately 13, 600 cm^"1 M^"1.

Since only reduced (RSH) thiols that are accessible to this water-soluble reagent are modified, it may be desirable to quantify the unreacted thiols in the presence of, for example, 6M guanidinum chloride.

The detection reagent may itself be provided as a column adjacent a column of the first reagent so that the sample passes through the first reagent and then through the column of the second reagent. The material that has passed through the reagents is then analysed.

The analysis of the sample after it has been contacted with the first reagent and/or detection reagent may be carried out in situ within for example the microfuged tube. Alternatively the reacted sample may be placed into a separate vessel such as a cuvette, for analysis.

The detection of the reacted thiol and/or unreacted thiol may be detected directly or indirectly, by photometric, chromatography, capillary electrophoresis or electrochemical methods.

Preferably the amount of thiol-containing compound reacted with the reagent is determined by detecting the absorption of the sample at 305-325, 310-315, especially 312 nm. This detects the absorption of the the MBT conjugate.

Alternatively the amount of unreacted thiol is preferably determined by reacting the unreacted thiol with Ellman's Reagent and detecting the released 5-mercapto-2-nitrobenzoic acid chromophore, as described above. Such detections may be carried out in situ within for example the Eppendorf or alternatively within a separate optical cuvette.

The reacted or unreacted thiols may be detected electrochemically. Preferably, potentiometric analysis utilising a two electrode system is employed within which one electrode serves as a reference electrode whilst the other acts as the indicating probe. A quinone or other suitable indicator is introduced to the system whereby it reacts with the species containing the free thiol groups. This leads to a disparity in the relative ratios of the oxidized/reduced ratio within the sample which can be recorded through monitoring the potential difference between the two electrodes. The quinone is the oxidised form and the resulting conjugate is initially in the reduced form. The greater the concentration of the free thiol introduced, the greater the concentration of the conjugate and hence the reduced species and the bigger the potential shift in the potential between the electrodes. The magnitude of the potential difference can be related to concentration and forms the basis of a calibration curve from which unknown samples can then be assessed. Preferably the quinone used is benzoquinone or most preferably napthoquinone.

In use with the device according to the invention, plasma or serum sample passes through the first reagent. The low molecular weight thiols are removed through exchange to form MBT conjugates. Proteins such as albumin do not react and pass through unmodified. The unreacted albumin therefore contains free thiols which are able to react with the quinone and produce a change in the potential difference between the two electrodes.

The amount of unreacted thiol may be compared with the total thiol in the original sample by repeating the assay with an aliquot of the original sample without contacting it with the first reagent.

Alternatively, a two or three electrode system may be used in which a counter electrode is used. A reference electrode such as a silver/silver chloride reference electrode may be used. Preferably a carbon electrode, such as a fibre, printed or composite electrode is used.

In the preferred embodiment the potential of the indicating electrode is controlled (relative to the reference electrode or a combined reference-counter electrode in a two electrode system). A potential range is swept using a given wave form (a linear sweep or a pulse wave form) such that an oxidation reaction is induced at the electrode surface at a particular potential characteristic of the system under investigation. The oxidation of the MBT conjugate species leads to a current which can be measured either by a chart recorder or a computer controlled potentiostat. The magnitude of the current being proportional to the concentration of free thiol initially present in the sample before it is reacted with the first reagent. In this exemplified case albumin would be silent as it does not react with the first reagent.

In amperometric analysis a fixed potential is used and the current is read as a function of time. The magnitude of the current is proportional to the concentration.

The invention also provides kits for use in the determination of the relative amount of a sulphydryl-containing protein compared with low molecular weight thiol-containing compounds comprising a substituted or non-substituted 2,2'-dithio(bis)benzothiazole reagent or a derivative thereof ("the first reagent").

Preferably the first reagent is provided in a column for passing a sample there through. The column may be provided in a microcentrifuge tube or a capillary tube, such as an Eppendorf tube as discussed above. The kit may additionally comprise one or more detection reagents, as defined above, for converting unreacted thiol and/or reacted thiol after a sample has been contacted with the first reagent, into detectable compounds. The detection reagent may also be used for the determination of the total thiol content in the initial sample.

