CA2007696A1 - Method and apparatus for the detection of paralytic shellfish poisons - Google Patents
Method and apparatus for the detection of paralytic shellfish poisonsInfo
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- CA2007696A1 CA2007696A1 CA 2007696 CA2007696A CA2007696A1 CA 2007696 A1 CA2007696 A1 CA 2007696A1 CA 2007696 CA2007696 CA 2007696 CA 2007696 A CA2007696 A CA 2007696A CA 2007696 A1 CA2007696 A1 CA 2007696A1
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Abstract
METHOD AND APPARATUS FOR THE DETECTION OF
PARALYTIC SHELLFISH POISONS IN SHELLFISH AND CRABS
ABSTRACT OF THE DISCLOSURE
This invention pertains to a novel method and apparatus for the detection of paralytic shellfish poisons. More particu-laxly, this invention relates to a method which utilizes as an indicator of paralytic shellfish poison a novel material protein which is correlative to the poison. An apparatus for the detection of the presence of paralytic shellfish poison in crustacean and mollusc species which comprises components for determining the existence of a high molecular weight saxtoxin induced protein in the tissues of crustacean and mollusc species after the species are exposed to saxitoxin.
PARALYTIC SHELLFISH POISONS IN SHELLFISH AND CRABS
ABSTRACT OF THE DISCLOSURE
This invention pertains to a novel method and apparatus for the detection of paralytic shellfish poisons. More particu-laxly, this invention relates to a method which utilizes as an indicator of paralytic shellfish poison a novel material protein which is correlative to the poison. An apparatus for the detection of the presence of paralytic shellfish poison in crustacean and mollusc species which comprises components for determining the existence of a high molecular weight saxtoxin induced protein in the tissues of crustacean and mollusc species after the species are exposed to saxitoxin.
Description
METNOD AND APPAR~TUB FOR ~H~ DsTEcTIoN OF
PARALYTIC ~NE~LFI~H POISON8 IN 8~E~LFI~H ~ND CRABS
FIELD OF THE INVENTION
This invention pertains to a novel method and apparatus for the detection of paralytic shellfish poisons. More particu-larly, this invention relates to a method and apparatus which utilize as an indicator of paralytic shellfish poison a novel material protein which is correlative to the poison.
BACKGROUND OF THE INVENTION
~' Paralytic shellfish poisoning (PSP) can occur in individuals who have consumed shellfish and crabs contaminated with dinoflagellate "Red Tide" blooms that possess a potent group of neurotoxins, that are collectively termed paralytic shellfish toxins.
In North America, PSP is a recurring summer time problem involving filter feeding shellfish that have been exposed ; to "Red Tide" blooms. The prospect of PSP represents a major economic deterrent to shellfish industry investment, a hazard to any existing shellfish industry and a serious concern to public health organizations. For instance, of the approximately 14,000 miles of Georgian Strait and Pacific Ocean coastline in British Columbia, Canada, approximately 70 p~rcent is closed to the commercial harvesting of shellfish because of the sporadic and unpredictable occurrence of toxic dinoflagellate blooms. A
` 30 similar problem exists with the Northwest Pacific coast and the Alaska Panhandle Pacific coast of the United States. Because of the unpredictability and hazardous consequences, and the absence of a reliable test for detecting dinoflagellates, a potentially lucrative shellfish industry is not utilized.
! A practical and reliable method for detecting PSP, in addition to the conventional mouse bioassay, would allow for . -- 1 -- .
t,p~`' ';
greater monitoring of the northwestern coastline of North America, and elsewhere, and provide initiatives for increased industry of shellfish produce. Methods for the detoxification of contaminated shellfish are unsatisfactory (Gill et al., 1984).
There are problems associated with poor precision of PSP
detection at lower limits of toxin concentration, using the conventional mouse bioassay. There is therefore a considerable need for the availability of sensitive, economical and relatively easy, but reliable monitoring procedures for the detection of PSP
in contaminated shellfish and crabs.
Since the early work of Sommer et al. with the sand crab, Emerita analo~a, (Sommer, ~., Wheldon, W.F., Kofoid, C.A.
and Stofler, R. Arch. Pathol. 24:537-559 (1937)), there have been many reports describing the accumulation of PSP in many genera and species of marine organisms (Foxall, T.L., Stoptaugh, N.H., Ikawa, M. and Sasner, J.J. in Toxic Dinoflaqellate Blooms (eds.
; T. Taylor, and H.H. Selinger) Elsevier/North Holland, NY 413-; 418 (1979); Onoue, Y., Noguchi, T. and Hashimoto, K. Bull. Jap.
Soc. Sci. Fish. 46:1031-1034 (1980); Hsu, C.P., Marchand, A. and Shimizu, Y. J. Fish. Res. Bd. Can. 36:32-36 (1979); Jonas-Davies, J. and Liston, J. in Toxic Dinoflaqellates (eds. D.M.
Anderson, A.W. White and D.G. Baden) Elsevier/New York 467-472 (1985)). Species that show resistance to saxitoxin (a type of PSP) generally also accumulate this toxin without adversity. PSP
sensitive species, on the other hand, do not retain the toxin in appreciable amounts and exhibit typical toxic effects when exposed to PSP (Adams, J.A., Seaton, D.D., Buchanan, J.B. and Longbottom, M.R. Nature 220:24-25 (1968); Twarog, B.M. and Yamaguchi, H. in Toxic Dinoflaqellate Blooms (eds. V.R. Lo Cicero) Massachusetts Science and Technology Foundation, Wakefield, Mass. 381-394 (1975); Cucci, T.L., Shumway, S.E., Newell, R.C. and Yentach, C.M. in Toxic Dinoflaqellates (eds.
D.M. Anderson, A.W. White and D.G. Baden) Elsevier. N.Y., Amsterdam, Oxford. 395-400 (1985). The phenomenon of PSP
resistance appears to be acquired in marine species. Support for this comes from studies that have been conducted on the small ".. ,;, . . . - . ' '. , ' ' ' , , .................. ~ ' ........... ' . ~ . . . , .: :' , - ~
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shore crab, H. oreaonesis, that sxhibited seasonal resistance to ; saxitoxin, Barber, K.G., Kitts, D.D., Townsley, P.M., Bull.
Envir. Contam. Toxicol. 40: 190-197, 1988, isolated nerves have been shown to be highly resistant to tetrodotoxin, but sensitive to saxitoxin (Kao, C.Y. and Fuhrman, F.A. Toxicon 5:25-34 (1967)). In both examples, resistance reported in these species was specific to the particular toxin that was present in the organism's environment.
Varying sensitivities o~ some marine organisms to PSP
has been attributed to behavioural changes in accumulating toxin (Price, R.J. and Lee, J.S. J. Fish. Res. Bd. Can. 29:1657-1658 ` (1972~), or a specific physiological protective mechanism that interferes with the expression of toxin at the target site on the nerve (Kao, C.Y. and Fuhrman, F.A. Toxicon 5:25-34 (1967), Twarog, M.B., Hidaka, T. and Yamaguchi, H. Toxicon. 10:273-278 (1972)). There is no evidence, however, to show that resistance to PSP is due to changes in sodium channel functions which ;~
generate action potentials (Twarag, B.M. and Yamaguchi, H. in Toxic Dinoflaqellate Blooms (eds. V.R. Lo Cicero) Massachusetts Science and Technology Foundation, Wakefield, Mass., 381-394 (1975)). A potential method for the detection of PSP in crustaceans and molluscs has been proposed (Kitts, D.D., Townsley, T.M. and Smith, D.S., 1989, The Northwest Environ. J.
5: 149-151.
