a competitive enzyme-linked immunoassay for domoic acid determination in human body fluids

~ Pergamon 0278-6915(94)00100-6

FdChem. Toxic'. Vol. 32. No. 12, pp. 1147-1154. 1994 Copyright ~ 1994 Elsevier Science Ltd

Printed in Great Britain, All rights reserved 0278-6915.94 $7 00 + 0.00

A COMPETITIVE E N Z Y M E - L I N K E D IMMUNOASSAY FOR DOMOI C ACID D E T E R M I N A T I O N IN H U M A N BODY

FLUIDS

D, S. SMITH and D. D. Ktr-rs*

Department of Food Sciences. University of British Columbia. 6650 Northwest Marine Dr. Vancouver. BC, Canada V6T I ZA

(Accepted 16 June 1994)

Abstract--A polyclonal antiserum was raised in mice against domoic acid. Two of three immunogens consisted of domoic acid coupled to ovalbumin (OVA) and keyhole limpet haemocyanin at molar ratios of 47:1 and 44:1, respectively using a carbodiimide reaction. Titres of both antisera exceeded 1/35,000 against domoic acid coupled to the non-relevant carrier. Domoic acid was also conjugated to bovine serum albumin at a molar ratio of 30:1 using N-hydroxysuccinimidyl-4-azidobcnzoate, a photoreactive compound. This immunogen, however, produced no measurable serum titres against domoic acid. The antiserum produced against the OVA conjugate displayed the highest affinity fur free domoic acid in competitive enzyme-linked immunosorbent assay (ELISA). Furthermore, this antiserum preparation did not significantly cross-react with glutamic acid, aspartic acid, the structural analogue kainic acid, or the paralytic shellfish toxin, saxitoxin. The competitive ELISA was used to quantify domoic acid concentrations in human body fluids spiked with pure domoate. The lower limits of accurate domoic acid determinations in competitive ELISA were 0.2 ,ug/ml in urine, 0.25 ~ug/ml in plasma and 10 ~ug/ml in milk. It was concluded that the competitive ELISA described herein could be used to quantitate directly the concentration of domoic acid in the body fluids of individuals with amnesic shellfish poisoning.

INTRODUCTION

Domoic acid (Fig. In) is a naturally occurring neuro- toxin that is produced by at least two species of red algae, Chondria arntata (Takemoto and Daigo, 1958) and Als id ium coral l ium (lmpellizzeria et al., 1975) as well as one species of marine diatom, Ni t : sch ia pungens (Subba Rao et al., 1988). Domoic acid commonly contaminates edible shellfish, and on ingestion by humans, causes an intoxication syndrome known as amnesic shellfish poisoning (ASP).

Like kainic acid (Fig. Ib), domoic acid is an excitatory amino acid and a potent agonist of gluta- matergic kainate receptors in the dorsal hippocampus of the brain (Debonnel et al., 1989 and 1990). The ASP syndrome is characterized by gastro-intcstinal symptoms such as vomiting, cramps and diarrhoea, and by neurological symptoms, characterized by severe headache, seizures, and either temporary or

*To whom correspondence should be addressed. Abbreviations : ASP = amnesic shellfish poisoning;

BSA = bovine serum albumin; DMSO = dimethyl sulfoxide; EDC = I-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride: ELISA = enzyme-linked immunosorbent assay; fc = final concentration; GAMIG-AP = goat anti-mouse immunoglobulin G-alkaline phosphatase conjugate; HSAB -- N-hydroxysuccinimidyl-4-azidobenzoate; IA = immunoassay; KLH = keyhole limpet haemocyanin; OVA = ovalbumin; PNPP --p-nitrophenyl phosphate; RIA = radio- immunoassay; UB = universal buffer.

permanent memory loss (Perl et al., 1990; Teitelbaum et al., 1990; Tryphonas et al., 1990a). The 1987 outbreak of domoic acid in mussels in eastern Canada (Wright et al., 1989) resulted in 107 confirmed cases of ASP, of which there were 12 cases of permanent memory loss and four fatalities (Teitel- baum et aL, 1990). Neuropathological studies of the patients who died revealed neuronal necrosis and loss, particularly in the hippocampal region (Teitel- baum et al., 1990). Cellular degeneration in the hippocampus as well as anterograde amnesia was also observed in rats injected with domoic acid (Suther- land et al., 1990).