Preferably the first reagent and the detection reagent are each arranged in a column so that, in use, a sample is passed firstly through the first reagent, followed by passing through the detection reagent.

The kit may additionally comprise two or more electrodes for detecting the reacted or non-reacted thiol-containing compounds after being contacted with the first reagent and optionally the detection reagent. The electrodes may be provided within a microfuge tube in which the first reagent has been provided as a column. Preferably they are arranged to assay a sample after it has passed through or been contacted with, the reagent. The microfuge tubes may additionally comprise the detection reagent in the form of a column. Alternatively, the detection reagent may be provided separately.

Kits according to the invention also include electrodes coated with the first reagent. In use the sample diffuses through the coating of the first reagent to the electrode, where the reacted or non-reacted thiols are detected.

Kits according to the invention which are provided with instructions for their use are also included within the scope of the invention.

The invention also provides use of a kit according to the invention in the detection of the relative amounts of sulphydryl containing proteins, such an albumin, compared with lower molecular weight thiol-containing compounds in a sample.

The invention will now be described by way of example only with reference to the following figures:

Figure 1 discloses a centrifugal filter device made in accordance with the invention.

Figure 2 shows spectroscopic profiles of centrifugal filter device processed MBT conjugates compared with that of an albumin sample.

Figure 3 shows typical response of a potentiometric device on the addition of albumin.

Figure 4 shows the voltammetric response to the addition of MBT conjugated to cysteine.

Figure 1 shows a centrifugal filter device (CFD). CFD (10) comprises a microfuge tube (12) with a screw cap (14).

Within the tube is provided a column of the first reagent (16). hi use the sample is placed above the first reagent (16) and passes through the reagent into the bottom of the microfuges tube (in the direction of the arrows). Micro fuged tube may comprise immediately below the first reagent (16) an additional layer, the additional layer comprising a detection reagent, such as Ellmans Reagent, through which the sample also passes.

The basic strategy is outlined in figure 1 and is highlighted by the passage of cysteine (I) through a centrifugal filter packed with a 2,2'-dithio(bis)benzothiazole (11) indicator. The latter is insoluble within aqueous solution and serves as a densely packed particulate filter, hi principle, the disulphide should react with free sulphydryl thiols through a mechanism analogous to that observed with Ellman's Reagent(ER) resulting in the formation of the mixed disulphide (TSI) and the release of the mercaptobenzothiazole anion (IV).

Spectroscopic investigation (uv/vis and ¹H NMR) of the filtrate revealed that the principal product in the filtrate was in fact the mixed disulphide(m) and not the free mercaptobenzothiazole (FV). It appears that the latter, like its disulphide parent, is insoluble within the buffer and is retained within the packing material. The addition of the amino acid functionality to the complementary thiazole component serves to aid solubilisation and, as such, is preferentially released. Similar results were obtained for homocysteine and glutathione. An assessment of the percentage conversion of the free thiol into the mercaptobenzothiazole (MBT) conjugate was conducted (employing a before and after spectroscopic methodology) with the recovery of the thiol constituents being in order of 100 + 2 % (based on 20 uM cysteine). The uv spectroscopic profiles for additions of CSH (40 uM), GSH (40 uM) and bovine albumin (0.82 mg/L) are shown in figure 2. The principal absorption bands for GSH and CSH moieties were observed at 312nm and correspond predominately to the MBT component of the conjugates. Significantly, the passage of albumin did not lead to any change in the absorption profile from that of the corresponding unfiltered control.

The molar absorptivities (CSH, Amax = 312nm, ε = 275 mol^"1 1 cm^"1 and GSH, a = 365 mor^"1 L cm^"1) are significantly less than that of ER (Amax = 412nm, e = 14150 mol^"1 lcm^"1) and can be attributed to the lack of the nitrothiolate chromophore. The prime advantage of this technique lies in the interaction of the thiol component with the particulate packing. As 2,2'-dithio(bis)benzothiazole is essentially insoluble, the exchange is dependent upon the target reacting directly with the solid packing as it is forced through the filter. One area for investigation revolved around whether sterically buried thiol functionalities, ie those within albumin, were capable of interacting with the solid. While such groups are accessible to freely diffusible derivatisation agents such as ER, it could be anticipated that the protein shield would prevent the intimate and specific contact required for reaction in the present case. The assay is therefore expected to allow a specificity, as yet, unattainable using conventional wet chemical techniques.

