In-situ reagent for detection of proteins

Abstract

The present invention relates to a stable protein and/or amino acid detecting composition that can be used as a reagent for in situ detection, such as on surfaces. The invention also relates to a method for detecting protein and/or amino acid on surfaces using the composition and kits comprising the composition.

Claims

1. A protein and/or amino acid detecting composition comprising: (a) about 0. 1 mmol/L to about 10 mmol/L or phthaldiaidenyde, (b) about 1 mmol/L to about 20 mmol/L of a C3-C6 thiol selected from the group consisting of N-Acetyl-L-Cysteine (NAC), N-acetyl-D-peniciliamine, N-acetyl-cysteamine, N-acetyl-homocysteine, and rnercaptosuccinic acid, (c) about 10 mmol/L to about 100 mmol/L of a buffer with a pH in the range of from about 7.5 to about 10, and (d) about 0.01% v/v to about 2% v/v of a surfactant, wherein the composition further comprises (e) about 0.05 mmol/L to about 5 mmol/L of a thiol reducing compound selected from Dithiothreitol, 2-mercaptoethylamine, and Tris (2-carboxyethyl) phosphine; wherein the composition is stable at 4 C. for 6 months and 3 months at room temperature.

2. The composition of claim 1, wherein the C.sub.3-C.sub.6 thiol is N-Acetyl-L-Cysteine.

3. The composition as claimed in claim 1, wherein the buffer has a pH in the range of pH 9 to pH 9.5.

4. The composition as claimed in claim 1, wherein the surfactant is a non-ionic surfactant.

5. The composition claimed in claim 1, further comprising a chelating agent.

6. The composition of claim 5, wherein the chelating agent is at a concentration of about 0.05 mmol/L to about 5 mmol/L.

7. The composition of claim 1, wherein the composition further comprises an organic solvent.

8. The composition of claim 7, wherein the solvent is selected from the group consisting of ethanol, methanol, acetone and acetonitrile.

9. The composition of claim 7, wherein the solvent is methanol.

10. A process for making the composition as claimed in claim 1, by mixing the components (a), (b), (d) and (e) into the buffer solution (c).

11. An in situ method for detecting protein and/or amino acid on a surface or substrate, comprising: a) applying the composition as claimed in claim 1 to a surface and/or substrate, and b) detecting fluorescence.

12. The method as claimed in claim 11, wherein the fluorescence is detected by any means or apparatus which is able to detect fluorescent light.

13. A kit for detecting protein and/or amino acid comprising: a) a composition as claimed in claim 1, and b) instructions for use.

14. A kit for detecting protein and/or amino acid in situ on a surface or substrate comprising: a) a composition as claimed in claim 1, b) means for applying the composition to a surface and/or substrate, and c) instructions for use.

Description

(1) The invention will now be further described by way of reference to the following Examples and Figures which are provided for the purposes of illustration only and are not to be construed as being limiting on the invention. Reference is made to a number of Figures in which:

(2) FIG. 1: shows the excitation and emission spectra of flourophores formed when reacting OPA/NAC reagents with BSA. FIG. 1a shows the excitation and emission peaks of the fluorophores when reacting unstable OPA/NAC reagent with BSA. FIG. 1b shows a 3D scan of the excitation and emission peaks of the fluorophores formed when reacting stable OPA/NAC reagent with BSA.

(3) FIG. 2: shows the stabilised OPA/NAC reagent's stability and sensitivity over a period of 6 months when reacted with BSA.

(4) FIG. 3: shows the stability of the fluorophore products formed when using the stabilised OPA/NAC reagent.

(5) FIG. 4: shows the sensitivity of the stabilised OPA/NAC reagent with proteins in aqueous solution.

(6) FIG. 5: shows the qualitative analysis obtained using the composition of the invention on a set of stainless steel forceps and screen shots taken with a G-BOX. FIG. 5 (a) shows the screen shot of the white light image of the forceps after SSD cleaning and before the composition of the invention is applied on the forceps; FIG. 5 (b) shows the screen shot of the fluorescence emitted after the composition of the invention has been sprayed onto the forceps; and FIG. 5 (c) shows the screen shot of the false colour overlay showing the protein present.