In 1987, the onset of gastro-intestinal symptoms in the ASP patients occurred within 24 hr of ingestion of toxic shellfish, while neurological symptoms began within 48 hr (Perl et al., 1990). The contaminated shellfish had to be traced, sampled and extensively analysed in order to confirm domoic acid intoxication as the cause of the symptoms (Wright et al., 1989). Although toxic shellfish poisonings in humans are rare in areas where extensive monitoring programmes are in place, the presence of domoic acid in regions with no previous record of outbreaks represents a potential for human ASP, given the conditions con- ducive to domoic acid accumulation in shellfish (Dickey et aL, 1992). In the event of other ASP outbreaks, direct measurement of domoic acid in the body fluids of affected individuals would aid in the diagnosis of ASP and provide useful information

1147

1148 D S. SMITH and D. D. KtTTS

/

CH3 COOH

CH3 /

/ COOH

" ' ~ COOH H

CH3 /

H2C ' .~ / COOH

COOH H

a . b .

Fig. I. Molecular structures of (a) domoic acid and (b) kainic acid.

regarding the toxicokinetics of this particular neuro- toxin.

Immunological methods offer a simple, sensitive and economical procedure for quantifying both large and small molecular compounds. Furthermore, because they can be adapted to formats that facilitate the rapid screening of many samples, specific anti- bodies are used in the diagnosis of a wide variety of physiological disorders, infectious diseases and in- toxications (Deshpande and Sharma, 1993), This work describes the successful production of a domoic acid immt.nogcn and the development of a competitive enzyme-linked immunosorbcnt assay (ELISA) for the accurate determination of domoic acid concentrations in human plasma, urine and milk.

MATERIAL~ AND METIIOI),~

Reagents

Domoic acid used as a standard in the competitive ELISAs was the domoie acid calibration solution (DACS-I) kindly supplied by Dr M. A. Quilliam, NRC, Halifax. Domoic acid used for sample spiking and antisera production, aspartic acid, glutamic acid, adjuvant, Na,HPO4, and p-nitrophenyl phosphate (PNPP), were all purchased from the Sigma Chemical Co. (St Louis, MO, USA). Ovalbumin (OVA), keyhole limpet haemocyanin (KLH), I-ethyl-3-(3- dimethylaminopropyl) carbodiimide hydrochloride (EDC) and conjugation and purification buffers were supplied as a kit by the Pierce Chemical Co. (Rock- ford, IL, USA). N-Hydroxysuccinimidyl-4-azidobenzoate (HSAB), was also purchased from Pierce. Kainic acid, dimethyl sulfoxide (DMSO) and bovine serum albumin (BSA) were obtained from ICN (Montreal. Province of Quebec, Canada). Tris and Tween-20 were purchased from Bio-Rad Labora- tories (Mississauga, Ontario, Canada), and NaCI, MgCI:, NaN~ and diethanolamine were obtained from BDH (Vancouver, British Columbia, Canada). The skim milk powder was manufactured by Carnation (Toronto, Ontario, Canada) and the goat anti-mouse IgG-alkaline phosphatase conjugate

(GAMIG-AP) was purchased from Bio/Can (Missis- sauga, Ontario, Canada). Saxitoxin was the generous gift of Dr Gilchrist, Food and Drug Administration, Cincinnati, OH, USA.

Human fluids

Human urine and milk were each provided by non-medicated, healthy female donors. Plasma was obtained from blood drawn from three healthy male donors and pooled.

Domoic acid conjugation

Domoic acid was conjugated to OVA and KLtl by method I, while conjugation of domoic acid to BSA was achieved using method 2.