A high molecular weight protein has been noted in the visceral tissues from crabs that have been exposed to PSP from "red tide" blooms, or artificially exposed by saxitoxin injec-;30 tions: Barker, K.G., Kitts, D.D., Townsley, P.M. and Smith, D.S., Toxicon 26: 1027-1034, I988. ;
SUMMARY OF THE INVENTION
., . ~
Relative resistance of the small shore crab, H.
oreqonesis, to acute injections of saxitoxin, a principal paralytic shellfish poison (PSP), has been correlated with the .
~- exposure of this crab to PSP. Electrophoretograms of crude - visceral homogenates from resistant crabs containing detectable amounts of PSP, have revealed the presence of a high molecular weight protein which is termed "saxitoxin induced protein" (SIP), which was absent in crabs known to be sensitive to saxitoxin.
Further experiments have shown that a high molecular weight protein (145,000 daltons) could also be induced in PSP sensitive crabs in a dose dependent manner by injection of saxitoxin. The appearance of this protein (SIP) co-migrates with the high moleGular weight protein present in saxitoxin resistant crabs.
A polyclonal antibody generated against crab SIP has been obtained and tested against both PSP contaminated crabs and shellfish, respectively. We have demonstrated, using immuno-blotting techniques, the presence of proteins in PSP contaminated bivalve molluscs which possess a degree of immunoreactivity to the SIP found in PSP resistant crabs.
, :
The invention is directed to a method for the detection of the presence of paralytic shellfish poison in a sea dwelling crustacean or mollusc species which comprises determining the existence of a high molecular weight saxtoxin induced protein complex in the tissues or extract o~ crustacean or mollusc , species after the crustacean or mollusc species has been exposed to saxitoxin or tetrodotoxin.
The invention is also directed to a method of determin-ing the presence of paralytic shellfisih poison in sea-dwelling crustaceans or molluscs which comprises: (a) placing an aliquot of crustacean or mollusc fluid or tissue in a container and diluting it with phosphate buffered saline solution; (b) placing a portion of the solution from step (a) into a container together with a saxitoxin or tetrodotoxin treated protein and incubating same for a reasonable period of time; (c) adding an enzyme conjugate to the solution in step (b) and incubating for a reasonable period of time; and (d) reading the absorbance of the ~ solution at 405 nm.
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DRAWINGS
In drawings which illustrate specific aspects and characteristics of the invention:
Figure 1 illustrates purified SIP in 6.5 percent SDS-PAGE. heft to right, (1) molecular weight standards, (2) purified 145,000 mol. vt. - 75,000 mol. wt. protein, (3) purified 145,000 mol. wt. protein after boiling 5 min., 100C reducing conditions.
Figure 2 illustrates reaction of soluble proteins from representative crab (a; water injected; saxitoxin injected); clam (b; PSP contaminated; control); oyster tc; PSP contaminated;
control) mussels (d; PSP contaminated; control) in ELISA assay with anti-SIP serum.
.:
Figure 3 illustrates SDS-PAGE (10 percent) gels (i) of control (11 and contaminated (2); (clam (a), oyster (b) and mussel (c) lysates with corresponding immuno-blot (ii).
Figure 4 illustrates a schematic flow sheet o~ a competitive ELISA assay for crab SIP.
, :
25Figure 5 illustrates a schematic flow sheet of a direct ELISA assay for shellfish and crab SIP.
.' .
DE~AILED DESCRIPTION OF SPECIFIC
;, EMBODIMENTS OF THE INVENTION
Recently, the inventors herein have noted the appear-ance of a high molecular weight protein in visceral tissues from i~ crabs that hava been naturally exposed to PSP from "red tide"
blooms, or artificially exposed by saxitoxin injections. In ; 35saxitoxin injected crabs, the appearance of the high molecular weight saxitoxin induced protein (SIP) occurs within minutes of - administration of the toxin and it is noteworthy that both crab .~
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... . .. . .. ~ . . , .. .. ... .; ,., . ;.. .... ~; . .. .. . . .. ....... ..... . . ...... . . ...... . .. .
resistance to saxitoxin as well as the presence of SIP are reduced within weeks when the crab is restored to toxin free water. Taken together, thess observations strongly suggest a direct association between the presence of SIP and the acquired resistance of the small shore crab to saxitoxinO
The presence of the saxitoxin induced protein can be determined by electrophoresis. Mobility of the saxitoxin induced protein complex with mol. wt. 75,000 - 160,000 dalton can be determined in a 6.5% polyacrylamide gel. The species may be a bivalve mollusc. The existence of the protein can be de~ermined by enzyme immunoassay.
The invention is also directed to an immuno-diagnostic test for determining the presence of paralytic shellfish poison ; in sea-dwelling crustaceans or molluscs which comprises removing from the crustacean or mollusc a determinative amount of body fluid or tissue and determining the presence of a high molecular weight saxitoxin induced protein complex in the crustacean or mollusc by immunodiagnostic test. ~
':
The high molecular weight protein may have a molecular weight of about 145,000 daltons. The existence of the high molecular weight protein can be determined by electrophoresis.
The molecular weight of the protein complex can be between about 75,000 and 160,000 daltons.
The saxitoxin induced protein can be purified using DEAE-sepharose. Soluble proteins obtained from the homogenized visceral ti~sue of the saxitoxin exposed ~rustacean can be fractionated by ammonium sulphate precipitation and applied to a Sephacryl S-300 gel filtration column, equilibrated with a phosphate-citrate buffer.
The molecular weight fraction obtained can be further fractionated by ion exchange chromatography using DEAE-Sepharose in a phosphate-citrate buffer, can be eluted with a linear :
:, "
gradient of sodium chloride and can be purified by repeated ethanol fractionation. The purity of the product can be assessed using discontinuous polyacrylamide electrophoresis followed by silver staining.
The presence of the saxitoxin induced protein(s) - complex can be used to prepare an enzyme-linked immunosorbent assay using diluted shellfish lysates. The presence of saxitoxin induced protein(s) complex in the crustaceans and molluscs can be determined by using immuno-blotting. The presence of the protein(s) can be determined by an antiserum.
The mollusc may be a bivalve mollusc, a clam or an oyster and the crustacean may be a crab. The crustacean may be a crab of the species Hemiqrapsus oregonoses or ~emiqrapsus nudas.
The saxitoxin induced protein (SIP) was isolated and purified from soluble proteins obtained from homogenized visceral ti3sue of saxitoxin resistant crabs as illustrated in Figure l.
The mobility of the SIP complex in a 6.5 percent polyacrylamide gel has been shown to correspond to an apparent molecular weight of 145,000 daltons. With the application of heat under non-reducing conditions, the mobility changes to 72,000 daltonsO
When heated under reducing conditions, the SIP migates at 79,000 daltons (see Figure l), thereby indicating that the true molecular weight of the SIP is probably 158,000 daltons. These results do not alter the distinct possibility that SIP is a polymer or association of smaller protein subunits. The decrease ! 30 in mobility due to heating under reducing conditions, compared to non-reducing conditions, suggests that the SIP subunits possess intra-chain disulphide bonds. `
The polyclonal antiserum raised to the pure SIP complex was tested for reactivity to saxitoxin contaminated crab and -; shellfish materials, respectively (see results depicted in Figures 2a, 2b, 2c and 2d and discussion below). Strong positive , ' - 7 - ~
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reactions with anti-SIP serum were obtained in Elisa tests with saxitoxin injected and wa~er injected control crab lysates (see Figure 2a). Saxitoxin crabs consistently produced higher absorption values over the entire dilution series, compared to water injected controls. These results reflect the prssence of the homologus protein detected in saxitoxin injected crabs.
