Comparison of Immunoassay, Cellular, and Classical Mouse Bioassay

Chapter 32

Comparison of Immunoassay, Cellular, and Classical Mouse Bioassay Methods for Detection of Neurotoxic Shellfish Toxins Downloaded by UNIV OF TEXAS AT AUSTIN on March 28, 2018 | https://pubs.acs.org Publication Date: May 5, 1996 | doi: 10.1021/bk-1996-0621.ch032

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Ian Garthwaite , Kathryn M . Ross , Mark Poli , and Neale R. Towers 1

AgResearch Ruakura, East Street, Private Bag 3123, Hamilton, New Zealand Toxinology Division, U.S. Army Medical Research Institute of Infectious Diseases, Fort Detrick, Frederick, MD 21702-5011

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Shellfish occasionally present a health risk to consumers due to bio-accumulation of natural marine toxins. One such class of toxin is the brevetoxins (PbTx) which are responsible for neurotoxic shellfish poisoning. Monitoring of commercial produce presently involves the evaluation of toxicity by intraperitoneal injection of a crude lipid extract of shellfish into mice. We have compared a newly developed ELISA assay for brevetoxins, with a detection limit of 0.25 mg brevetoxin-2 (PbTx-2) per kg shellfish flesh (2.5 ng/mL in the assay), a neuroblastoma-based sodium channel enhancement assay (detection limit 15 ng/mL), and the standard mouse bioassay for evaluation of shellfish samples collected during recent New Zealand algal blooms. Discrepancies among the assays were observed due to the presence of a 'new' lipid soluble, sodium channel active biotoxin. Neurotoxic shellfish poisoning (NSP) occurs when shellfish that have accumulated the lipid-soluble brevetoxins are eaten. Symptoms include incoordination, respiratory distress, paralysis and convulsions. To protect human health, many governments have set maximum permissible limits for the toxins, with the regulations set by the US F D A being the most widely accepted. Attempts are currently being made worldwide to replace the nonspecific mouse bioassays specified in these regulations with more humane, specific assays. Here we compare two possible alternatives. In the summer of 1992-3, New Zealand (NZ) experienced its first NSP event (7), with shellfish samples showing positive in the mouse bioassay. A portion of the samples collected during this and subsequent outbreaks, including a number of samples which gave irregular results in the mouse bioassay, have now been analysed for brevetoxin by ELISA and by a neuroblastoma assay which detects toxins activating the cellular sodium channel. 0097-6156/96/0621-O404$15.00/0 © 1996 American Chemical Society Beier and Stanker; Immunoassays for Residue Analysis ACS Symposium Series; American Chemical Society: Washington, DC, 1996.

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Methods for Detecting Neurotoxic Shellfish Toxins 405

Materials and Methods

Downloaded by UNIV OF TEXAS AT AUSTIN on March 28, 2018 | https://pubs.acs.org Publication Date: May 5, 1996 | doi: 10.1021/bk-1996-0621.ch032

Shellfish Material. Shellfish samples were collected at a number of sites around the New Zealand coastline as part of the Marine Biotoxin Monitoring Programme. The majority of samples represented mussel {Perna canaliculus), scallop (Placopecen magellanicus), and oyster (Tiostrea chilensis, Crassostrea gigas), although some samples of tuatua (Paphies subtriangulata subtriangulata), pipi (Paphies australis) and cockles {Bassina yaîeï) were also analysed. Extraction. Shellfish material was prepared according to (2). Briefly, shellfish were shucked and the flesh (100 g) homogenised with 300 mL acetone for 20 s. After filtration through Whatman 541 paper the solid residue was reextracted with a further 200 mL acetone, and the filtered extracts were pooled. Acetone was removed by rotary evaporation at 35 °C and the aqueous residue partitioned twice against dichloromethane. The lower (dichloromethane) fraction was passed through anhydrous NaS0 and dried in vacuo to a constant weight. This fraction was termed the lipid extract. 2

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Mouse Bioassay. The mouse bioassay, which involves the evaluation of toxicity by intraperitoneal injection of the crude lipid extract of shellfish into mice, was performed according to the method of (3) except that the lipid extract was suspended in a solution of 1 % Tween 60 in 0.85% NaCl or in vegetable oil and diluted to a fin concentration equivalent to 10 g sample per mL for intraperitoneal injection. Results were calculated as mouse units as described by (3). All animal manipulations were approved by animal ethics committees established under the Animal Protection (code of ethical conduct) Regulations Act, 1987. t

