Patent Application: US-45035103-A

Abstract:
a method of detecting and / or measuring the amount of a paralytic shellfish toxin present in a sample , comprising the steps of : 1 ) providing an isolated and purified saxiphilin , or fragment thereof which contains a saxitoxin binding site ; 2 ) contacting it with the sample ; 3 ) mearsuring binding of pst contained in the sample to said isolated and purified saxiphilin ; and correlating the amount of binding with either the presence or absence of psts in the sample or with the pst concentration in the sample .

Description:
the invention will now be described in detail by way of reference only to the following non - limiting examples and drawings . crude saxiphilin was obtained by homogenising specimens of the centipede ethmostigmus rubripes in 10 mm tris - hcl , 0 . 2 mm edta ( ph 7 . 4 ) ( 2 × 10 sec bursts with a waring blender at maximum setting ; 3 ml buffer : 1 g centipede ) containing a cocktail of protease inhibitors ( 5 mm edta , 1 μm pepstatin , 1 μm aprotonin , 100 μm phenylmethylsulfonyl fluoride ). after centrifuging the homogenate at 24 , 000 g for 20 min , the pellet was rehomogenised and centrifuged as above . the two supernatants were combined and passed through a 0 . 2 μm cellulose acetate filter ( nalgene ). the saxiphilin was then precipitated from this supernatant by exposure to 40 - 60 % ammonium sulphate , followed by removal of non - saxiphilin molecules from this precipate by redissolving into a buffered solution of ph 5 . 0 - 6 . 5 and centrifuging to leave a supernatant containing saxiphilin . this supernatant was then exposed to a glass fibre - polyethylene imine ( pei ) support matrix , prepared by incubation of glass fibre with 0 . 3 % pei in water solution ( v / v ) for at least 1 hour and removal of the pei by draining or aspiration under vacuum . saxiphilin was eluted from the matrix using high salt , with the saxiphilin typically being eluted by nacl or kcl at a concentration from 600 mm to saturation , at ph 5 - 9 . the protein has also been subjected to chromatofocussing on pbe 94 resin equilibrated with 25 mm imidazole - hcl ph 7 . 4 and eluting with a solution containing 25 ml polybuffer 74 , brought to a final volume of 200 ml and a ph of 4 . 0 with hcl . polybuffer removal and buffer exchange can then be achieved by size exclusion chromatography or desalting columns , such as pd - 10 columns from amersham pharmacia biotech . one diagnostic kit for qualitative detection of psts using the purified saxiphilin of the invention is in the form of a test strip . the kit uses a solid matrix , or “ wick ”, upon which the reaction occurs . the kit has a band of saxitoxin at one end of this solid matrix , applied using a method known as “ printing ”. this immobilises the saxitoxin , which then acts as an anchor for modified saxiphilin ( described below ) as it flows past the “ printed ” saxitoxin . if the modified saxiphilin is already bound to a pst from a test sample , then it will be unable to bind to “ printed ” saxitoxin , and will continue to flow , preventing colour development . if the test sample has no psts , then the modified saxiphilin will bind to the band of “ printed ” saxitoxin , forming a coloured spot . to generate a coloured spot upon anchorage of saxiphilin , saxiphilin is conjugated to colloidal gold or coloured latex microbeads . the principle is that the colloidal gold , an intensely coloured reagent , is aggregated into a spot obvious to the human eye when the conjugated saxiphilin binds the stx immobilised on to the membrane . as it flows past the band of printed saxitoxin , it will stop and aggregate , or continue and not form a band visible to the human eye . this is illustrated schematically in fig1 . thus this form of assay provides a qualitative “ yes / no ” assay for the presence of psts in a sample . the test strip provides a positive control . an alternative approach is to use a liposome encapsulated - dye . liposomes provide instantaneous enhancement , and have considerable potential for automated assays . the experimental system is a competitive receptor assay and consists of a wicking reagent containing saxitoxin / biotin - tagged liposomes with entrapped dye and a plastic - backed nitrocellulose strip that has an immobilized saxiphilin competition zone and a liposome avidin capture zone in an ascending sequence ( fig2 ). a mixture of the wicking reagent and a sample containing an unknown quantity of psts is allowed to migrate along