Since SIP appears within minutes of saxitoxin injection/ the high cross-reactivity in water control crabs to anti-SIP may be partially explained by the presence of pre-formed precursors of SIP which are assembled guickly in the crab following exposure to saxitoxin, in addition to the presence of cross-reacting antigens. Similarly, frozen-thawed contaminated bivalve molluscs collected at Okeovar Arm, on the British Columbia, Canada, Pacific Ocean coast during an incider.t of "red tide" in Septem-ber, 1986, tested positively to the inventors' crab anti-SIP
antibody (see Figures 2b, 2c and 2c). Reactions of contaminated clam (see Figure 2b) and oyster (see Figure 2c) gave stronger absorbance readings in the Elisa assay compared to their respective controls at both 10 and 10l dilution levels. There was little quantitative difference observed between the control and PSP-contaminated mussels (see Figure 2d).
: The possibility that the immunological activityobserved in both contaminated and control shellfish could be due to a greater population of SIP-immunoreactive proteins or a higher concentration of a single SIP immunoreactive protein in contaminated shellfish, was tested with crab anti-SIP using SDS
PAGE according to Laemmli, U.K. Nature 227:680-685 ~1970), and immuno-blotting according to Towbin, H., Stahelin, T. and Gordon, J. Proc. Natl. Acad. Sci. U.S.A. 76:~350-4354 (1979) procedures, respectively. Substantial qualitative di~ferences in the soluble protein profiles were observed in contaminated shellfish compared to their respective controls (see Figure 3a). The various protein profiles in contaminated shellfish that cross-react with . crab anti-SIP vary considerably between the individual species of shellfish (see Figure 3b). Moreover, bands which appear in : .:
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contaminated shellfish are absent in control samples (these are marked with an arrow). The inventors suggest, for purposes of understanding the invention, although they do not wish to be bound or disadvantaged by any adverse findings, that the change in protein profile as well as the higher level of cross-reactiv-ity in PSP contaminated oysters and clams to the SIP antisera may reflect biochemical changes in the shellfish due to the PSP
intoxication. Moreover, the cross-reactivit:y of these bands in PSP contaminated shellfish with crab anti-SIP provides compelling evidence that they share some degree of structural similarity with crab SIP. Since crab SIP appears to be a protein aggregate, it is the inventors' hypothesis that different PSP induced ; proteins in shellfish are composed of protein subunit(s) which are common to the SIP found in the small shore crab.
At present, saxitoxin can be directly detected using homologus antisera (see procedures in Carlson, R.E., Lever, M.L., Lee, B.W. and Guire, P.E. in Seafood Toxins (ed. E.P. Ragelis) ACS Symposium Series 262, American Chemical Society, Wash. D.C., 181-191 (1984) Davio, S.R. Toxicon 23:669-675 (1985); Chu, E'.S.
and Fan, T.S.L. J. Assoc. vf Anal. Chem. 66:13-16 (1985)).
~owever, these antisera are limited to saxitoxin only, and do not cross-react with other structurally related toxins (eg., neosaxitoxin) that are also associated with PSP. Antiserum directed against antigens in shellfish expressed as a consequence of PSP intoxication represents a novel approach to the problem of PSP detection and has potential value in the rapid screening of shellfish for PSP contamination. The discovery of PSP-induced components in bivalve shellfish provides insights into mechanisms of PSP-resistance, and is the basis for the immuno-diagnostic test for PSP-contaminated shellfish of the invention.
.:
Example ` ~
`i ' :' A discussion of the data and results pictorialized in ~
the Figures and the methods used to obtain the data appears `
below. Figure 1 demonstrates purified SIP in 6.5 percent SDS- `
.~ '' '' _ g _ ~ '~
, PAGE. Left to right, (1) molecular weight standards, (2) purified 145,000 mol. wt. - 75,000 mol. wt. SIP (saxitoxin induced protein), (3) purified SIP tsaxitoxin induced protein, 145,000 mol. wt. - 75,000 mol. wt.~ 5 min. 100C reducing conditions.
The methods used to obtain this data were as ~ollows.
The SIP was purified using DEAE-Sepharose. Briefly, soluble ~ proteins from homogenized visceral tissue of saxitoxin resistant - 10 H. oreqonesis were ~ractionated by ammonium sulphate precipita-tion and applied to a Sephacryl S-300 gel filtration column (1.5 x 26 cm~ equilibrated with 40 mM phosphate-citrate buffer, pH 5.0 with 170 mM NaCl. ~he large molecular weight fraction was further fractionated by ion exchange chromatography using DEAE-15 Sepharose in 40 mL phosphate citrate buffer, pH 5.0 with 170 mM
NaCl. The SIP was eluted with a linear gradient of 170 mM to 1.7 M NaCl and purified by repeated ethanol fractionation. Purity was assessed using discontinuous polyacrylamide electrophoresis (Laemmli, U.K. Nature 227:680-685 (1970)) followiny visualization by silver staining (Merril, C.R., Goldman, S., Sedman, S.A. and Ebert, M.H. Science 211:1437-1438 (1981)).
;; Figure 2 depicts the reaction of soluble proteins from representative crab (a water injected; saxitoxin injected); clam (b; PSP contaminated; control); oyster (c; PSP contaminated;
control) mussels (d; PSP contaminated; control) in ELISA assay with anti-SIP serum.
- The methods used were as follows. An enzyme-linkèd immunosorbent assay (ELIS~) procedure was developed using diluted crab and shellfish lysates. Homogenates were diluted 1:4 with water and protein concentrations were determined (Bradford, M.M.
Analytical Biochem. 72:248-254 (1976)) and adjusted to ensure a uniform protein concentration in the ELISA. All ELISA's were performed using flat-bottom (96 well, Immulon II DynatechTM) micro-titer plates. Anti-SIP serum was diluted with phosphate buffered saline and 0.2 percent skim milk powder. Dilutions of ... .
" - 10 -,''`' ~ " '.
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anti-SIP serum of l:lO00 for crab and l:lO0 for bivalve mollusc samples were chosen. The enzyme conjuyate was goat anti-rabbit IgG alkaline phosphatase conjugate, diluted l:lO00 and the substrate was p-nitrophenyl phosphate (Sigma). ELISA plates were incubated at room temperature for l.0 hr. and ~bsorbance values were recorded at 405 nm using a EAR 400 ELISA plate reader (SLT-Lab instruments). Within assay coefficient of variation was ~ g.57+0.38%.
.;
Figure 3 illustrates SDS-PAGE (lO]percent) gels (i) of control (l) and contaminated (2); (clam (a), oyster (b) and mussel (c~ lysates with corresponding immuno-blot (ii~.
Methods employed were as follows. Gels were stained with coomassie Blue R250 or blotted to nitrocellulose by the method of Towbin (Towbin, H., Stahelin, To and Gordon, J. Proc.
Natl. Acad. Sci. U.S.A. 76:435Q-4354 (1979)) at 38 volts, 160 mA, for 4.5 hr using a BioRad Trns Blot Cell. Immuno-blots were dried and incubated for 2 hr in 2% skim milk powder. Anti-SIP
serum was diluted l:100 in phosphate buffered saline containing 0.2% skim milk powder. Goat anti-rabbit IgG alkaline phosphatase was used at a dilution of 1:1000. The blots were washed in buffered saline and 100 mMTris-HCl (pH 7.8), respectively. The substrate solution. AS-MX napthophosphate in 100 mM Tris-HCl buffer containing Fast Red TR salt was added until the bands could be clearly visualized.
~ -Figure 4 illustrates schematically a competitive EhISA
assay for crab 5IP. This assay includes a diagnostic stick 2 which can be used as part of a six component kit and competitive ELISA assay procedure for practising the invention.
.
The Competitive ELISA assay is useful when there is a shortage of purified SIP antigen. It can be inferred that an individual trained in ELIS~ methodologies would choose the Competitive procedure for a homologous assay only. Figure 4 (a--:
:
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c) illustrates the diagnostic kit and the components that can be used in practising the invention.
As seen in Figure 4, the diagnostic stick is con-structed so that it has an untreated finger gripping portion 4and an area 6 which contains the SIP antigen. The Competitive ELISA kit 10 is composed of SIP diagnostic stick 2, a first Reagent A containing container 12, a second Reagent B contain-ing container 14 and a third Reagent C containing container 16.