(7) FIG. 6: shows a contaminated surgical scalpel blade sprayed with a 4 month old reagent. Image captured and optically enlarged with a G-BOX.

(8) FIG. 7: shows the stability of the fluorophores formed when the stabilised OPA/NAC reagent of this invention reacts with protein. The blade was left on a bench top in open air for 1 month. Image was captured with a G-BOX without re-spraying the instrument with the reagent.

(9) FIG. 8: shows the visualisation of protein matter and semi-quantitative analysis of (the stabilised OPA/NAC reagent) sprayed onto a BSA standard. Image is captured using a G-BOX and analysed using D-Plot software.

(10) FIG. 9: shows the visualisation of protein matter and quantitation of BSA (0 to 100 nanograms) using the stabilised OPA/NAC reagent on a surgical blade. Image is captured using G-BOX and analysed using D-Plot software. The regression and linearity is calculated in Excel 2003.

(11) FIG. 10: shows the visualisation and quantification of protein residues on surgical instruments.

(12) FIG. 11: shows the quantitative analysis obtained using the composition of the present invention on a set of stainless steel forceps; FIG. 10 (a) shows the screen shot of a false colour overlay of a set of forceps after SSD cleaning showing the residual protein present at different intensities on different sites of the forceps; FIG. 10 (b) shows a 3-D plot which is generated showing the varying quantities of residual protein present on the forceps; and FIG. 10 (c) shows the plot of protein intensities.

(13) FIG. 12: shows the results from a Fibrinogen standard. FIG. 12 (a) shows the fluorescence emitted and captured in a false colour overlay screen shot of the varying standards of Fibrinogen; FIG. 12 (b) shows the 3 D plot which is generated showing the varying quantities of the standard; and FIG. 12(c) shows the standard curve for Fibrinogen.

EXAMPLES

Example 1

Preparation of OPA/NAC Reagent

(14) Sodium borate buffer pH 9.23 was prepared by dissolving 9.5 g of Di-sodium tetraborate decahydrate in 500 ml of deionised H2O with several drops of 1 M NaOH to adjust the pH. Then 60 mg of N-Acetyl-L-Cysteine were added to the buffer solution and stirred with a magnetic stirrer until completely dissolved. The OPA solution was prepared by dissolving 60 mg of OPA in 5 ml of methanol and then this solution was added into the borate buffer solution. The OPA/NAC reagent solutions were prepared weekly and stored refrigerated in the dark at +4 C. for 24 h before use in order to reduce the background fluorescence.

Example 2

Preparation of the Stabilised OPA/NAC for Protein Detection

(15) Sodium borate buffer pH 9.23 was prepared by dissolving 19 grams of Di-sodium tetraborate decahydrate in 900 ml of deionised H.sub.2O with several drops of 1 M NaOH to adjust the pH. Then 1 ml of Triton X-100, 1.63 g of N-Acetyl-L-Cysteine, 0.077 g of DTT were added to the buffer solution and stirred with a magnetic stirrer until completely dissolved. The OPA solution was prepared by dissolving 0.268 g OPA in 10 ml methanol and then the OPA solution was added into the borate buffer solution. The solution was made up to 1 L. The OPA/NAC solution was stored refrigerated in the dark at +4 C. for 24 hours before use in order to reduce the background fluorescence.

Example 3

Preparation of the Stabilised OPA/NAC Reagent Further Comprising Sodium EDTA

(16) The same OPA solution as described in Example 2 was prepared with the addition of 0.32 g of disodium EDTA were added to the buffer solution and stirred with a magnetic stirrer until completely dissolved.

Example 4

Determining the Stability of the Stabilised OPA/MAC Reagent

(17) To evaluate the stability and sensitivity of the stabilised OPA/NAC reagent inter assays with BSA were performed over a period of 6 months.

