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Marine Laboratory Report No 13/00

COLORIMETRIC PROTEIN PHOSPHATASE ASSAY FOR THE DETECTION OF THE DIARRHETIC SHELLFISH TOXIN, OKADAIC ACID S O’Neill, I M Davies and C F Moffat November 2000

Fisheries Research Services Marine Laboratory Victoria Road Aberdeen AB11 9DB

TABLE OF CONTENTS SUMMARY........................................................................................................................

1

1.

INTRODUCTION...................................................................................................

1

2.

DEVELOPMENT OF A COLORIMETRIC PROTEIN PHOSPHATASE ASSAY FOR USE IN A MONITORING SITUATION.............................................

1

2.1 2.2 2.3

1 4 5

3.

Historical Perspective of the Project.......................................................... The Tubaro et al. (1996) Protein Phospatase Inhibition Assay ................. Assay Parameters ..................................................................................... 2.3.1 Modification and Optimisation of the Tubaro et al. (1996) Protein Phosphatase Assay........................................................... 2.3.2 Optimisation of O.D. Readings ...................................................... 2.3.3 Assay Condtions: pH, Enzyme Concentration, Incubation Time and Temperature................................................. 2.3.3.1 pH........................................................................... 2.3.3.2 Enzyme concentration............................................ 2.3.3.3 Temperature ........................................................ 2.3.3.4 Incubation time....................................................... 2.3.4 Freeze Thawing and Pre-Incubation of Enzyme and Reagents ............................................................................... 2.3.5 Standard Curves and Associated Precision Data.......................... 2.3.6 Discussion: Assay Conditions, Temperature, pH, Enzyme Concentration and Incubation Time............................................... 2.3.6.1 Temperature ........................................................ 2.3.6.2 pH........................................................................... 2.3.6.3 Enzyme quality....................................................... 2.3.6.4 Incubation time....................................................... 2.3.6.5 Choice of assay parameters ..................................

5 5 7 7 7 7 9 9 10 12 12 12 12 13 13

TOXIN DETECTION EXPERIMENTS................................................................... 13 3.1 3.2 3.3

3.4 3.5

Detection Requirements ............................................................................ Detection of Toxin From Standard Reference Material MUS-2 ................. Spiking Experiments Using Okadaic Acid Standard.................................. 3.3.1 Method........................................................................................... 3.3.2 Spiking Results .............................................................................. 3.3.3 Detection of Okadaic Acid by the Different Treatments and Extraction Methods.............................................. 3.3.4 Spiking Summary Tables............................................................... Useability................................................................................................... 3.4.1 PP2A as Screening Technique...................................................... 3.4.2 Future Work on Choice of Protocol................................................ Quantification of Okadaic Acid by PP2A Inhibition Assay .........................

13 15 16 16 16 19 20 21 21 21 21

4.

CONCLUSIONS .................................................................................................... 22

5.

ACKNOWLEDGEMENTS ..................................................................................... 23

6.

REFERENCES...................................................................................................... 23

FIGURES ANNEXES 1. 2. 3. 4. 5. 6.

PP2A Protein Phosphatase Enzyme Inhibition Assay for the Detection of Okadaic Acid. DSP Shellfish Extraction and Clean Up. Okadaic Acid Shellfish Spiking Procedure. Extraction of DSP Toxins from MUS-2 CRM. Derivation of the Equation for the Light Path of a Falcon Microtitre Plate. Note Concerning Quality of PP2A Enzyme.

COLORIMETRIC PROTEIN PHOSPHATASE ASSAY FOR THE DETECTION OF THE DIARRHETIC SHELLFISH TOXIN, OKADAIC ACID S O’Neill, I M Davies and C F Moffat FRS Marine Laboratory Aberdeen

SUMMARY FRS Marine Laboratory (FRS ML) is contracted to MAFF and SERAD to undertake research into alternative methods to that of the mouse bioassay, for the detection of the Diarrhetic Shellfish Poisoning (DSP). The detection level of the bioassay is 200 ng toxin/g shellfish. Colorimetric protein phosphatase assays have been developed which can detect the DSP toxin okadaic acid. A published colorimetric method using PP2A enzyme was assessed and subsequently modified for use as an okadaic acid screening method. Spiking experiments with autoclaved mussels (Mytilus edulis) demonstrated a detection limit of 33 ng okadaic acid per gram of shellfish. Toxin recovery of ≥73% was achieved at 0.25 to 3 ng toxin/ml reaction mixture using the certified reference material (MUS-2).