The absence of the MBT spectroscopic profile from the centrifugal filter device (CFD) processed albumin, figure 2, would suggest that the protein did not react. This preliminary result was followed up by passing a series of albumin solutions (0.02 to 0.82 mg/L) through the modified filter with the filtrates analysed for thiol through the addition of ER. In contrast to the previous experiments with the monomolecular thiols, positive results were obtained. The recovery of ER active albumin was typically 1OO ± 1% (RSD=I.5%, N=3 based on 0.82 mg/L AIbSH) and serves to confirm the inventions supposition that direct and specific interaction at the solid interface is required for disulphide exchange to occur.

The clinical efficacy of the system for discriminating between albumin, low molecular weight thiols and indeed other physiological components was then assessed through examining the plasma thiol profiles of three individuals (2M, IF). Plasma samples (500 uL) were passed directly through the filter without any form of pretreatment. The filtrate was first analysed using direct uv spectroscopic analysis as indicated previously (figure 2). The absence of any appreciable absorbance within the plasma at 312nm should therefore allow any increase at this wavelength, subsequent to CFD processing, to be attributed predominantly to the presence of MBT conjugates. The magnitude of the latter providing an immediate estimation of low molecular weight thiol concentration. However, there is an inherent limitation to this approach in that the spectroscopic properties of the conjugate will differ depending on the nature of the amino acid substituent. This is evident from the molar absorptivity data presented previously for the GSH, CSH and HCSH variants. The data obtained is presented in table 1 and was analysed using the regression data from both GSH and CSH calibration runs, the difference between the two estimates in practice have a negligible bearing on the measurement with the differing determinations of total low molecular weight thiols being well within the experimental error for either process.

Direct CFD Subject

Analysis _{M1 M2 F 1}

Ann (3 l2 nm, Corr) 0.046 0.052 0.060

^RSH(OSH) ^{/ mM} 0.147 0.164 0.186

RSH_(CSH)/ mM 0.142 0.164 0.193

Each based on 3 replicate measurements Highest RSD value obtained was 5.38

Table 1. Direct uv/vis analysis of plasma RSH concentration

A series of secondary experiments were then conducted using ER before and after the passage of plasma through the modified filter device. The results are detailed in table 2. Ellman analysis of the plasma before filtration yields the total plasma thiol concentration (PSH) with the subsequent filtrate providing albuminSH - given that the low molecular weight species will have reacted to form the ER inactive MBT conjugates.

CFDfER. Subject Analysis Ml M2 Fl

(A) ER Before CFD

^Abs(412 nm) 0,170 0.243 0.203 Total PSH / mM 0.530 0.778 0.650

(B) ER After CFD

^Abs(412 nm) 0.122 0.192 0.143

AIbSH / mM 0.391 0.615 0.457

RSH ₍A-B₎ / mM 0.139 0 163 0.193 jϊach based on 3 replicate measurements Highest RSD value obtained was 538

Table 2 CFD/ER uv/vis analysis of plasma RSH concentration It can be assumed that the substrate of the albuminSH from the PSH result should yield the low molecular weight thiol concentration. The estimates shown in table 2 are in excellent agreement with the direct uv spectroscopic determination. This again confirms that, despite the minor variation in uv/vis sensitivty between different MBT-SR conjugates, the procedure is indeed clinically robust.

Preferably the first reagent is manipulated to incorporate a chromophore, such as a nitro group common to Ellmans Reagent. Alternatively, an electro responsive redox substituent may be provided to enhance the assay performance.

Preferably the detection of the reactive or non-reactive thiol is carried out in situ within the Eppendorf. However the detection may also take place by removing the sample after it passed through the column and placing it into a suitable optical cuvette or other container for further analysis.