Materials Reagent A: TRIS buffered saline (container 12) Reagent B: AntiSIP-alkaline P04 conjugate (con-15tainer 14~;
Reagent C: Chromagen substrate solution, p-nitro-phanyl phosphate (container 16);
Diagnostic stick: SIP coated Procedure Step 1 (Crab extract dilution): In a plastic disposable test tube 18, add an aliquot of crab extract and ; dilute the sample with 1.0 ml of Reagent A from contain~r 12.
Step 2: Transfer 0.5 ml from test tube 18 of step 1 to plastic disposable test tube 20. Introduce 0.5 ml of contents from Reagent B (container 14) and insert the SIP diagnostic stick. Allow to sit and incubate for 10 minutes.
Step 3: Remove the SIP diagnostic stick from test tube 20 and wash the stick with the contents of Reagent A (container 12). Reintroduce the SIP diagnostic stick into test tube 22 and add 1.0 ml of Reagent C (container 16).
., 1 . ' . . .... . . . . . . . ., . . `, . .
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.
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- Measure quantitatively and qualitatively as follows:
Quantitatively - Measurs by absorbance at 405 nm. Qualitatively - Measure by visual inspection of the colour change.
Preamble: The Direct ELISA methodology for SIP
, detection is the procedure of choice since it can be used for both crab and shellfish SIP detection, respectively. There must be no shortage of purified SIP antigen available to perform the . .
test.
;`, 10 Figure 5 illustrates the components for sample dilution and measurement. The Direct E~IS~ kit ll is composed of anti-SIP immunoglobin coated cuvette 10, a first Reagent A container 30, a second reagent B container 32 and a third Reagent C
container 34. ;
Materials . . I .
;; Reagent A: TRIS buffered saline (container 30);
l 20 Xeagent B: SIP-alkaline phosphatase conjugate ,~ (container 32):
, Reagent C: Chromagen substrate solution; p-; nitrophenyl phosphate.
Procedure ; Step l ~Crab/shellfish extract dilution): In a plastic ! disposable test tube 38, add an aliquot of crab/shellfish extract ;`
and dilute the sample with l.0 ml of Reagent A from container 30.
Step 2: ~ransfer 0.5 ml from the test tube 38 to the , SIP-antiyen coated cuvette lO. Incubate for 10 minutes and decant off the contents of cuvette lO.
Step 3: Add to cuvette lO 0.5 ml of Reagent B SIP -enzyme conjugate 32. Incuba~e for 10 minutes. Decant off the ~: .:
" ,A"
contents of cuvette lO. Wash the cuvette lO with Reagent A
(container 30).
.
Step 4: Add 0.5 ml of Reagent A 30 to cuvette lO and decant off the contents as in step 3.
Step 5: Add l.O ml Reagent C 34 to the cuvette lO and incubate for 30 minutes.
~hen measure quantitatively by absorbance at 405 nm or qualitatively by visual inspection of the colour change of the contents.
As will be apparent to those skilled in the art in the light of the foregoing disclosure, many alterations and modifica-tions are possible in the practice of this invention without departing from the spirit or scope thereof. Accordingly, the scope of the invention is to be construed in accordance with the substance defined by the following claims.
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PARALYTIC ~NE~LFI~H POISON8 IN 8~E~LFI~H ~ND CRABS
FIELD OF THE INVENTION
This invention pertains to a novel method and apparatus for the detection of paralytic shellfish poisons. More particu-larly, this invention relates to a method and apparatus which utilize as an indicator of paralytic shellfish poison a novel material protein which is correlative to the poison.
BACKGROUND OF THE INVENTION
~' Paralytic shellfish poisoning (PSP) can occur in individuals who have consumed shellfish and crabs contaminated with dinoflagellate "Red Tide" blooms that possess a potent group of neurotoxins, that are collectively termed paralytic shellfish toxins.
In North America, PSP is a recurring summer time problem involving filter feeding shellfish that have been exposed ; to "Red Tide" blooms. The prospect of PSP represents a major economic deterrent to shellfish industry investment, a hazard to any existing shellfish industry and a serious concern to public health organizations. For instance, of the approximately 14,000 miles of Georgian Strait and Pacific Ocean coastline in British Columbia, Canada, approximately 70 p~rcent is closed to the commercial harvesting of shellfish because of the sporadic and unpredictable occurrence of toxic dinoflagellate blooms. A
` 30 similar problem exists with the Northwest Pacific coast and the Alaska Panhandle Pacific coast of the United States. Because of the unpredictability and hazardous consequences, and the absence of a reliable test for detecting dinoflagellates, a potentially lucrative shellfish industry is not utilized.
! A practical and reliable method for detecting PSP, in addition to the conventional mouse bioassay, would allow for . -- 1 -- .
t,p~`' ';
greater monitoring of the northwestern coastline of North America, and elsewhere, and provide initiatives for increased industry of shellfish produce. Methods for the detoxification of contaminated shellfish are unsatisfactory (Gill et al., 1984).
There are problems associated with poor precision of PSP
detection at lower limits of toxin concentration, using the conventional mouse bioassay. There is therefore a considerable need for the availability of sensitive, economical and relatively easy, but reliable monitoring procedures for the detection of PSP
in contaminated shellfish and crabs.
Since the early work of Sommer et al. with the sand crab, Emerita analo~a, (Sommer, ~., Wheldon, W.F., Kofoid, C.A.
and Stofler, R. Arch. Pathol. 24:537-559 (1937)), there have been many reports describing the accumulation of PSP in many genera and species of marine organisms (Foxall, T.L., Stoptaugh, N.H., Ikawa, M. and Sasner, J.J. in Toxic Dinoflaqellate Blooms (eds.
; T. Taylor, and H.H. Selinger) Elsevier/North Holland, NY 413-; 418 (1979); Onoue, Y., Noguchi, T. and Hashimoto, K. Bull. Jap.
Soc. Sci. Fish. 46:1031-1034 (1980); Hsu, C.P., Marchand, A. and Shimizu, Y. J. Fish. Res. Bd. Can. 36:32-36 (1979); Jonas-Davies, J. and Liston, J. in Toxic Dinoflaqellates (eds. D.M.
Anderson, A.W. White and D.G. Baden) Elsevier/New York 467-472 (1985)). Species that show resistance to saxitoxin (a type of PSP) generally also accumulate this toxin without adversity. PSP
sensitive species, on the other hand, do not retain the toxin in appreciable amounts and exhibit typical toxic effects when exposed to PSP (Adams, J.A., Seaton, D.D., Buchanan, J.B. and Longbottom, M.R. Nature 220:24-25 (1968); Twarog, B.M. and Yamaguchi, H. in Toxic Dinoflaqellate Blooms (eds. V.R. Lo Cicero) Massachusetts Science and Technology Foundation, Wakefield, Mass. 381-394 (1975); Cucci, T.L., Shumway, S.E., Newell, R.C. and Yentach, C.M. in Toxic Dinoflaqellates (eds.
D.M. Anderson, A.W. White and D.G. Baden) Elsevier. N.Y., Amsterdam, Oxford. 395-400 (1985). The phenomenon of PSP
resistance appears to be acquired in marine species. Support for this comes from studies that have been conducted on the small ".. ,;, . . . - . ' '. , ' ' ' , , .................. ~ ' ........... ' . ~ . . . , .: :' , - ~
, . :
shore crab, H. oreaonesis, that sxhibited seasonal resistance to ; saxitoxin, Barber, K.G., Kitts, D.D., Townsley, P.M., Bull.
Envir. Contam. Toxicol. 40: 190-197, 1988, isolated nerves have been shown to be highly resistant to tetrodotoxin, but sensitive to saxitoxin (Kao, C.Y. and Fuhrman, F.A. Toxicon 5:25-34 (1967)). In both examples, resistance reported in these species was specific to the particular toxin that was present in the organism's environment.