1. INTRODUCTION Diarrhetic Shellfish Poisoning (DSP), a gastrointestinal illness, can be caused by the consumption of marine shellfish that contain DSP toxins. Symptoms of DSP are diarrhoea, abdominal pain, nausea and vomiting (Morton and Tindall, 1996). The method currently used for detecting the presence of DSP toxins in shellfish involves a solvent extraction of the toxin, and presentation of the extract, in a detergent, to a mouse by intraperitoneal injection (i.p.) (Yasumoto et al., 1984). This mouse bioassay, or variations of it, is used world-wide. Many other methods have been developed to detect DSP, including protein phosphatase enzyme inhibition assays, as discussed in Marine Laboratory Report 02/00 (O’Neill and Moffat, 2000). MAFF has funded research (project DH67) into alternative methods for monitoring shellfish toxins to that of the mouse bioassay. Under the current MAFF project, in conjunction with SERAD, a protein phosphatase enzyme inhibition assay has been investigated and modified for the detection of okadaic acid, the main causative toxin identified in occurrences of DSP. This report highlights progress, up to May 2000, on the development of a PP2A protein phosphatase assay for use as a screening technique for okadaic acid.

2. DEVELOPMENT OF A COLORIMETRIC PROTEIN PHOSPHATASE ASSAY FOR USE IN A MONITORING SITUATION 2.1

Historical Perspective of the Project

In 1996, under MAFF project DH67, research at the Central Science Laboratory’s Food Science Laboratory Torry commenced into techniques that could replace or reduce the use of the DSP mouse bioassay. Several options were explored including an HSP 70 reporter gene assay (CAT-Tox Assay Version 1.2), creation of an HSP 70 ELISA, the bioluminescence monitoring system MICROTOX and three protein phosphatase enzymatic