Method I. Approximately 2mg KLH and OVA were each dissolved in 200;tl deionized water. Do- moic acid (I mg in 520#1 conjugation buffer) was added to each of the protein solutions and gently mixed. To the vial containing the KLH, 50/~1 of an EDC stock solution (10 mg/ml) was added. The entire contents of the vial containing the OVA, however, were added directly to a second vial containing 10mg EDC. Both preparations were incubated for 2hr at room temperature. Precipitates were removed from both conjugates by centrifugation (800g, 10 min) and free domoic acid was separated using a desalting column (Pierce) equilibrated with conjugation buffer. Protein-containing fractions of both conjugates were concentrated to about 0.5 ml with Centricon-30 ultrafiltration devices (Amicon, Beverly, MA, USA).

Method 2. To a 1 ml solution of BSA (250 ltg, ml), 501~1 HSAB (401tg/ml DMSOI was added and the mixture was incubated for 2 hr in the dark at room temperature. Precipitates were removed by centrifugation (800g, 10 rain)and the HSAB-activated BSA was separated from free HSAB using a desalting column equilibrated with 10ram Na:HPO4, pH7.4. The protein-containing fractions were pooled and mixed in a quartz cuvette with 500 fig domoic acid in 0.5ml 10ram Na:HPO4 pH7.4. The cuvctte was irradiated with long-wave ultraviolet light for 50 scc

ELISA for domoic acid in human fluids 1149

at 9cm from the source. Free domoic acid was remo~,ed using a centricon-30 ultrafiltration device.

To ensure complete removal of free domoic acid. all retentates were re-run through a desalting column equilibrated with purification buffer. Protein-containing fractions were pooled, filter sterilized, aliquotted into sterile vials, and stored at 4 C. These domoic acid-protein conjugates are referred to as the OVA-DA. the KLH-DA, and the BSA-DA. Protein concentrations of all conjugates were determined by the method of Bradford (1976).

Free domoic acid concentrations were determined by measuring absorbance at 242 nm using a Shi- madzu model UV-160 spectrophotometer. Conju- gation efficiency was determined by comparing the relative amounts of free domoic acid before and aftcr conjugation.

Antisera production

Male Balb/C mice were each given one injection with one of the conjugates mixed I : I with Freund's incomplete adjuvant followed by repeated ip injections, spaced 10-14 days apart, containing no adjuvant. Tail bleeds were performed to obtain a preliminary measurement of titre, and serum was collected from the heart after the animals had becn humanely killed. The mice injected with the KLtl DA and OVA DA conjt,gates were killed after four and live ip injections, respectively. The scra obtained are referred to herein as the anti-OVA-DA, the anti-KLll -DA and the anti-BSA-DA.

Determination t~" titre

Coming (Coming, NY, USA) or Linbro (Flow, ICN, Montreal, Province of Quebec, Canada) immunoassay (IA) plates were coated with domoic acid conjugate or carrier protein alone (I,ug/ml) in 100 mM NaFICO3 pH 9.6 and incubated overnight at 4 C. Plates were blocked with a l-hr incubation at 37 C in 200/~1 universal buffer (UB) well containing 10 mM Tris-HCI pH 8.0, 250 mM NaCI, 0.05% NAN,, I mM MgCI., and 0.7% Tween-20 and supplemented with 5% skim milk powder. Anti-OVA-DA, anti- KLH-DA and anti-BSA-DA were diluted between

I 500 and I 40,000 in UB supplemented with I°o skim milk powder. The anti-BSA-DA serum was cross-absorbed with 100/tg BSAml in UB and I% skim milk powder for 45 min prior to testing it against the BSA-DA and BSA alone. A non-relevant mouse serum, also diluted 1,1000 in UB with I% skim milk powder, was used as a blank. The diluted sera were addcd to the plates at 1001~1 well and incubated at 37 C for 2 hr. Following a wash in UB, G A M I G - A P diluted 1/3000 in UB with I% skim milk powder was added (100/tl, well) and incubated at 37 C for I hr. PNPP (0.5mg/ml) in 100mM diethanolamine pH 10.0 was added (100/~l/well) after a thorough washing and incubated at 37 C for 30-60min. Absorbances were measured at 405 nm with a reference wavelength of 655 nm using a Model 450 microplate reader (Bio-Rad. Mississauga. On- tario, Canada). The serum titre was calculated as the dilution of antiserum to yield an absorbance of 0.05 in ELISA after a 1-hr incubation in PNPP.