Varying sensitivities o~ some marine organisms to PSP
has been attributed to behavioural changes in accumulating toxin (Price, R.J. and Lee, J.S. J. Fish. Res. Bd. Can. 29:1657-1658 ` (1972~), or a specific physiological protective mechanism that interferes with the expression of toxin at the target site on the nerve (Kao, C.Y. and Fuhrman, F.A. Toxicon 5:25-34 (1967), Twarog, M.B., Hidaka, T. and Yamaguchi, H. Toxicon. 10:273-278 (1972)). There is no evidence, however, to show that resistance to PSP is due to changes in sodium channel functions which ;~
generate action potentials (Twarag, B.M. and Yamaguchi, H. in Toxic Dinoflaqellate Blooms (eds. V.R. Lo Cicero) Massachusetts Science and Technology Foundation, Wakefield, Mass., 381-394 (1975)). A potential method for the detection of PSP in crustaceans and molluscs has been proposed (Kitts, D.D., Townsley, T.M. and Smith, D.S., 1989, The Northwest Environ. J.
5: 149-151.
A high molecular weight protein has been noted in the visceral tissues from crabs that have been exposed to PSP from "red tide" blooms, or artificially exposed by saxitoxin injec-;30 tions: Barker, K.G., Kitts, D.D., Townsley, P.M. and Smith, D.S., Toxicon 26: 1027-1034, I988. ;
SUMMARY OF THE INVENTION
., . ~
Relative resistance of the small shore crab, H.
oreqonesis, to acute injections of saxitoxin, a principal paralytic shellfish poison (PSP), has been correlated with the .
~- exposure of this crab to PSP. Electrophoretograms of crude - visceral homogenates from resistant crabs containing detectable amounts of PSP, have revealed the presence of a high molecular weight protein which is termed "saxitoxin induced protein" (SIP), which was absent in crabs known to be sensitive to saxitoxin.
Further experiments have shown that a high molecular weight protein (145,000 daltons) could also be induced in PSP sensitive crabs in a dose dependent manner by injection of saxitoxin. The appearance of this protein (SIP) co-migrates with the high moleGular weight protein present in saxitoxin resistant crabs.
A polyclonal antibody generated against crab SIP has been obtained and tested against both PSP contaminated crabs and shellfish, respectively. We have demonstrated, using immuno-blotting techniques, the presence of proteins in PSP contaminated bivalve molluscs which possess a degree of immunoreactivity to the SIP found in PSP resistant crabs.
, :
The invention is directed to a method for the detection of the presence of paralytic shellfish poison in a sea dwelling crustacean or mollusc species which comprises determining the existence of a high molecular weight saxtoxin induced protein complex in the tissues or extract o~ crustacean or mollusc , species after the crustacean or mollusc species has been exposed to saxitoxin or tetrodotoxin.
The invention is also directed to a method of determin-ing the presence of paralytic shellfisih poison in sea-dwelling crustaceans or molluscs which comprises: (a) placing an aliquot of crustacean or mollusc fluid or tissue in a container and diluting it with phosphate buffered saline solution; (b) placing a portion of the solution from step (a) into a container together with a saxitoxin or tetrodotoxin treated protein and incubating same for a reasonable period of time; (c) adding an enzyme conjugate to the solution in step (b) and incubating for a reasonable period of time; and (d) reading the absorbance of the ~ solution at 405 nm.
.:
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DRAWINGS
In drawings which illustrate specific aspects and characteristics of the invention:
Figure 1 illustrates purified SIP in 6.5 percent SDS-PAGE. heft to right, (1) molecular weight standards, (2) purified 145,000 mol. vt. - 75,000 mol. wt. protein, (3) purified 145,000 mol. wt. protein after boiling 5 min., 100C reducing conditions.
Figure 2 illustrates reaction of soluble proteins from representative crab (a; water injected; saxitoxin injected); clam (b; PSP contaminated; control); oyster tc; PSP contaminated;
control) mussels (d; PSP contaminated; control) in ELISA assay with anti-SIP serum.
.:
Figure 3 illustrates SDS-PAGE (10 percent) gels (i) of control (11 and contaminated (2); (clam (a), oyster (b) and mussel (c) lysates with corresponding immuno-blot (ii).
Figure 4 illustrates a schematic flow sheet o~ a competitive ELISA assay for crab SIP.
, :
25Figure 5 illustrates a schematic flow sheet of a direct ELISA assay for shellfish and crab SIP.
.' .
DE~AILED DESCRIPTION OF SPECIFIC
;, EMBODIMENTS OF THE INVENTION
Recently, the inventors herein have noted the appear-ance of a high molecular weight protein in visceral tissues from i~ crabs that hava been naturally exposed to PSP from "red tide"
blooms, or artificially exposed by saxitoxin injections. In ; 35saxitoxin injected crabs, the appearance of the high molecular weight saxitoxin induced protein (SIP) occurs within minutes of - administration of the toxin and it is noteworthy that both crab .~
_ 5 --: A~
. .. ..... . ... .. . . .... . . .. . ~, .. . .. ... .. , . .. ... , .. . . . ~. . . ~ .... .. . .. .. . . .... .
... . .. . .. ~ . . , .. .. ... .; ,., . ;.. .... ~; . .. .. . . .. ....... ..... . . ...... . . ...... . .. .
resistance to saxitoxin as well as the presence of SIP are reduced within weeks when the crab is restored to toxin free water. Taken together, thess observations strongly suggest a direct association between the presence of SIP and the acquired resistance of the small shore crab to saxitoxinO
The presence of the saxitoxin induced protein can be determined by electrophoresis. Mobility of the saxitoxin induced protein complex with mol. wt. 75,000 - 160,000 dalton can be determined in a 6.5% polyacrylamide gel. The species may be a bivalve mollusc. The existence of the protein can be de~ermined by enzyme immunoassay.
The invention is also directed to an immuno-diagnostic test for determining the presence of paralytic shellfish poison ; in sea-dwelling crustaceans or molluscs which comprises removing from the crustacean or mollusc a determinative amount of body fluid or tissue and determining the presence of a high molecular weight saxitoxin induced protein complex in the crustacean or mollusc by immunodiagnostic test. ~
':
The high molecular weight protein may have a molecular weight of about 145,000 daltons. The existence of the high molecular weight protein can be determined by electrophoresis.
The molecular weight of the protein complex can be between about 75,000 and 160,000 daltons.
The saxitoxin induced protein can be purified using DEAE-sepharose. Soluble proteins obtained from the homogenized visceral ti~sue of the saxitoxin exposed ~rustacean can be fractionated by ammonium sulphate precipitation and applied to a Sephacryl S-300 gel filtration column, equilibrated with a phosphate-citrate buffer.
The molecular weight fraction obtained can be further fractionated by ion exchange chromatography using DEAE-Sepharose in a phosphate-citrate buffer, can be eluted with a linear :
:, "
gradient of sodium chloride and can be purified by repeated ethanol fractionation. The purity of the product can be assessed using discontinuous polyacrylamide electrophoresis followed by silver staining.
The presence of the saxitoxin induced protein(s) - complex can be used to prepare an enzyme-linked immunosorbent assay using diluted shellfish lysates. The presence of saxitoxin induced protein(s) complex in the crustaceans and molluscs can be determined by using immuno-blotting. The presence of the protein(s) can be determined by an antiserum.
The mollusc may be a bivalve mollusc, a clam or an oyster and the crustacean may be a crab. The crustacean may be a crab of the species Hemiqrapsus oregonoses or ~emiqrapsus nudas.
The saxitoxin induced protein (SIP) was isolated and purified from soluble proteins obtained from homogenized visceral ti3sue of saxitoxin resistant crabs as illustrated in Figure l.