1

Colorimetric Protein Phosphatase Assay for the Detection of the Diarrhetic Shellfish Toxin methods. After initial investigations into the various methods, it was decided to adopt measurement of the inhibition of protein phosphatase by okadaic acid as the preferred method for detecting DSP because it offered a sensitive method, was a relatively straightforward procedure to carry out and could allow a high throughput of shellfish samples. At 100 ng okadaic acid/ ml reaction mixture, only marginal protein induction was observed using the HSP 70 reporter gene assay and okadaic acid did not appear to be cytotoxic in the human liver cell line (HepG2) at up to 10 ng okadaic acid/ml reaction mixture. The in-house creation of an HSP 70 ELISA method required several incubation steps and more time efficient methods were available in the form of the protein phosphatase assays. The MICROTOX toxicity test was found to lack sensitivity to okadaic acid. Information on various DSP detection methods, including those utilising protein phosphatase inhibition, can be found in Marine Laboratory Report No 02/00, (O’Neill and Moffat, 2000). In eukaryotic organisms, protein phosphatase enzymes remove the phosphate group from intracellular target proteins (MacKintosh and MacKintosh, 1993). This is part of the processes of phosphorylation and dephosphorylation that regulate many intracellular functions. In eukaryotes, protein tyrosine phosphatase (PTPase) removes phosphate from proteins or peptides containing phosphotyrosine and the protein serine/threonine phosphatase (PSPase) removes phosphate from phosphoserine or phosphothreonine. Until recently the serine/threonine phosphatases have been categorised into four principal catalytic subunits; PP1, PP2A, PP2B and PP2C. Honkanen et al. (1995) stated that three further serine/threonine phosphatases (PP3, PP4 and PP5) have subsequently been identified. The two major classes, PP1 and PP2, can be distinguished by their ability to dephosphorylate phosphorylase kinase and their sensitivity to two endogenous thermostable proteins, inhibitor 1 and inhibitor 2 (Honkanen et al., 1995). PP1 is potently inhibited by inhibitor 1 and inhibitor 2, and dephosphorylates the ∃ subunit of phosphorylase kinase, specifically. PP1 is also ATP and Mg2+ dependent. PP2 subunits are unaffected by the inhibitor 1 and inhibitor 2 proteins, and dephosphorylate the ∀ subunit of phosphorylase kinase preferentially. The three subunits of PP2 can be distinguished by their requirement for divalent cations; PP2A has no absolute requirement for divalent cations, PP2B has an absolute requirement for Ca2+/calmodulin and PP2C requires Mg2+ (Kendrew, 1994). The diarrhetic shellfish toxin, okadaic acid (OA), was the first substance discovered to be a potent inhibitor of PP1 and PP2A (Bialojan and Takai, 1988), and subsequently has become widely used as a tool to investigative the role of protein phosphatase. Dinophysis toxin 1 (DTX-1) is also a potent inhibitor of PP1 and PP2A. Okadaic acid completely inhibits PP1 and PP2A at nanomolar concentrations, has little effect on PP2B and no effect on PP2C, PTPases or any protein kinases tested (Honkanen et al., 1995). PP2A from skeletal muscle is inhibited about 200 times more strongly than PP1 (Falconer, 1993). PP3, PP4 and PP5 are also affected by okadaic acid (Honkanen et al., 1995). The inhibitory action of okadaic acid and DTX-1 on the dephosphorylation of the serine/threonine phosphatases PP1 and PP2A has given rise to assay methods with the potential for the detection of the DSP phosphatase inhibitors in shellfish. All of the phosphatase methods under consideration at the Food Science Laboratory Torry (FSL Torry) were based around the same principle of measuring the inhibition of an enzyme reaction by okadaic acid. Initially, a commercial kit available from the Promega UK Ltd (Southampton, UK) for detecting the release of free phosphate was tested. Although not devised for measuring okadaic acid, the kit was applied to this task and okadaic acid standard curves could be

2

Colorimetric Protein Phosphatase Assay for the Detection of the Diarrhetic Shellfish Toxin produced. However, this method was abandoned due to problems with phosphate contamination (O’Neill, S., unpublished data). A colorimetric protein phosphatase assay for detection of DSP in mussels had been developed and published by Simon and Vernoux in 1994. This assay did not rely on measuring the release of free phosphate and therefore phosphate contamination was not a problem. This method utilised semi-purified PP2A that the authors had prepared themselves from rabbit. This protein phosphatase inhibition assay was tested at FSL Torry using commercially available PP2A, supplied by Calbiochem-Novabiochem (UK) Ltd (Nottingham, UK). The enzyme reaction was carried out in a 1 ml cuvette, and the absorbance was measured in a spectrophotometer. The published method (Simon and Vernoux, 1994) was found to work, using the commercial PP2A enzyme. The main limitation of the method was that only a single sample could be tested at any time. Carrying out the assay in a 1 ml cuvette format as described in Simon and Vernoux (1994) using a commercial source of enzyme, required at lot of enzyme. As the enzyme costs account for the majority of the cost of the assay it was a logical step to decrease the volume of the reaction. The protocol was modified and developed in order that the reaction could be carried out in a microtitre plate format. This had several benefits. It allowed a standard curve to be set up on the plate and this in turn allowed several shellfish samples to be tested simultaneously and the toxicity of the shellfish to be calculated from their optical density (O.D.) values in relation to the specific standard curve. In addition to the work on the colorimetric assay, a further investigation using a 32P radio labelled protein phosphatase inhibition assay was carried out (Reece and O’Neill, 1996). The 32P method had been previously used at Dundee University to measure microcystins, potent PP1 and PP2A inhibitors found in freshwater (MacKintosh and MacKintosh, 1993). The suitability of the 32P radio labelled protein phosphatase assay for DSP detection was tested at FSL Torry (Reece and O’Neill, 1996). It was concluded that the 32P radio isotope PP2A inhibition assay was the most desirable of all the methods tested, combining sensitivity with high sample throughput and low cost. More information on this assay can be found in CSL Report TD 2792 (Reece and O’Neill, 1996). During 1996, the work on the alternative methods was transferred to the SOAEFD Marine Laboratory, Aberdeen (renamed Fisheries Research Services, Marine Laboratory (FRS ML). Due to a general ethos at FRS ML to minimise radio isotope work, the use of the 32P assay at FRS ML was reconsidered and the colorimetric method re-visited. Although work on the colorimetric assay had halted at FSL Torry, advances in the development of a colorimetric assay had been made elsewhere. Dr Nunez, of the PHLS Colindale Laboratory (London, UK), had been working on a protein phosphatase method for DSP detection. A visit to the PHLS Colindale Laboratory allowed Dr Nunez’s assay, which was carried out in a microtitre plate, to be attempted and a protocol for the technique was obtained. Unfortunately, the publication of Dr Nunez’s work did not include the microtitre plate version of the protein phosphatase assay but instead referred to the more traditional cuvette format (Nunez and Scoging, 1997). A colorimetric method based around the Simon and Vernoux (1994) method, carried out in a microtitre plate format, had been published by Tubaro et al. (1996). This published method had some similarity in format to the assay that had been developed at CSL. This was an end-point assay carried out with a standard curve on a microtiter plate utilising para-nitrophenyl phosphate (pNPP) as the substrate and PP2A as the enzyme. The Tubaro et al. (1996) method used different buffers, source of enzyme, incubation time and temperature from the CSL microtitre plate assay (O’Neill, S. unpublished). The main benefit of the Tubaro et al. (1996) method was a reduction in the amount of enzyme required to run the assay. This was achieved by opting not to measure in the initial linear rate of the reaction but to allow the reaction to progress and record the absorbance after 60 minutes incubation. It appeared that this method met the objectives of the programme and was more economical in its use of enzyme. It was