(18) A standard assay with Bovine Serum Albumin was prepared. The standard BSA solutions were prepared and diluted with deionised water and made up to concentration ranges of 0-100 g ml.sup.1. Blank measurements and standard assays with BSA were performed with freshly prepared and stored stabilised OPA/NAC reagent solutions (as described in Examples 2 and 3) stored both at room temperature and in the refrigerator, at 4 C. for various periods of time.

(19) Reaction assays using the stabilised OPA/NAC reagent (mentioned in Example 2 and 3) with BSA were prepared in 1 cm acrylic PMMA or silica cell fluorescent cuvettes, that is 300 l of the BSA concentration mixed thoroughly with 200 l of stabilised OPA/NAC reagent allowed the reaction mixture to stand for 5 minutes at room temperature before the fluorescence was measured at Excitation/Emission=350/450 nanometers.

(20) The fluorescence emitted from the reaction from the isoindole products formed was measured with a HITACHI F4500 Fluorescence Spectrophotometer. The Spectrophotometer was set at excitation and emission 350 nanometers and 450 nanometers wavelengths respectively with band widths of 10 nanometers excitation and 2.5 nanometers emission and a PM voltage of 700 Volts. The data was then used to calculate the limit of detection of the reagent with respect to protein detection.

(21) The reagent sensitivity was stable at room temperature for up to 3 months at room temperature. The results shown in FIG. 2 indicate that the reagent sensitivity was stable for up to 6 months at 4 C. The linearity of the BSA standards is 0.99 over the six months period and the sensitivity is 363.2 g mL.sup.1/RFU (Table 1).

(22) TABLE-US-00001 TABLE 1 Stability and sensitivity of stabilised OPA/NAC reagent with BSA over 6 month storage period. Time Linearity R.sup.2 L.o.D g/mL @S/N = 2 Day 2 y = 39.9x + 77.1 0.99 1.9 1 month y = 39.5x + 80.7 0.99 2.0 2 months y = 37.8x + 112.2 0.99 2.9 3 months y = 36.3x + 145.5 0.99 4.0 4 months y = 34.9x + 172.7 0.99 4.9 5 months y = 32.3x + 173.3 0.99 5.3 6 months y = 32.0x + 66.09 0.99 2.1

(23) Reaction rate of the stabilised OPA/NAC with proteins and the development of fluorescence is very rapid with a completion time of <3 minutes. In fact, in the imaging system fluorescence development was complete by the time the image was first captured.

Example 5

Determining the Stability of the Stabilised OPA/NAC Reagent Products Formed after Reaction with Protein Matter

(24) To evaluate the stability of the isoindole products formed, assays of 10 replicates of a known concentration were prepared and mixed with stabilised OPA/NAC reagent (as mentioned in Example 4) in a fluorescent cuvette. This was achieved by measuring the fluorescence from the isoindole products at time intervals to compare the products loss in fluorescence detection.

(25) The fluorescence emitted from the reaction mixture was detected after allowing the reaction mixture to stand for 5 minutes (as mentioned in Example 4) at room temperature before measuring the fluorescence at Excitation/Emission=350/450 nanometers.

(26) The reaction mixture was left for 360 minutes (6 hours) and the fluorescence from the reaction mixture measured at intervals of an hour to detect the stability of the isoindole products.

(27) This example shows that not only the stabilised OPA/NA reagent is stable over 6 months but the isoindole products in solution are stable over a period of up to 6 hours.

Example 6

Specificity of the Stabilised OPA/NAC Reagent

(28) Protein standard solutions (0 to 100 g/ml) containing BSA, Haemoglobin, Fibrinogen, Cytochrome-C, Globulin, Myoglobin, were prepared in deionised water as mentioned in Example 2. The reproducibility of the method was evaluated by repeated analysis of BSA and cytochrome-C with 10 replicates of each.