The mobility of the SIP complex in a 6.5 percent polyacrylamide gel has been shown to correspond to an apparent molecular weight of 145,000 daltons. With the application of heat under non-reducing conditions, the mobility changes to 72,000 daltonsO
When heated under reducing conditions, the SIP migates at 79,000 daltons (see Figure l), thereby indicating that the true molecular weight of the SIP is probably 158,000 daltons. These results do not alter the distinct possibility that SIP is a polymer or association of smaller protein subunits. The decrease ! 30 in mobility due to heating under reducing conditions, compared to non-reducing conditions, suggests that the SIP subunits possess intra-chain disulphide bonds. `
The polyclonal antiserum raised to the pure SIP complex was tested for reactivity to saxitoxin contaminated crab and -; shellfish materials, respectively (see results depicted in Figures 2a, 2b, 2c and 2d and discussion below). Strong positive , ' - 7 - ~
: .;
~'' ~`;.
reactions with anti-SIP serum were obtained in Elisa tests with saxitoxin injected and wa~er injected control crab lysates (see Figure 2a). Saxitoxin crabs consistently produced higher absorption values over the entire dilution series, compared to water injected controls. These results reflect the prssence of the homologus protein detected in saxitoxin injected crabs.
Since SIP appears within minutes of saxitoxin injection/ the high cross-reactivity in water control crabs to anti-SIP may be partially explained by the presence of pre-formed precursors of SIP which are assembled guickly in the crab following exposure to saxitoxin, in addition to the presence of cross-reacting antigens. Similarly, frozen-thawed contaminated bivalve molluscs collected at Okeovar Arm, on the British Columbia, Canada, Pacific Ocean coast during an incider.t of "red tide" in Septem-ber, 1986, tested positively to the inventors' crab anti-SIP
antibody (see Figures 2b, 2c and 2c). Reactions of contaminated clam (see Figure 2b) and oyster (see Figure 2c) gave stronger absorbance readings in the Elisa assay compared to their respective controls at both 10 and 10l dilution levels. There was little quantitative difference observed between the control and PSP-contaminated mussels (see Figure 2d).
: The possibility that the immunological activityobserved in both contaminated and control shellfish could be due to a greater population of SIP-immunoreactive proteins or a higher concentration of a single SIP immunoreactive protein in contaminated shellfish, was tested with crab anti-SIP using SDS
PAGE according to Laemmli, U.K. Nature 227:680-685 ~1970), and immuno-blotting according to Towbin, H., Stahelin, T. and Gordon, J. Proc. Natl. Acad. Sci. U.S.A. 76:~350-4354 (1979) procedures, respectively. Substantial qualitative di~ferences in the soluble protein profiles were observed in contaminated shellfish compared to their respective controls (see Figure 3a). The various protein profiles in contaminated shellfish that cross-react with . crab anti-SIP vary considerably between the individual species of shellfish (see Figure 3b). Moreover, bands which appear in : .:
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' ' ' , , . ' , , ,. . ' ; . ' : ~, ' .
contaminated shellfish are absent in control samples (these are marked with an arrow). The inventors suggest, for purposes of understanding the invention, although they do not wish to be bound or disadvantaged by any adverse findings, that the change in protein profile as well as the higher level of cross-reactiv-ity in PSP contaminated oysters and clams to the SIP antisera may reflect biochemical changes in the shellfish due to the PSP
intoxication. Moreover, the cross-reactivit:y of these bands in PSP contaminated shellfish with crab anti-SIP provides compelling evidence that they share some degree of structural similarity with crab SIP. Since crab SIP appears to be a protein aggregate, it is the inventors' hypothesis that different PSP induced ; proteins in shellfish are composed of protein subunit(s) which are common to the SIP found in the small shore crab.
At present, saxitoxin can be directly detected using homologus antisera (see procedures in Carlson, R.E., Lever, M.L., Lee, B.W. and Guire, P.E. in Seafood Toxins (ed. E.P. Ragelis) ACS Symposium Series 262, American Chemical Society, Wash. D.C., 181-191 (1984) Davio, S.R. Toxicon 23:669-675 (1985); Chu, E'.S.
and Fan, T.S.L. J. Assoc. vf Anal. Chem. 66:13-16 (1985)).
~owever, these antisera are limited to saxitoxin only, and do not cross-react with other structurally related toxins (eg., neosaxitoxin) that are also associated with PSP. Antiserum directed against antigens in shellfish expressed as a consequence of PSP intoxication represents a novel approach to the problem of PSP detection and has potential value in the rapid screening of shellfish for PSP contamination. The discovery of PSP-induced components in bivalve shellfish provides insights into mechanisms of PSP-resistance, and is the basis for the immuno-diagnostic test for PSP-contaminated shellfish of the invention.
.:
Example ` ~
`i ' :' A discussion of the data and results pictorialized in ~
the Figures and the methods used to obtain the data appears `
below. Figure 1 demonstrates purified SIP in 6.5 percent SDS- `
.~ '' '' _ g _ ~ '~
, PAGE. Left to right, (1) molecular weight standards, (2) purified 145,000 mol. wt. - 75,000 mol. wt. SIP (saxitoxin induced protein), (3) purified SIP tsaxitoxin induced protein, 145,000 mol. wt. - 75,000 mol. wt.~ 5 min. 100C reducing conditions.
The methods used to obtain this data were as ~ollows.
The SIP was purified using DEAE-Sepharose. Briefly, soluble ~ proteins from homogenized visceral tissue of saxitoxin resistant - 10 H. oreqonesis were ~ractionated by ammonium sulphate precipita-tion and applied to a Sephacryl S-300 gel filtration column (1.5 x 26 cm~ equilibrated with 40 mM phosphate-citrate buffer, pH 5.0 with 170 mM NaCl. ~he large molecular weight fraction was further fractionated by ion exchange chromatography using DEAE-15 Sepharose in 40 mL phosphate citrate buffer, pH 5.0 with 170 mM
NaCl. The SIP was eluted with a linear gradient of 170 mM to 1.7 M NaCl and purified by repeated ethanol fractionation. Purity was assessed using discontinuous polyacrylamide electrophoresis (Laemmli, U.K. Nature 227:680-685 (1970)) followiny visualization by silver staining (Merril, C.R., Goldman, S., Sedman, S.A. and Ebert, M.H. Science 211:1437-1438 (1981)).
;; Figure 2 depicts the reaction of soluble proteins from representative crab (a water injected; saxitoxin injected); clam (b; PSP contaminated; control); oyster (c; PSP contaminated;
control) mussels (d; PSP contaminated; control) in ELISA assay with anti-SIP serum.
- The methods used were as follows. An enzyme-linkèd immunosorbent assay (ELIS~) procedure was developed using diluted crab and shellfish lysates. Homogenates were diluted 1:4 with water and protein concentrations were determined (Bradford, M.M.
Analytical Biochem. 72:248-254 (1976)) and adjusted to ensure a uniform protein concentration in the ELISA. All ELISA's were performed using flat-bottom (96 well, Immulon II DynatechTM) micro-titer plates. Anti-SIP serum was diluted with phosphate buffered saline and 0.2 percent skim milk powder. Dilutions of ... .
" - 10 -,''`' ~ " '.
:. ,. . ,, . . ~ ~ , . . . . . . .
anti-SIP serum of l:lO00 for crab and l:lO0 for bivalve mollusc samples were chosen. The enzyme conjuyate was goat anti-rabbit IgG alkaline phosphatase conjugate, diluted l:lO00 and the substrate was p-nitrophenyl phosphate (Sigma). ELISA plates were incubated at room temperature for l.0 hr. and ~bsorbance values were recorded at 405 nm using a EAR 400 ELISA plate reader (SLT-Lab instruments). Within assay coefficient of variation was ~ g.57+0.38%.