3

Colorimetric Protein Phosphatase Assay for the Detection of the Diarrhetic Shellfish Toxin decided that this method should be pursued and work at the FRS ML concentrated on this procedure.

2.2

The Tubaro et al. (1996) Protein Phosphatase Inhibition Assay

Tubaro et al. (1996) described the protocol for a protein phosphatase inhibition assay, carried out in a microtitre plate, using PP2A. An okadaic acid standard curve was prepared on the microtitre plate and, on the same plate, the enzyme reaction was carried out in the presence of extracts of shellfish. The reaction was started and the absorbance of the wells recorded after 60 minutes at room temperature. The level of inhibition due to the shellfish extracts was calculated from the absorbance reading obtained in relation to the okadaic acid standard curve. Toxicity of the shellfish was expressed as ng OA/g shellfish. Attempts to reproduce the Tubaro et al. (1996) colorimetric protein phosphatase inhibition assay encountered many problems. Tubaro et al. (1996) used Upstate Biotechnology (UBI), based in the USA, as the source of PP2A. UBI supplied a recipe for the enzyme buffer recommended which had been omitted from the published method of Tubaro et al. (1996). Although UBI could supply the buffer, at £30 ml, it was expensive especially if enzyme buffer was to be included in blank controls with every assay. It was decided to follow the UBI enzyme-buffer recipe and make it on site at FRS ML. When the assay was attempted, including the enzyme buffer, problems were encountered and it was not possible to produce a useable standard curve. Personal communication from Dr Tubaro confirmed that they have had similar problems with the PP2A obtained from UBI. Work at FRS ML on the assay conditions indicated that the Tubaro et al. (1996) protocol was not, or was no longer, optimal for the current enzyme. It transpired that the suppliers of the enzyme (UBI), had abandoned some of their QC procedures, including using a protein phosphatase inhibition assay as one of their methods for defining enzyme activity. Despite obvious quality issues the company would not re-instate their QC procedures. The PP2A from UBI is sold as 10 units in 50 µl of storage buffer; a unit is defined as releasing 1 nmole of phosphate per min from 15 µM [32P] labelled Phosphorylase A at 30°C. The company redefined the activity for each new batch of enzyme, stating how many nmole of phosphate per minute that 0.01 Unit PP2A released from a phosphopeptide, KRpTIRR, as determined using a Ser/Thr Phosphatase Assay Kit. The quality control data supplied with the batches of enzyme purchased from UBI indicated that activity varied, with 0.01 Unit PP2A releasing from 25.3 nmole to 8.4 nmole of phosphate per minute from KRpTIRR. Therefore, purchasing 10 units of enzyme did not ensure that the activity (as determined by the Ser/Thr phosphatase assay kit) would be the same in different batches of enzyme. It was therefore necessary to enquire about the activity of each batch prior to purchase. Initially, this was relatively easy as the activity data was supplied with the enzyme and the UK distributors could be contacted for the information. In order to ensure the suitability of the enzyme, each batch of enzyme was tested at FRS ML to determine the quantity necessary to carry out the assay. Increasing the quantity of enzyme added to a reaction, to counter for low activity, resulted in a lateral shift in the standard curve and a consequential loss in sensitivity. As the concentrations of okadaic acid used in the standard curve did not change, increasing the enzyme concentration altered the ratio between the two. The overall inhibition of the enzyme was less and more enzyme was available to drive the reaction forward. A conflict of interest arose between the manufacturers of the PP2A enzyme and the distributors, UBI. The manufacturer, objected to UBI re-testing and reporting the activity of