the strip by capillary action . in the saxiphilin zone , competitive binding with the psts receptor occurs . the unbound liposomes , proportional to the amount of saxitoxin in the sample , are carried into the liposome capture zone where they are concentrated . the color intensity of the saxiphilin zone and the avidin zone are estimated either visually or by scanning densitometry . the amount of immobilized saxiphilin must be as low as possible to increase sensitivity to psts , but sufficient to allow visual detection of liposomes . a typical migration assay requires approximately 100 μl sample solution and should reach the ppb detection level in less than 10 minutes , corresponding to psts detection limits in the low ng range . liposomes are highly stable molecules that can be stored at least one year at + 4 ° c . and several months at room temperature . this assay would be easily used in to field testing , without any special equipment or technical skills required . the kit would include special holders for the individual strips , in which openings are provided for sample application and optical readout . this low cost saxiphilin migration sensor allows easy and rapid screening of environmental samples and constitute an unparalleled and reliable tool for the psts risk assessment . establishing the assay in a microtitre plate format allows its more sophisticated use , and enables quantitative results to be obtained . one preferred format is a binding inhibition asay . saxitoxin is coated on a 96 - well microtitre plate . test samples are mixed with labelled saxiphilin and added to the 96 well plate . toxin - free samples do not prevent the labelled saxiphilin from binding to the saxitoxin - coated surface of the wells of the 96 well plate , forming a coloured region . toxin - containing samples inhibit colour formation with the degree of inhibition being proportional to the amount of toxin present . the plate is then read in a spectrophotometic plate reader and the amount of toxin quantified . a further possible technique for quantification of psts involves high - performance liquid chromatography coupled to a post - column oxidation system and a fluorescence detector . this procedure is complex and requires expensive psts standards . however , the combination of highly specific saxiphilin - based identification and sensitive detection by means of surface plasmon resonance ( spr ), overcomes the drawbacks related to chromatographic techniques . the device requires a pst such as saxitoxin to be coupled to an activated gold surface , which is exposed to a liquid sample during the analysis . the spr sensor detects changes in the reflection of laser light caused by the change of refractive index at the metal - liquid interface . thus , when saxitoxin is coupled to the activated gold surface of the sensor , a refractive index alteration is induced by saxiphilin binding ( fig4 ). in the case where psts are present in the sample , a competition occurs between the free psts and bound saxitoxin , and the resulting signal decrease can be quantified . this flow - injection receptor assay consists of i ) reagent pumping and sample injection systems , ii ) a mixing cell where the competitive receptor assay occurs and iii ) the spr sensor including a flow cell and optical devices ( fig5 ). this system can be fully automated , such as in the biacore 2000 ™ available from biacore ab . by linking saxiphilin to a solid phase , its ability to bind saxitoxin may be used to separate saxitoxin from liquid samples passed over the saxiphilin linked solid support . since binding to centipede saxiphilin is ph dependent , the bound toxin can then be eluted . isolated saxiphilin was used to prepare an affinity column using the ultralink ™ tm kit manufactured by pierce chemical company . this resin relies upon azolactone coupling chemistry and uses an inert semi - rigid resin with medium to fast flow characteristics . 1 . the ammonium sulphate precipitated saxiphilin was resuspended into the pierce supplied coupling buffer of buph citrate - carbonate buffer ( ph 9 .). 2 . this resuspended saxiphilin was added to 0 . 15 g of 3m emphaze biosupport medium ab 1 ( supplied by pierce ) which hydrates the resin and binds available proteins . the resin swelled to 1 ml . 3 . after 1 hour of gentle mixing , the resin and saxiphilin preparation was packed into a mini - column , and the resin was allowed to settle 4 . the column was then washed with phosphate buffered saline ( 15 mls ) 5 . 