.;
Figure 3 illustrates SDS-PAGE (lO]percent) gels (i) of control (l) and contaminated (2); (clam (a), oyster (b) and mussel (c~ lysates with corresponding immuno-blot (ii~.
Methods employed were as follows. Gels were stained with coomassie Blue R250 or blotted to nitrocellulose by the method of Towbin (Towbin, H., Stahelin, To and Gordon, J. Proc.
Natl. Acad. Sci. U.S.A. 76:435Q-4354 (1979)) at 38 volts, 160 mA, for 4.5 hr using a BioRad Trns Blot Cell. Immuno-blots were dried and incubated for 2 hr in 2% skim milk powder. Anti-SIP
serum was diluted l:100 in phosphate buffered saline containing 0.2% skim milk powder. Goat anti-rabbit IgG alkaline phosphatase was used at a dilution of 1:1000. The blots were washed in buffered saline and 100 mMTris-HCl (pH 7.8), respectively. The substrate solution. AS-MX napthophosphate in 100 mM Tris-HCl buffer containing Fast Red TR salt was added until the bands could be clearly visualized.
~ -Figure 4 illustrates schematically a competitive EhISA
assay for crab 5IP. This assay includes a diagnostic stick 2 which can be used as part of a six component kit and competitive ELISA assay procedure for practising the invention.
.
The Competitive ELISA assay is useful when there is a shortage of purified SIP antigen. It can be inferred that an individual trained in ELIS~ methodologies would choose the Competitive procedure for a homologous assay only. Figure 4 (a--:
:
, ~ ~ `' -' . ' . .. : ': ' '; ; ' . ~ . ' .;
c) illustrates the diagnostic kit and the components that can be used in practising the invention.
As seen in Figure 4, the diagnostic stick is con-structed so that it has an untreated finger gripping portion 4and an area 6 which contains the SIP antigen. The Competitive ELISA kit 10 is composed of SIP diagnostic stick 2, a first Reagent A containing container 12, a second Reagent B contain-ing container 14 and a third Reagent C containing container 16.
Materials Reagent A: TRIS buffered saline (container 12) Reagent B: AntiSIP-alkaline P04 conjugate (con-15tainer 14~;
Reagent C: Chromagen substrate solution, p-nitro-phanyl phosphate (container 16);
Diagnostic stick: SIP coated Procedure Step 1 (Crab extract dilution): In a plastic disposable test tube 18, add an aliquot of crab extract and ; dilute the sample with 1.0 ml of Reagent A from contain~r 12.
Step 2: Transfer 0.5 ml from test tube 18 of step 1 to plastic disposable test tube 20. Introduce 0.5 ml of contents from Reagent B (container 14) and insert the SIP diagnostic stick. Allow to sit and incubate for 10 minutes.
Step 3: Remove the SIP diagnostic stick from test tube 20 and wash the stick with the contents of Reagent A (container 12). Reintroduce the SIP diagnostic stick into test tube 22 and add 1.0 ml of Reagent C (container 16).
., 1 . ' . . .... . . . . . . . ., . . `, . .
. ~ . .. .. . . .
... , ... ~ . . . . ..
.
. i., ` . . ~ .
- Measure quantitatively and qualitatively as follows:
Quantitatively - Measurs by absorbance at 405 nm. Qualitatively - Measure by visual inspection of the colour change.
Preamble: The Direct ELISA methodology for SIP
, detection is the procedure of choice since it can be used for both crab and shellfish SIP detection, respectively. There must be no shortage of purified SIP antigen available to perform the . .
test.
;`, 10 Figure 5 illustrates the components for sample dilution and measurement. The Direct E~IS~ kit ll is composed of anti-SIP immunoglobin coated cuvette 10, a first Reagent A container 30, a second reagent B container 32 and a third Reagent C
container 34. ;
Materials . . I .
;; Reagent A: TRIS buffered saline (container 30);
l 20 Xeagent B: SIP-alkaline phosphatase conjugate ,~ (container 32):
, Reagent C: Chromagen substrate solution; p-; nitrophenyl phosphate.
Procedure ; Step l ~Crab/shellfish extract dilution): In a plastic ! disposable test tube 38, add an aliquot of crab/shellfish extract ;`
and dilute the sample with l.0 ml of Reagent A from container 30.
Step 2: ~ransfer 0.5 ml from the test tube 38 to the , SIP-antiyen coated cuvette lO. Incubate for 10 minutes and decant off the contents of cuvette lO.
Step 3: Add to cuvette lO 0.5 ml of Reagent B SIP -enzyme conjugate 32. Incuba~e for 10 minutes. Decant off the ~: .:
" ,A"
contents of cuvette lO. Wash the cuvette lO with Reagent A
(container 30).
.
Step 4: Add 0.5 ml of Reagent A 30 to cuvette lO and decant off the contents as in step 3.
Step 5: Add l.O ml Reagent C 34 to the cuvette lO and incubate for 30 minutes.
~hen measure quantitatively by absorbance at 405 nm or qualitatively by visual inspection of the colour change of the contents.
As will be apparent to those skilled in the art in the light of the foregoing disclosure, many alterations and modifica-tions are possible in the practice of this invention without departing from the spirit or scope thereof. Accordingly, the scope of the invention is to be construed in accordance with the substance defined by the following claims.
.
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Claims (27)
1. A method for the detection of the presence of paralytic shellfish poison in a sea dwelling crustacean or mollusc species which comprises determining the existence of a high molecular weight saxtoxin induced protein complex in the crustacean or mollusc species after the crustacean or mollusc species is exposed to saxitoxin or tetrodotoxin.
2. A method as claimed in claim 1 wherein the presence of the saxitoxin induced protein is determined by electrophore-sis.
3. A method as claimed in claim 2 wherein mobility of the saxitoxin induced protein complex with mol. wt. 75,000 - 160,000 dalton is determined in a 6.5% polyacrylamide gel.
4. A method as claimed in claim 3 wherein the species is a bivalve mollusc.
5. A method as claimed in claim 3 wherein the species is a crustacean.
6. A method as claimed in claim 4 or 5 wherein the existence of the protein is determined by enzyme immunoassay.
7. An immuno-diagnostic test for determining the presence of paralytic shellfish poison in sea-dwelling crustaceans or molluscs which comprises removing from the crustacean or mollusc a determinative amount of body fluid or tissue and determining the presence of a high molecular weight saxitoxin induced protein complex in the crustacean or mollusc by immunodiagnostic test.
8. A method as claimed in claim 7 wherein the high molecular weight protein has a molecular weight of about 145,000 daltons.
- Page 1 of Claims -
- Page 1 of Claims -
9. A method as claimed in claim 8 wherein the existence of the high molecular weight protein is determined by electropho-resis.
10. A method as claimed in claim 7 wherein the molecular weight of the protein complex is between about 75,000 and 160,000 daltons.
11. A method as claimed in claim 10 wherein the saxitoxin induced protein is purified using DEAE-sepharose.
12. A method as claimed in claim 7 wherein soluble proteins are obtained from the homogenized visceral tissue of a saxitoxin exposed crustacean and are fractionated by ammonium sulphate precipitation and applied to a Sephacryl S-300 gel filtration column, equilibrated with a phosphate-citrate buffer.
13. A method as claimed in claim 12 wherein the molecular weight fraction obtained from fractionation is further fraction-ated by ion exchange chromatography using DEAE-Sepharose in a phosphate-citrate buffer, is eluted with a linear gradient of sodium chloride and is purified by repeated ethanol fractiona-tion.
14. A method as claimed in claim 13 wherein the purity of the product is assessed using discontinuous polyacrylamide electrophoresis followed by silver staining.
15. A method as claimed in claim 7 wherein the presence of the saxitoxin induced protein(s) complex is used to prepare an enzyme-linked immunosorbent assay using diluted shellfish lysates.
16. A method as claimed in claim 15 wherein the presence of saxitoxin induced protein(s) complex in the crustaceans and molluscs is determined by immuno-blotting.