4

Colorimetric Protein Phosphatase Assay for the Detection of the Diarrhetic Shellfish Toxin the PP2A using a Ser/Thr phosphatase assay kit as they felt that this misrepresented the activity of the enzyme. UBI no longer carry out this enzyme activity check and consequently no longer report this information. Discussions have been held between UBI and FRS ML, and UBI have indicated a willingness to reinstate their previous QC procedures.

2.3

Assay Parameters

2.3.1

Modification and Optimisation of the Tubaro et al. (1996) Protein Phosphatase Assay

Following the initial problems encountered with the published method, and a modified protocol supplied by Dr Tubaro, it was decided to undertake experiments to confirm that the assay conditions were optimal. In the initial attempts to reproduce the Tubaro et al. (1996) protocol, O.D. values at 405 nm of less than 0.4 and as low as 0.2 were obtained for the uninhibited reaction. Producing a standard curve with such a low O.D. range is problematic, as it has the effect of magnifying the errors arising from imprecision of the points on the calibration curve. These errors could be reduced by increasing the O.D. working range. If the assay was not optimised then it would be possible to increase the working O.D. range by alteration of the assay conditions. If the assay was already at optimal conditions then an alternative approach would be to increase the concentration of enzyme used with, if necessary, a corresponding increase in substrate. It was a priority to increase the O.D. range of the enzyme substrate reaction. This in turn would allow a suitable O.D. range in which to produce a standard curve. Although published work seldom refers to the actual O.D. range for standard curves, experience suggests that an O.D. working range between 0.6-1.6 is acceptable. The upper limit is constrained by the detection capabilities of the instruments employed for the measurement. In some plate reader this upper O.D. limit is 2 while other can measure up to 3.0. The production of high O.D. values is undesirable, for routine use, as it indicates an inefficient use of enzyme. The following were investigated to find out how the working O.D. range could be increased: 1.

the optimum wavelength for recording the assay;

2.

the effect of altering the environmental conditions under which the assay was conducted, including temperature, pH and length of incubation;

3.

the effect of varying enzyme concentration; and

4.

the effect of repeated freeze thawing of the enzyme and the effect of pre-incubation of the enzyme and reagents.