Neuroblastoma Assay. The neuroblastoma assay was performed as described by (4) with minor modifications. Stock cultures of the mouse neuroblastoma cell lines Neuro-2A and NB41A3 were maintained in a humidified incubator (5% C0) at 37 °C in RPMI medium (RPMI 1640, Gibco BRL 31800-022, New York, NY) supplemented with 10% fetal calf serum, 2 mM glutamine, 1 mM pyruvate, 100 U/mL penicillin and 100 U/mL streptomycin. Monolayer cultures (5xl0 cells/well) were prepared and incubated for 9-25 h with the sample extract (10 \\L) or brevetoxin standard (PbTx-2) in both the presence (test wells) and absence (controls) of ouabain (final assay concentration of 600 μΜ for Neuro-2A, 850 μΜ for NB41A3) and veratridine (final assay concentration of 60 μΜ for Neuro-2A and 85 μΜ for NB41A3); total test volume was 230 μL/well. Cell viability was determined by aspiration of the medium and incubation of the cell layer with 3-[4,5-dimethylthiazol2-yl]-5,5-diphenyltetrazolium bromide (MTT) dye solution. A 5 mg/mL stock solution of MTT in 0.01 M phosphate buffer pH 7.4 containing 0.15 M NaCl (PBS) was diluted 1:6 with RPMI and 60 μΐ. added to each well. The cells were incubated for 20 min with the MTT solution, which was then removed by aspiration. The precipitated dye was resuspended in 100 μίΛνεΙΙ DMSO and absorbance at 595 nm determined using a microplate spectrophotometer. Lipid extracts of shellfish material were either applied to the assay as a suspension in 1 % Tween 60 solution (as pe 2

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Beier and Stanker; Immunoassays for Residue Analysis ACS Symposium Series; American Chemical Society: Washington, DC, 1996.

IMMUNOASSAYS FOR RESIDUE ANALYSIS

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mouse bioassay), or resuspended in methanol to a concentration representing 1 g original shellfish meat per mL and diluted 1:9 in serum-free medium. A minimum of three replicates of standards and samples were used for each determination of toxin concentration. E L I S A . Antibodies were raised against brevetoxin-BSA conjugate (PbTx-3-BSA) as described in (5). Brevetoxin standard (PbTx-2) and brevetoxin-horseradish peroxidase conjugates (PbTx-3-HRP) were purchased from Chiral Corp, Miami, F L . Amplification reagent (ELAST) was obtained from Dupont-NEN, Germany. The direct competitive ELISA was performed as follows: ELISA plates (NUNC Immunoplate I, Denmark) were coated with antiserum in 0.05 M sodium bicarbonate buffer pH 9.4 (50 uL, 50 μg/mL) overnight at 20 °C. After a wash in PBS, additional binding sites were blocked by incubation with 1% B S A solution (300 μ ί , l h , 20-25 °C). Plates were washed once in PBS and either used immediately or stored at 4 °C for up to 7-days. For assay, 50 uL sample or standard was added to the wells together with 50 pL brevetoxin-horseradish peroxidase solution (100 μg/mL). After incubation at 20-25 °C for 3 h wells were washed four times with PBS + 0.05% Tween 20 (PBST), four times with PBS, and the amplification/substrate reagents sequentially added to the wells as follows. Biotinyl tyramide solution (100 pL/well) was incubated for 15 min, wells were aspirated, washed four times with PBST and four times with PBS. Streptavidin-horseradish peroxidase (100 uL/well) was then added and incubated for 30 min, before the wells were again aspirated and washed four times with PBST and four times with PBS. 3, 3', 5, S'-Tetramethylbenzidine (TMB) substrate solution, prepared by adding 100 pJL T M B stock (10 mg/rnL in DMSO) to 10 mL sodium acetate buffer 0.1 M , pH 5.5 containing 0.005% H 0 , was then added and incubated for 15 min, before the reaction was stopped by the addition of 50 uX 2 M H S 0 and absorbance at 450 nm determined using a microplate spectrophotometer. Standards and samples were prepared for ELISA by dilution of a methanol suspension with PBS to a maximum methanol concentration of 10% (v:v). Samples were analysed in triplicate over a range of dilutions. 2