(29) The fluorescence measured with the spectrophotometer when using the stabilised OPA/NAC reagent with the different proteins gave a linear response with all the proteins tested (FIG. 4). The sensitivity varied with the nature of the protein. The limit of detection for BSA in the system was 0.3 g/mL (Table 2)

(30) TABLE-US-00002 TABLE 2 Sensitivity of stabilised OPA/NAC reagent with proteins in aqueous solution. Sensitivity Protein Linearity R.sup.2 (g/mL/RFU) Fibrinogen y = 8.705x + 122.1 0.99 9 Cyt-C y = 19.69x + 203.2 0.97 20 Globulin y = 23.84x + 295.8 1.00 24 BSA y = 34.84x + 22.69 1.00 35 Haemoglobin y = 46.27x + 206.1 1.00 46 Myoglobin y = 49.23x + 292.9 0.99 49

Example 7

In Situ Detection of Protein Residues on Surgical Instruments

(31) The stabilised OPA/NAC reagent (as described in Example 2 and 3) was used to detect protein residues in situ on surfaces. The advantage of detecting protein residues in situ means that all amino acid and ammonia residues have been removed by a simple water wash.

(32) A standard assay with BSA was prepared (as mentioned in Example 4).

(33) Protein visualisation experiments were carried out using a modified gel documentation system (G-BOX) from SYNGENE (Cambridge, UK). The system using mercury lamps with phosphorescence tubes optimised to give a excitation wavelength of 350 nm very close the optimal for the OPA/NAC reagent. The emitted light was captured by a cooled CCD camera after passing through a 440 nm interference filter (i.d. 7 cm). Spray bottles were from a high street pharmacy. All spraying was done on sheets of black non-fluorescent art paper, which were changed after each use.

(34) Semi-quantitative analysis of the G-BOX images was done using a software programme D-Plot from HydeSoft Computing, Vicksburg, USA.

(35) Protein spots were pipetted on to stainless steel surfaces and allowed to air dry at room temperature for at least 4 hours before investigation. The instrument and/or surface was placed on the platform and sprayed with a fine mist of the stabilised OPA/NAC reagent. The chamber of the platform was closed and a series of subsequent images captured with first white light and then UV (350 nanometers) at exactly the same setting on the camera. The images are superimposed with a false colour and the protein spots are used for qualitative (FIG. 5) and semi quantitative analysis (please refer to Example 8).

Example 8

Semi Quantitative Analysis

(36) The images captured were imported into the D-Plot software program. The images were converted from a bitmap to 3D using the software L plug-in. The plug-in maps pixel values of images to z values in a surface plot. The volume under the surface of the fluorescent spots is measured as a measure of the intensity of the fluorescence. The value is used for the quantitative analysis.

(37) A: Stability of the Dry Fluorescent Derivatives.

(38) The reagent is stable up to 6 months at 4 C. A 4 month old reagent was used in detecting protein on a surgical scalpel blade (FIG. 6). Surprisingly, the formed fluorophores fluoresced even after 9 months when left in open air. This is shown in FIG. 7.

(39) B: Sensitivity of the Stabilised OPA/NAC Reagent in Detecting Protein Spots in Situ.

(40) Standard protein (BSA) was used to determine the sensitivity of the system in detecting protein residues on instrument stainless steel surfaces. BSA (0 to 1200 ng) in 1 l spots (2 mm.sup.2) were pipetted on a clean stainless steel surface and dried at room temperature. The spots were then sprayed with the stabilised OPA/NAC reagent and analysed as described in Examples 5 and 6. The volume under surface was plotted against the concentration to assay the linearity of the reagent for in situ protein visualisation (FIG. 8). The regression R.sup.2 of the standard calibration was 0.98. The limit of detection using the defined system, is currently 500 picograms of protein per spot or 250 pg/mm.sup.2. (FIG. 9)

(41) C: Semi Quantitative Analysis of Protein Residues in Surgical Instruments.

(42) In order to test the applicability of the developed system in detecting protein residues after the surgical instruments have been through a wash a hydrophobic protein (Fibrinogen) was used on a pair of scissors. The instrument was sprayed with OPA/NAC/DTT after contamination and washed in a typical SSD. The washed instrument was photographed and re-sprayed and subsequently photographed at the same settings. The image was analysed to quantify the protein residues remaining.