- Page 2 of Claims -
- Page 2 of Claims -
17. A method as claimed in claim 7 wherein the presence of the protein(s) is determined by an antiserum.
18. A method as claimed in claim 7 wherein the mollusc is a bivalve mollusc.
19. A method as claimed in claim 7 wherein the mollusc is a clam.
20. A method as claimed in claim 7 wherein the mollusc is an oyster.
21. A method as claimed in claim 7 wherein the crustacean is a crab.
22. A method as claimed in claim 7 wherein the crustacean is a crab of the species Hemigrapsus oregonoses or hemigrapsus nudes.
23. A method of determining the presence of paralytic shellfish poison in sea-dwelling crustaceans or molluscs which comprises:
(a) placing an aliquot of crustacean or mollusc fluid or tissue in a first container and diluting it with phosphate buffered saline solution;
(b) placing a portion of the step (a) first container solution into a second container together with a saxitoxin or tetrodotoxin induced protein antisera-alkaline phosphate conjugate, and a saxitoxin or tetrodotoxin induced protein diagnostic agent, and incubating the combination for a reasonable period of time;
(c) removing the saxitoxin or tetrodotoxin induced protein diagnostic agent and washing it with phosphate buffered saline solution;
- Page 3 of Claims -(d) introducing the saxitoxin or tetrodotoxin induced protein diagnostic agent into a third container and adding to it a chromagen substrate solution: and (e) measuring the absorbance of the resultant solution at 405 nm.
(a) placing an aliquot of crustacean or mollusc fluid or tissue in a first container and diluting it with phosphate buffered saline solution;
(b) placing a portion of the step (a) first container solution into a second container together with a saxitoxin or tetrodotoxin induced protein antisera-alkaline phosphate conjugate, and a saxitoxin or tetrodotoxin induced protein diagnostic agent, and incubating the combination for a reasonable period of time;
(c) removing the saxitoxin or tetrodotoxin induced protein diagnostic agent and washing it with phosphate buffered saline solution;
- Page 3 of Claims -(d) introducing the saxitoxin or tetrodotoxin induced protein diagnostic agent into a third container and adding to it a chromagen substrate solution: and (e) measuring the absorbance of the resultant solution at 405 nm.
24. A method as claimed in claim 23 wherein the chromagen substrate solution is p-nitrophenyl phosphate.
25. An apparatus for determining the presence of paralytic shellfish poison in sea dwelling crustaceans or molluscs which comprises:
(a) a first container containing phosphate buffered saline solution;
(b) a second container containing saxitoxin or tetrodotoxin induced protein antisera-alkaline phosphate conjugate;
(c) a third container containing chromagen substrate solution, p-nitrophenyl phosphate; and (d) a substrate medium which has thereon a saxitoxin or tetrodotoxin induced protein.
(a) a first container containing phosphate buffered saline solution;
(b) a second container containing saxitoxin or tetrodotoxin induced protein antisera-alkaline phosphate conjugate;
(c) a third container containing chromagen substrate solution, p-nitrophenyl phosphate; and (d) a substrate medium which has thereon a saxitoxin or tetrodotoxin induced protein.
26. A method of determining the presence of paralytic shellfish poison in sea-dwelling crustaceans or molluscs which comprises:
(a) placing in a first container an aliquot of crustacean or mollusc fluid or tissue and diluting it with a phosphate buffered saline solution;
(b) placing a portion of the step (a) first container solution into an anti-saxitoxin or tetrodotoxin induced protein immunoglobin-coated second container and incubating the contents of the second container for a reasonable period of time, and then decanting the contents of the second container;
(c) placing in the second container a saxitoxin or tetrodotoxin induced protein-alkaline phosphate conjugate and incubating the conjugate for a reasonable period of time;
- Page 4 of Claims -(d) decanting the contents of the second container according to step (c) and washing the second container with phosphate buffered saline solution;
(e) adding to the second container a phosphate buffered saline solution and decanting off the contents as in procedure (d);
(f) adding to the second container chromagen substrate solution, p-nitrophenyl phosphate and incubating the contents until a chemical reaction occurs; and (g) reading the absorbance of the incubated solution obtained by procedure (f) at 405 nm.
(a) placing in a first container an aliquot of crustacean or mollusc fluid or tissue and diluting it with a phosphate buffered saline solution;
(b) placing a portion of the step (a) first container solution into an anti-saxitoxin or tetrodotoxin induced protein immunoglobin-coated second container and incubating the contents of the second container for a reasonable period of time, and then decanting the contents of the second container;
(c) placing in the second container a saxitoxin or tetrodotoxin induced protein-alkaline phosphate conjugate and incubating the conjugate for a reasonable period of time;
- Page 4 of Claims -(d) decanting the contents of the second container according to step (c) and washing the second container with phosphate buffered saline solution;
(e) adding to the second container a phosphate buffered saline solution and decanting off the contents as in procedure (d);
(f) adding to the second container chromagen substrate solution, p-nitrophenyl phosphate and incubating the contents until a chemical reaction occurs; and (g) reading the absorbance of the incubated solution obtained by procedure (f) at 405 nm.
27. An apparatus for determining the presence of paralytic shellfish poison in sea-dwelling crustaceans or molluscs which comprises:
(a) a first container containing phosphate buffered saline solution;
(b) a second container containing saxitoxin or tetrodotoxin induced protein-alkaline phosphatase conjugate;
(c) a third container containing chromagen substrate solution, p-nitrophenyl phosphate; and (d) a fourth container coated with anti-saxitoxin or tetrodotoxin induced protein immunoglobin.
- Page 5 of Claims -
(a) a first container containing phosphate buffered saline solution;
(b) a second container containing saxitoxin or tetrodotoxin induced protein-alkaline phosphatase conjugate;
(c) a third container containing chromagen substrate solution, p-nitrophenyl phosphate; and (d) a fourth container coated with anti-saxitoxin or tetrodotoxin induced protein immunoglobin.
- Page 5 of Claims -
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CA 2007696 CA2007696A1 (en) | 1990-01-12 | 1990-01-12 | Method and apparatus for the detection of paralytic shellfish poisons |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CA 2007696 CA2007696A1 (en) | 1990-01-12 | 1990-01-12 | Method and apparatus for the detection of paralytic shellfish poisons |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2007696A1 true CA2007696A1 (en) | 1991-07-12 |
Family
ID=4144010
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA 2007696 Abandoned CA2007696A1 (en) | 1990-01-12 | 1990-01-12 | Method and apparatus for the detection of paralytic shellfish poisons |
Country Status (1)
| Country | Link |
|---|---|
| CA (1) | CA2007696A1 (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0857972A1 (en) * | 1997-01-24 | 1998-08-12 | Tepual, S.A. | Immunoassay for the detection and quantitation of toxins causing paralytic shellfish poisoning |
| ES2155396A1 (en) * | 1999-06-04 | 2001-05-01 | Consejo Superior Investigacion | Identification of PSP toxins involves using mass spectrometry with nanospray ionization |
| CN105259292A (en) * | 2015-11-12 | 2016-01-20 | 上海市农业科学院 | Method for measuring paralysis shellfish poison in aquatic products |
-
1990
- 1990-01-12 CA CA 2007696 patent/CA2007696A1/en not_active Abandoned
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0857972A1 (en) * | 1997-01-24 | 1998-08-12 | Tepual, S.A. | Immunoassay for the detection and quantitation of toxins causing paralytic shellfish poisoning |
| ES2155396A1 (en) * | 1999-06-04 | 2001-05-01 | Consejo Superior Investigacion | Identification of PSP toxins involves using mass spectrometry with nanospray ionization |
| CN105259292A (en) * | 2015-11-12 | 2016-01-20 | 上海市农业科学院 | Method for measuring paralysis shellfish poison in aquatic products |
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