4 mls of quench buffer ( 3m ethanolamine ph 9 . 0 ) was then added and the resin gently mixed in this buffer for 2 . 5 hours 6 . the column was then washed with 15 ml phosphate buffered saline 7 . the top of the resin was then sealed with a porous disc insert 9 . wash the column with 15 ml 100 mm hepes - naoh ( ph 7 . 4 ) and it is ready for testing for ability to bind saxitoxin tritiated saxitoxin ( amersham pharmacia biotech ) was used to measure the columns ability to bind saxitoxin . three 2 μl aliquot of the 3 h - stx ( 60 nm ) was counted in a scintillation counter to measure how much radioactivity was going to be applied to the column . these aliquots contained 2113 ± 42 counts per minute ( cpm ). 200 μl of 3 h - stx (= 211 , 300 cpm — see point above ) was added to the column and allowed to flow into the resin . the 3 h - stx is prepared by 150 - fold dilution of the commercially supplied 3 h - stx ( in 0 . 01 m acteic acid containing 2 % ethanol ) into 1 mm citrate buffer ( ph 5 . 0 ). the column was then washed with 5 ml 100 mm hepes - naoh ( ph 7 . 4 ). the flow through was collected . the column was then washed successively with 5 ml of the following with each sample collected separately : as can be seen , there are two major peaks of radioactivity . the first elutes in the first fraction and is essentially unbound by the column . the second peak is eluted by 100 mm hepes - naoh ph 5 . 0 . tritiated saxitoxin contains free tritium and so these two peaks were tested for biological activity by measuring their ability to bind to the two known receptors for saxitoxin , namely the sodium channel and saxiphilin . sodium channel : 100 mm mops - naoh ( ph 7 . 4 ), 100 mm choline chloride , 100 μl of fractions 1 and 5 ( wash and first ph 7 . 4 wash , ph 5 . 0 wash respectively ), 10 μl rat brain vesicles containing sodium channels in a final volume of 250 μl . samples were done in duplicate and a negative control containing an excessive amount of unlabelled tetrodotoxin was also performed to define specific levels of any bound 3 h - stx . saxiphilin : 100 mm mops - naoh ( ph 7 . 4 ), 100 mm sodium chloride , 100 μl of fractions 1 and 5 ( wash and first ph 7 . 4 wash , ph 5 . 0 wash respectively ), 1 . 5 μl characterised centipede saxiphilin preparation in a final volume of 250 μl . samples were done in duplicate and a negative control containing an excessive amount of unlabelled saxitoxin was also performed to define specific levels of any bound 3 h - stx . as can be seen in fig7 only the ph 5 . 0 eluted peak of radioactivity from the column retained biological activity ie it could bind to the two known saxitoxin receptors . the ph 7 . 4 peak did not contain any such biological activity ( except for a minimal amount of receptor binding activity in the saxiphilin assay ) indicating that the radioactivity was tritium unincorporated into saxitoxin ( a known property of commercially supplied 3 h - stx ). after the final 0 . 5n hcl wash , the column was washed with 20 ml 100 mm hepes - naoh ( ph 7 . 4 ) and stored overnight at 4 ° c . this column was removed and allowed to return to room temperature and the above elution experiment was repeated and the profile shown in fig8 was obtained . as can be seen , the second peak of activity has shifted to be eluted by the 3rd wash with hepes - naoh ( ph 7 . 4 ) which may indicate degradation of the saxiphilin . these peaks were not tested for biological activity . thus it will be appreciated that saxiphilin can be bound to a solid phase chromatography resin such as pierce &# 39 ; s emphaze biosupport medium ab 1 . on this column saxitoxin is bound by the saxiphilin and separated from other material ( eg free tritium ), then the saxitoxin can be eluted from the column with ph 5 . 0 . the eluted saxitoxin retains biological activity . treatment with acid ( eg 0 . 5 n hcl ) may degrade the linked saxiphilin . non - specific retention of the 3 h - stx by the treated resin is not apparent . it will be apparent to the person skilled in the art that while the invention has been described in some detail for the purposes of clarity and understanding , various modifications and alterations to the embodiments and methods described herein may be made without departing from the scope of the inventive concept disclosed in this specification . references cited herein are listed on the following pages , and are incorporated herein by this reference . anderson d . m . 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