2.3.2

Optimisation of O.D. Readings

This colorimetric protein phosphatase inhibition assay measures the hydrolysis of para nitrophenyl phosphate (p-NPP) to para nitrophenol (p-NP) and free phosphate, in the presence of the PP2A. The generation of the p-NP, as the reaction progresses, results in the formation of a yellow colour and the optical density of this colour can be measured at a specified wavelength. The methods of Simon and Vernoux (1994) and Tubaro et al. (1996) measure the liberation of p-NP by the change in absorbance at 405 nm, whereas Nunez and Scoging (1997) measure the optical density at 400 nm. The uninhibited reaction was set up in a 1 ml cuvette against a reaction blank, in a Carey scanning spectrophotometer. The scanning spectrophotometer measured the change in the

5

Colorimetric Protein Phosphatase Assay for the Detection of the Diarrhetic Shellfish Toxin optical density of the reaction and the reaction blank as the enzyme reaction progressed. Measurements were recorded in the visible spectrum and part of the infra red spectrum. This allowed investigation of the wavelengths that resulted in the maximum O.D. values. The uninhibited, blank corrected PP2A reaction was run in a cuvette at 18°C (room temperature that day) using 0.05 U PP2A/ml reaction. The highest absorbance readings for the reaction were obtained between 385-420 nm (Table 1). The O.D. values dropped off steeply below 390 nm and tailed off above 420 nm. The effect of temperature and enzyme concentration were investigated and demonstrated that it was possible to increase the O.D. values by altering these parameters (Table 1). Working at 30°C using 0.1U PP2A/ml, blank corrected absorbance values of 1.2 were achievable after a 60 minute incubation and >2 if the reaction was incubated for two hours. The optical density of the combined reagents of the assay, including reaction buffer, enzyme buffers and the pNPP substrate is used as a reaction blank. The O.D. obtained for a solution of these reagents is then subtracted from the O.D. obtained from the enzyme reaction. The reagent blank appears clear to the naked eye but measurements by scanning spectrophotometry, using 0.8 ml in a cuvette, revealed that the reagent blank has a high O.D., >2.5, at and below 380 nm at 30°C. Above 390 nm, the O.D. of the reagent blank drops steeply, approaching zero absorbance at 420 nm. The microtitre plate reader identified for the work contained a 405 nm filter. Even though this would not achieve the maximum O.D. for the reaction, this was the nearest to the maximum O.D. value which could be obtained for the machine without having a customised filter made. The 405 nm filter was capable of measuring between 400-410 nm. All future measurements where made with the 405 nm filter. The typical O.D. value for the reaction blank, measured at 405 nm in a 250 µl reaction in a microtitre well, is ~0.6. All results are therefore blank corrected against a reagent blank. For use in plate readers that can measure O.D. up to a maximum value of 2, the non-blank corrected values of the reaction must not exceed this value and the blank corrected O.D. value would have to be 0.6 following pre-incubation of the reagents, there was ~50% reduction in the O.D. as a result of the freeze thawing. It was decided that in future, all PP2A enzyme would be diluted and stored in ampoules suitable for single use. Partially used ampoules would be discarded after use. Table 2

Effect of pre-incubation times on precision of O.D. values obtained after 60 minutes

Pre-incubation (min)

Precision (% cv) of reaction Enzyme ampoule used straight from deep freeze at 20°C

Enzyme ampoule used after freeze thawing

0

9.4

14.8

5

6.7

6.6

10

5.9

9.1

15

4.2

6.6

The duration of the pre-incubation did not enhance the O.D. values for the freshly thawed ampoule. With regard to the freeze thawed enzyme, it did benefit from a pre-incubation of

9

Colorimetric Protein Phosphatase Assay for the Detection of the Diarrhetic Shellfish Toxin five minutes but increasing the duration of the incubation up to 15 minutes did not convey any further benefit. The precision of replicates of a newly thawed ampoule of enzyme and an ampoule that had undergone freeze thawing both benefited from a pre-incubation. For the freshly thawed ampoule of PP2A, precision improved with increased duration of the pre-incubation (Table 2). Even though the 15 minute pre-incubation gave greater precision, it was avoided in later experiments after it was found to have a detrimental effect for one batch of enzyme, whereas the 10 minute incubation did not. Due to this, a 10 minute pre-incubation was selected. 2.3.5