Preferably the microfuged tube is provided with one or more electrodes which extend into the bottom of the cuvette. Electrodes at the bottom of the cuvette to allow direct analysis of the sample after it has passed through the column through potentiometric, voltometric or amperometric news. The advantage of this is that it allows the detection and analysis of the sample to be simplified.

The potentiometric response was recorded using a two electrode system. The electrode assembly consisted of an indicating electrode (typically carbon) and a silver / silver chloride (Ag/AgCl) reference electrode. The electrode were immersed in a solution containing naphthoquinone (NQ) which gives rises to the initial potential. Upon the addition of albumin to the system, the albumin reacts with the naphthoquinone resulting in a change in redox potential. This is recorded as a change in the potential difference between the two electrodes - the magnitude of which can then be related to the concentration of the albumin through either standard calibration methods or through the method of Standard Addition. Figure 4. The detection of the MBT-cysteine conjugate can also be determined through either amperometric or voltammetric means. Upon passing the sample of reduced thiol through the filter - the conjugates are released and then interrogated by means of either amperometric (fixed potential) or voltammetric (sweep/pulse) methods. A three electrode system is normally employed in which the oxidation of the conjugate occurs at the working electrode at a particular voltage. In this case the oxidation of cysteine can be observed as a sharp peak in the current - potential profile (at approximately +0.34V vs 3M Ag/ AgCl Reference). The height of the peak (magnitude of the current) is proportional to the concentration of the conjugate and thus allows quantitative measurements to be made. Amperometric systems can be employed in which the potential of the working electrode is fixed at a value more positive than the peak potential observed for the conjugate. The current recorded at the electrode is proportional to the concentration of conjugate present.

Table 3 shows a comparison between direct spectroscopic assay and the potentiometric system described previously. The sample was passed through the centrifugal filter device (CFD) to remove low molecular weight thiols. The remaining reduced thiol component can then be attributed solely to the presence of albumin. The latter was then determined using conventional spectroscopic (Ellmans Reagent/Direct UV/Vis) or potentiometric (ImM Naphthaquinone). The responses were then compared against the conventional bromocreseol green (BCG) assay.

BCG is one of the most commonly used methods in the art for the detection of serum albumin. Albumin forms a complex with bromocresol green at, for example, pH 4.2, which is green. The increased absorption on complex formation is proportional to albumin concentration. Absorption is typically measured at 630 to 640 nm or bichromically 600/800 nm. Commercially available kits for BCG assays are available from a number of sources, including Olympus Life and Material Science GmbH, O'Callaghan's Mills, Co. Claire, Eire. Spectroscopic Assays

Controls M1 M2 M3 F1

UV/Vis after CFD 33 45 45 44

BCG Assay 33 42 42 39

Potentiometric Assays .

Subject

Controls M1 M2 M3 F2

Electrochem after CFD 35 43 39 46

UV/Vis after CFD 33 45 45 42

BCG

33 42 42 39

Table 3. Albumin concentration (g/L) from clinical control samples.

Reference:

(1) West, LC. Diabetic Medicine, 2000 17, 171

(2) Moriarty S.E., Shah J.H., Lynn, M., Jiang, S., Openo K., Jones D.P., Sternberg P., Free Rad Biol Med., 2003, 35, 1582

(3) Robertson R.P., Harmon, J., Tran P.O., Tanaka Y., Takahashi H., Diabetes, 2003, 52, 581

(4) VanderJagt D. J., Harrison J.M., Ratliff D.M., Hunsaker L. A., Vander Jagt D.L., Clin Biochem, 1999, 34, 265

(5) Ellman, G. Archives of Biochemistry and Biophysics, 1959, 82, 70-77

Claims

1. Method for determining the relative amount of a sulphydryl-containing protein, such as albumin, compared with lower molecular weight thiol-containing compounds in a sample containing both sulphydryl containing protein and lower molecular weight thiol-containing compounds, comprising the step of contacting the sample with substituted or non-substituted 2.2'-dithio(bis)benzothiazole reagent (the first reagent) or a derivative thereof and determining directly or indirectly an amount of reacted thiol compounds that have reacted with the first reagent (reacted thiols) and/or thiol-containing compounds that have not reacted (unreacted thiol) with the first reagent.