(43) Results show that any remaining protein can be visualised and quantified. In this particular case there was 140 ng of fibrinogen remaining as calculated from the calibration of fibrinogen (FIG. 9). In order to achieve this, the protein area was manually outlined with D-Plot. The volume of the fluorescence within the protein spot was calculated by D-plot and the actual amount of protein is calculated by reference to a series of standard spots as shown in FIG. 12. It is clear that with suitable edge detection software and a protein calibrant, this process could be automated and simplified. The regression and linearity is calculated in Excel 2003.

(44) Discussion

(45) Instability of the OPA/NAC solution/reagent is caused by the formation of disulphide bridges between thiols present in the reagent mixture; this does not allow OPA to combine with the thiol in the solution to form the reacting reagent hence its loss in sensitivity and stability over time. The stability of the solution/reagent was achieved by the addition of thiol-reducing compounds such as DTT (dithithreitol) or TCEP (Tris 2-carboxyethyl phosphine hydrochloride).

(46) Stabilisation of Isoindoles Formed by Stabilised OPA/NAC Reagents

(47) The secondary limitation of the OPA/NAC reagent is its production of unstable isoindole derivatives which lose the fluorescent signal rapidly in time. This causes time constraints when performing assays with this reagent.

(48) Calculations and Statistics

(49) Intra and inter assays of the reagents showed the reproducibility of the reactions and hence the validation of the reagents. Descriptive statistics (Mean, Standard deviation and Coefficient of variance) were calculated to show the significant improvement of the limit of detection of the stabilised OPA/NAC reagent. The limit of detection and quantification took into consideration 95% confidence intervals and were calculated with the following formula:
LoD=((CV2)/100))mean concentration
LoQ=LoD2.5

(50) Statistical calculations were performed using MS-Excel 2003.

(51) The long term stability of the OPA/NAC reagent exhibited a loss in sensitivity from 76 g ml.sup.1.sup.1/RFU observed after 24 hours from its preparation down to 16 g ml.sup.1/RFU 72 hours later. This confirmed the constant decay in the reagents stability.

(52) The instability of the reagent originates from the thiol compound forming disulphide bridges within the reagent mixture rather than binding with OPA to react with the protein and/or amino acid to produce the isoindole derivatives of the reaction. In addition, the isoindole fluorophore derivatives are themselves unstable and give rise to loss of a signal after 30 minutes of the reaction taken place which causes a major limitation to the precision of any analytical study.

(53) Introducing a thiol reducing compound into the reagent mixture has effectively reduced the formation of the disulphide bridges between the thiols in the reagent. The results show that it has even improved the long term stability of the reagent. In addition, the composition of this invention has shown to increase the sensitivity and stability of the reagent when reacted with BSA and the stability over time of the isoindole derivatives produced from the reaction mixture.

(54) The stabilised OPA/NAC reagent enhances the long term stability of the reagent in comparison to the original and unstable OPA/NAC by maintaining a constant sensitivity signal over time.

(55) The LoD's of the OPA/NAC reagents of the invention were calculated based upon the coefficient of variance from the intra assay variability performed. These confirmed that the addition of a thiol reducing agent and a chelating agent to the OPA/NAC reagent increased the limit of detection in reactions with BSA. The original OPA/NAC showed a LoD of 840 ng ml.sup.1, the addition of DTT and EDTA at the required amounts to the OPA/NAC reagent showed a range of 750-530 ng ml.sup.1 and the addition of TCEP showed a significant increase of LoD for the reagent which ranged from 700 down to 10 ng ml.sup.1.

(56) Isoindole Stabilisation

(57) The original OPA/NAC showed 33% decay in fluorescence signal of its isoindole products 30 minutes after the initial measurement. In contrast the addition of TCEP showed 2-80% increase of isoindole fluorescence signal whereas DTT demonstrated a range of 1-27% decrease. The time dependency significantly showed variability with the amount and type of thiol reducing agent which is introduced into the OPA/NAC reagent mixture.

(58) These results show that long term stability of the OPA/NAC reagent can be achieved by the addition of a thiol reducing agent. The addition of thiol reducing compounds show a greater LoD, less signal fluorescence variability and a constant stability with age of the reagent and its products and with a detection limit at least 300 times more sensitive than ninhydrin in respect of protein detection.