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Results - Part I - Assay Performance Mouse Bioassay. With the exception of a small number of assays run at Ruakura to confirm the toxicity of bulk extracts prepared in our laboratory for oral dosing trials and other work, all mouse bioassays were run by the Communicable Disease Centre, Institute of Environmental Science & Research, Porirua, as part of the N Z biotoxin monitoring programme. Subsamples of extracts prepared for mouse bioassay were obtained and used for the comparative assays described below. Neuroblastoma Assay. The neuroblastoma assay gave reproducible curves for the brevetoxin PbTx-2 standard against which the toxin concentration of sample extracts could be assessed. The standard assay could be completed with incubation times as



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Methods for Detecting Neurotoxic Shellfish Toxins

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brief as 9 h, with low intraassay variability. Extended incubation times up to 24 h gave parallel standard curves (Figure 1). The standard working range was slightly wider for the NB41A3 cell line than for the Neuro-2A. Interassay and intraassay variances were similar for the two lines (Table I). NB41 A3 was the preferred cell line because it gave the most reproducible monolayer culture. NB41A3 cells exhibited neuroblast-like morphology in comparison to the mixed neuron-like and amoeboidlike cell morphology of the Neuro-2A line. When analyzing shellfish material, resuspension of the lipid extract in methanol was found to be the best method of sample preparation. The use of a Tween suspension of sample increased the viable cell count in control wells, in a non reproducible manner, above that determined for the no-toxin standard wells. The methanol procedure did not give these artifacts in the control and was thus adopted for the remainder of this study. Employing this methanol resuspension, reproducible interassay results were obtained for both assay (with ouabain/veratridine) and control (no ouabain/veratridine) samples. Coefficients of variance for these samples were in the order of 10-20%. Samples were quantified in these assays as 'brevetoxin (PbTx2) equivalents' with a detection limit of 15 ng/mL in the sample extract, equivalent to 1.5 mg/kg in the shellfish tissue. The ELISA assay (Figure 2) was optimised by titrating concentrations of all reagents and amplification system components to give the final assay setup described above. The standard working range and detection limits were determined, along with interassay and intraassay variances (Table I). Methanol resuspension of samples were employed in all ELISA analyses. Methanol was found to give a matrix effect, although this was consistent for all samples and standards. The use of a methanol concentration of 10% throughout for both samples and standards was therefore sufficient to give reproducible results. Brevetoxin-free shellfish material showed no cross-reaction in the ELISA. Spike recovery experiments have yet to be performed. ELISA.

Table I. Comparison of Assay Sensitivity and Precision.

Toxin concentration ng/mL

Neuro 2A

NB41A3

ELISA

Coefficient of variance (%)



Intraassay

1000 100 10 1 Working range

11.65 15.86 8.87

Interassay

12.15 16.38 10.43

15-200 ng/mL

Intraassay

21.57 14.56 8.22 -

Interassay

19.63 15.68 8.23 -

15-200 ng/mL

Intraassay

Interassay

10.38 5.21 3.29 5.47

11.96 5.07 5.89 11.37

2.5-75 ng/mL



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Figure 1. Effect of Incubation Time on Neuroblastoma Assay For Brevetoxin. Brevetovin standard was incubated with the NB41A3 cell line for 8.5 to 24 h. Parallel curves are observed for PbTx-2 over this period indicating consistent response. Absorbance was normalized as percentage of no-toxin sample (including ouabain/veratridine). Absorbance for this Zero sample ranged from 0.822 Absorbance units (8.5 h) to 0.556 (24 h). 2.0

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Figure 2. Standard curve for Brevetoxin ELISA. The brevetoxin (PbTx-2) standard curve prepared in 10% methanol/PBS has linear working range (I„ to l ) of 2.5 to 75 ng/mL, I, = 15 ng/mL. Maximum absorbance is 1.5 Absorbance units. l5

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32. GARTHWAITE ET AL.


409

Interassay and intraassay coefficients of variation (calculated as standard deviation/mean χ 100 (%)), was assessed over triplicate samples for each assay. The linear working range and minimum detection limits are also shown. Results - Part II - Analysis of Shellfish Material

Extracts prepared from shellfish collected during the summer of 1993-4 and found to contain NSP toxins by the mouse bioassay were strongly positive in both the ELISA and the neuroblastoma assay systems, indicating the presence of compounds with both brevetoxin-like structure and biological activity. Brevetoxin derivatives were subsequently isolated from NZ shellfish and purified to homogeneity by Prof. Yasumoto (Yasumoto, T., Tohoku University, Japan, personal communication, 1994). Table Π shows the result of analysis of two of these toxins in the two assay systems. The ELISA data shows that both toxins cross-react with the antibody, and thus have a considerable degree of structural homology with the brevetoxin standard PbTx-2. The neuroblastoma assay indicates that the new toxins have a very much higher biological activity, the 'slow acting' toxin being some 90 times more potent than brevetoxin (PbTx-2).