Standard Curves and Associated Precision Data

The following concentrations of okadaic acid standard (NRC, Canada) were used to create a standard curve: 25.3 ng OA/ml, 15 ng OA/ml, 7.5 ng OA/ml,1.5 ng OA/ml, 1.0 ng OA/ml, 0.75 ng OA/ml, 0.5 ng OA/ml, 0.25 ng OA/ml, 0.125 ng/OA/ml, 0.064 ng OA/ml, 0.032 ng/ml and 0.0 ng OA/ml. The 25.3 ng OA/ml concentration was introduced to lengthen the lower tail of the standard curve. The accompanying software for the MRX temperature control plate reader was used to fit a logit curve, which plots optical density against the log of ng OA/ml. For ease of comparison of results, optical density readings can be expressed as % absorption (Fig. 7). Precision data was obtained for each okadaic acid standard concentration used in the calibration curve. The Revelation software, used in conjunction with the MRX plate reader, enables the predicted concentrations to be obtained for the points used to create the standard curve. The programme, following fitting of the calibration curve to the data, calculates the predicted toxin concentration for each point on the standard curve (Fig. 7). This provided useful data on the errors associated with the results generated from different sections on the curve. These data, viewed in conjunction with the curve, indicated the section of the curve that could most reliably be used to report toxicity. It was commonly observed that the O.D. readings for the 3.0 ng OA/ml and 1.5 ng OA/ml standards did not fall on the predicted calibration curve (Fig. 7). The lack of definition between the O.D. values for these two standard concentrations affects the calibration curve, resulting in large error values for the predicted toxin concentrations (see data in Fig. 7). As this was a recurring problem and was probably due to a systematic procedural error in creating the calibration curve. Further work should allow this issue to be resolved and reduce the error at the lower end of the curve. Precision of the individual standard concentrations used to create a standard curve was investigated over an 180 minute incubation period and was shown to improve with the duration of the incubation (Fig. 8). During the first 30 minutes of incubation, there was a large imprecision associated with many of the okadaic acid standard concentrations. Reading of the reaction should therefore only be done after a minimum of 30 minutes incubation. There is only minimal benefit, with respect to precision, in using an incubation time greater than 60 minutes (Fig. 8). As the incubation time of the reaction was lengthened, the reaction progressed and the O.D. values of the uninhibited reaction increased. The absorbance values of the standard points on the curve also increased. It was likely that this was because the enzyme that was not completely inhibited by okadaic acid had longer to cleave the free phosphate from the pNPP, resulting in more colour being generated by the reaction. The percentage O.D readings of individual OA concentration on the curve increased as incubation time was lengthened. In effect, the assay became less sensitive as incubation time was increased. Table 3a shows

10

Colorimetric Protein Phosphatase Assay for the Detection of the Diarrhetic Shellfish Toxin the increase in the OA concentration required to produce O.D. values of 90%, 50% and 10% of the maximum O.D. at 30 minute, 45 minute and 60 minute incubation times. The loss in sensitivity that occurs in-conjunction with an increase in incubation can be utilised in the reporting of data. If more than one dilution of a test sample is presented to the assay, it is normal practice to report data from the dilution which produces an O.D. reading nearest to the middle of the calibration curve. Due to the shift in the detection limit with incubation time three time measurements of the same reaction are taken. The position of the O.D. value on the calibration curve from a test sample will alter with the incubation time. Using incubations at 30, 45 and 60 minutes produces three sets of data for a test sample. The toxicity data can then be reported from curve in which the O.D. for a shellfish sample being tested falls within the area of a curve with the greatest accuracy. The large number of points used in the standard curve takes into account the movement of the curve with incubation time. However, this means that there is a trade-off between the precision of the individual OA concentrations in a standard curve (see Fig. 8) and the detection limit. Table 3a Calibration curve data at three incubation times

30 min

Incubation time 45 min

60 min

Max O.D. of standard curve

0.964

1.281

1.546

Min O.D. of standard curve

0.057

0.070

0.078

ng OA/ml detection levels at three sections of the standard curve at 30, 45 and 60 minute incubations 30 min

45 min

60 min

10 % O.D. of standard curve

6.539 ng OA/ml

6.601 ng OA/ml

6.791 ng OA/ml


0.947 ng OA/ml

1.052 ng OA/ml

1.117 ng OA/ml


0.131 ng OA/ml

0.148 ng OA/ml

0.160 ng OA/ml

Many calibration curves were generated during the development and use of the assay. These curves followed the same general pattern of a loss of sensitivity with increased incubation time. However, each individual curve must be looked at to determine the specific detection limits. Differences in detection limits between batches of enzyme may occur. If specific batches of PP2A result in high O.D. values then reducing the concentration of PP2A being presented to the assay may be considered. Altering the enzyme concentration of the assay affects the sensitivity of the standard curve (Table 3b).