2. A method according to claim 1, wherein the first reagent is provided in a column and the sample is passed through the column.

3. A method according to claim 1 or claim 2 wherein the sample is whole blood, plasma or serum, saliva or urine.

4. A method according to any preceding claim, wherein the amount of low molecular weight thiol containing compound that has reacted with the first reagent is determined.

5. A method according to any preceding claim, wherein the amount of thiol containing compounds remaining in the sample after contacting it with the first reagent is determined.

6. A method according to any preceding claim, additionally comprising the step of determining the total amount of thiol containing compounds in an aliquot of the sample which has not been contacted with the first reagent and comparing this total to the amount of thiol-containing compounds that have reacted with the first reagent and/or the amount of thiol-containing compounds that have not reacted with the first reagent in the sample contacted with the reagent.

7. A method according to claim 2, wherein the column is a microfuge tube or a capillary tube.

8. A method according to any one of claims 1 to 5, wherein the First reagent is provided on the surface of one or more electrodes.

9. The method according to any preceding claim, wherein the low molecular thiols react with the reagent to produce a mercaptobenzothiazole (MBT) conjugate and the amount of MBT conjugate is detected.

10. A method according to any one of claims 1 to 8, additionally comprising the step of providing one or more detection reagents to convert the reacted thiol and/or unreacted thiol into detectable compounds.

11. A method according to any preceding claim wherein the reacted thiol and/or unreacted thiol are detected, directly or indirectly, photometrically, chromatographically, electrophoretically or electrochemically.

12. Method according to any preceding claim wherein the amount of thiol containing compound reacted with the reagent is determined by detecting the absorption of the sample at 305-325 nm.

13. Method according to any preceding claim wherein the amount of unreacted thiol is determined by reacting the unreacted thiol with Ellmans Reagent and release the chromophone 5-mercepto-2-nitrobenzoic acid, and determining the amount of chromophore released.

14. Method according to any one of claims 1 to 11 comprising reacting the unreacted thiol with a quinone to produce detectable sample mixture and measuring the potential difference of the detectable sample mixture.

15. Method according to any one of claims 1 to 11, wherein a counter electrode is provided and the current produced by oxidation of one or more components in the sample after it has been contacted by the first reagent is detected voltammetrically.

16. Method according to any one of claims 1 to 11 or 15 comprising detecting the reacted thiols and/or unreated thiol amperometrically.

17. A kit for use in the determination of the relative amount of a sulphydryl-containing protein such as albumin compared to lower molecular weight thiol containing compounds comprising a substituted or non-substituted 2,2'-dithio(bis)benzothiazole reagent (the first reagent).

18. A kit according to claim 17, wherein the first reagent is provided in a column for passing a sample through.

19. A kit according to claim 18, wherein the column is provided in a microcentrifuge tube or a capillary tube.

20. A kit according to any one of claims 17 to 18 additionally providing one or more detection reagents for converting unreacted thiol and/or reacted thiol, after a sample has been contacted with the 2,2-dithio(bis)benzothiazole reagent, into detectable compounds.

21. A kit according to claim 20, wherein the 2,2-dithio(bis)benzothiazole reagent and detection reagent are each arranged in a column so that, in use, a sample is passed through the 2, 2-dithio(bis)benzothiazole reagent, and then passed through the detection reagent.

22. A kit according to any one of claims 17 to 21, additionally comprising two or more electrodes for detecting reacted or non-reacted thiol-containing compounds after being contacted with the first reagent.

23. A kit according to claim 22, wherein the electrodes are provided within a micro fuge tube in which the first reagent is provided as a column as defined in claim 19.

24. A kit according to claim 23, wherein the microfuge tube additionally comprises a detection reagent according to claim 21.

25. A kit according to any one of claims 17 to 24, in which an electrode is coated with the first reagent.

26. A kit according to any one of claims 17 to 25 additionally comprising instructions for using the kit to detect the relative amount of a sulphydryl containing protein compared with lower molecular weight thiol-containing compounds in a sample.

27. Use of a kit according to any one of claims 17 to 26 to detect relative amounts of sulphydryl containing proteins, such as albumin, compared with lower molecular weight thiol-containing compounds in a sample.