Are The New Zealand NSP Toxins Brevetoxins ?

Table II. Analysis of Purified N Z NSP Toxins by E L I S A and Neuroblastoma Assay. 100 μβ 'Fast acting' Toxin ELISA Neuroblastoma

65 μg PbTx-2 Equiv. 457 [ig PbTx-2 Equiv.

100 μβ 'Slow acting' Toxin

20 μg PbTx-2 Equiv. 9000 μg PbTx-2 Equiv.

Values indicate the equivalent amount of brevetoxin required to give the same level of response in the assay. Irregular Results...Non-Brevetoxin NSP Toxins?

In 1994-5 a number of oyster samples collected from the South Island of New Zealand gave results in the mouse bioassay indicative of NSP toxin levels greater than 660 mouse units per 100 g shellfish flesh, a level 33 times the action limit. These results were questioned, however, as the observed symptoms were different from those normaly associated with NSP toxins. Further, unlike the brevetoxin-containing extracts, these samples did not show the expected proportional reduction in toxicity on dilution. Indeed, sample extracts diluted beyond 1:5 did not lead to death. A number of these samples were analysed by ELISA and although the ELISA detected the presence of brevetoxins in some extracts, the concentrations were extremely low and could not account for the positive mouse bioassay results. In the neuroblastoma assay no effect was observed over incubation periods ranging from 9




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to 18 h, an incubation period normally sufficient to detect brevetoxins. In assays incubated for 25 h, however, marked sodium channel activity was observed. To resolve the apparently conflicting results with these samples, the regulators repeated the mouse bioassay analyses in parallel using the N Z acetone extraction (2) and the A P H A ether extraction (3) procedures. The high toxicities of the acetone extracts were not observed in the ether extracts of the same shellfish sample. Indeed, most of the ether extracts analyzed returned toxicities below the action level. Furthermore, the results of the mouse bioassay using ether extraction now agreed with the more specific ELISA and short incubation neuroblastoma assays. A bulk extract of shellfish material containing 200 MU/100 g tissue was prepared and used to compare the three assay methods. The toxin content of 100 g oyster flesh was determined to be 208 M U by the mouse bioassay (acetone); less than 10 ng of PbTx-2 equivalents by ELISA; and 455 μg of PbTx-2 equivalents by long incubation neuroblastoma assay. It should be noted that in preliminary studies this material produced no signs of toxicity when administered to rats (up to 63 g/100 g liveweight) and mice (up to 126 g/100 g liveweight) by gavage (Towers, N . ; Garthwaite, I.; Smith, B . L . ; Munday, R ; AgResearch Ruakura, New Zealand, unpublished data) The acetone soluble 'biotoxin' has subsequently been isolated by Yasumoto, and its structure is under investigation. Preliminary results indicate that the toxin is not a poly-ether and is structurally unrelated to the brevetoxins (Yasumoto, T., Tohoku University, Japan, unpublished data.). Samples of this purified material have been analysed by ELISA and neuroblastoma assay. The antibody used in the ELISA was shown to have no cross-reactivity with the material. The neuroblastoma assay showed no effect over standard incubation periods of 9 to 18 h, but marked sodium channel activity at 25 h (Figure 3).

Conclusions In this paper we have described the development of a brevetoxin-specific ELISA, and compared this and the recently described neuroblastoma-based assay with the classical mouse bio-assay for the analysis of the compounds responsible for neurotoxic shellfish poisoning. These analytical methods have helped us understand the toxins present in recent algal bloom incidents around the N Z coastline and have exemplified some of the problems in the use of nonspecific bioassays in assessing the potential toxicity of shellfish samples. Ideally the procedure used in screening shellfish for harmful toxins should show a direct correlation between the toxicity (or toxin concentration) recorded in test systems and severity of the toxic effects on ingestion of the contaminated food or extracts of the food. However, published data relating test results with oral toxicity are not available for any of the three assay systems for NSP (brevetoxins) and all three assays circumvent a major component of the 'real' situation - the digestive process leading to uptake of toxin. Currently the mouse bioassay is the procedure most widely accepted by regulatory authorities for the detection of brevetoxins in shellfish. However, the


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GARTHWAITE ET AL.


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Comparison of Immunoassay, Cellular, and Classical Mouse Bioassay

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