11

Colorimetric Protein Phosphatase Assay for the Detection of the Diarrhetic Shellfish Toxin Table 3b 50% inhibition levels of two calibration curves with different enzyme concentrations. (mean O.D. of both curves >1)

2.3.6

Incubation time (min)

Concentration of PP2A/ml reaction

30 minutes

45 minutes

60 minutes

0.04 U/ml

0.867 ng OA/ml

1.021 ng OA/ml

1.138 ng OA/ml

0.05 U/ml

1.197 ng OA/ml

1.288 ng OA/ml

1.385 ng OA/ml

Discussion: Assay Conditions, Temperature, pH, Enzyme Concentration and Incubation Time

2.3.6.1 Temperature The advantageous effect of increasing the incubation temperature to 37°C contradicted the findings of Dr Tubaro (pers. comm.). A possible explanation may be that the dri-block heater and the Falcon plates are specifically designed to maximise the thermal transfer to the truncated conical wells, which make intimate contact with the recessed holes on the aluminium heating block. The poor O.D. values obtained using the incubation oven may indicate that the lack of thermal contact between the oven racks and the microtitre plate result in the contents of the microtitre wells taking longer to reach the desired temperature. Consequently, if Dr Tubaro used an incubation oven for her temperature experiments, that may explain why an increase in the incubation temperature did not result in enhanced performance of the enzyme. However, this does not explain why Dr Tubaro found 37°C to be detrimental to the assay. Further data will be collated on the effect of temperature on the kinetics of the reaction. It was concluded that the assay should be carried out at 37°C using the dri-block heater to ensure efficient heat transfer during the early stages of the assay. 2.3.6.2 pH The effect of pH (Fig. 1.) indicated that the published value of pH 8.4 was favourable and should be retained in the method. The use of pH 7.5 had a detrimental effect on the O.D. obtained for the reaction. The influence of pH was only tested at 22°C. However, the assay was found to perform well at higher temperatures using pH 8.4. It was concluded that pH º 8.4 was satisfactory for a range of incubation temperatures between 22 and 37 C. 2.3.6.3 Enzyme quality Increasing the concentration of enzyme in conjunction with an increase in the incubation temperature resulted in higher O.D. values. Blank corrected O.D. values of >0.6, for the uninhibited reactions, could typically be obtained using an enzyme concentration 0.05 U/ml reaction. The Dynex MRX-II plate reader can measure O.D. values up to 3. However, working at such high values is an inefficient use of the enzyme. High O.D. values obtained by using enzyme concentrations greater than 0.05 U/ml resulted in a decrease in the sensitivity of the assay to OA. It was possible to lower the enzyme concentration by 20% from 0.05 U/ml to 0.04 U/ml for some particularly active batches of enzyme which in turn increased the sensitivity of the assay to OA.

12

Colorimetric Protein Phosphatase Assay for the Detection of the Diarrhetic Shellfish Toxin As the activity of the purchased enzyme from UBI differs from batch to batch, it is not practical to state the exact enzyme concentration (U/ml) required for the assay. It is necessary to test each batch of enzyme on receipt and then determine what concentration should be used to give a suitable blank corrected O.D. ≥0.6. 2.3.6.4 Incubation time As the incubation time is increased, so the enzyme has longer to work on the substrate and produce the colour forming reaction product. In general, increasing the incubation time results in a greater O.D. being obtained. However, the reaction also slows as the enzyme utilises the substrate. Doubling the O.D. value, achieved from a 60 minute incubation, required the incubation to be extended to ~180 minutes (Fig. 4). In a monitoring situation, lengthening the incubation time may reduce the throughput of shellfish samples being tested. Very short incubation times of