Competitit'e ELISA

All incubation steps were carried out at 37 C and plates wcre washed with U13 between every stcp. Anti-OVA -DA serum was diluted 1/2000 in UB plus I% skim milk powder and inclt, dcd in the assay as a positive control and a =ncasurc of maximum absorbancc (i.e. 0% inhibition). Pure domoic acid standards were diluted appropriately into this preparation and analysed in triplicate. Concentrations of the domoic acid standards ranged from 0.2 to 100 ng/ml. Test samples were prepared as described below and were also analysed in triplicate.

Falcon (Bccton Dickson, Lincoln Park, N J, USA) or Coming IA plates were coated with 0.5/~g/ml KLII -DA for 2 hr. Plates wcre blockcd with 5% skim milk powder in UB without Twecn-20 (200/~1/wc11) for 30 min. Standard and test sample preparations wcrc applied to the plate at 100 ill/well and incubated for 2 hr. G A M I G - A P and PPNP were subsequently addcd, and absorbances were mcasurcd its dcscribcd in the titre determination experiment. A sample con- raining a non-relevant mouse serum instead of the anti-OVA-DA serum with no domoic acid served as

Table I. Conjugat ion efficiency o f domoic acid (DA) to o' .a lhumin (OVA). kcyh,.fle limpet hacmocyanin (K Li t ) and bovine serum albumin (BSA). and titr¢ cstimatu)n tff the anti-dome, it acid sera again..t the varut~.us

conjugates and carrier pr,.:.tcins in ¢n.,yme-linked immunos,:~rbcnt a..say (ELISA)

Text ant iserum" Conjugath 'm ¢tficiency . . . . . . . . . . . .

Antigen (molar ratio c a r r i e r : D A ) ant i -OVA DA anti-Kl+l! DA anti-13gA DA

O V A DA 1:47 - - * " I 35,04X) <~ I 500+" OVA~ I :0 - - <l 1 10IX) - - K L H DA 1:44 I 50.0(X) - - q~ I 500 KLH,* I : 0 <I I, 10(X) - - - -

BSA+ DA I : 30 < I 500 .,t I 500 ~ I 5t)4~ BSA, + I :0 - - - - << I 5o+~

"Serum tutrc was estimated by the maximum serum ddutwm that allov.cd an ah,,orhancc o f greater than 0.050 in the EL/SA.

' ' N o t te..ted 't',,t. indicates no ahsorbance generated in the E L I S A The dilutH',n indicated is the Iov..csl dilution of

ant iserum lesled. .*Unconjugated carrier pro te in ,~Th¢ ant=-BSA DA '.a.as cross-absorbed v, ith 104+)pg BSA m l

F('T t2 12 O

1150 D.S. SMITH and D. D KITTS

a blank (,4~ --- 0.000). Inhibition was expressed as a percentage (%1) and calculated from blanked absorbance values as follows: %1 = 100[1--(A,/A,.)], where A, is the absorbance of the test sample and ,4, is the absorbance generated by the anti-OVA-DA (I/20003 preparation ~'ithout added domoic acid (i.e. the positive control).

Determination of cross-reactirity

The cross-reactivity of the anti-OVA-DA serum was tested in the competitive ELISA as described above. Anti-OVA-DA was diluted 1/2000 in UB with I% skim milk powder. Kainic acid, aspartic acid and glutamic acid were added to 500-#1 volumes of the anti-OVA-DA preparation at a final concentration (fc) of 50~ug/ml. Saxitoxin and domoic acid were added at 1.0pg/ml (fc) and 5.0 ng/ml (fc), respectively. Absorbances (405 nm) obtained with the test compounds were directly compared with that generated by the positive control (anti-OVA-DA alone) using Student's t-test for paired observations.

Preparation of test samplt's

Test samples consisted of human urine, plasma and milk spiked with pure domoic acid at known concentrations. All test samples were diluted into a preparation of the anti-OVA-DA serum. This preparation consisted of the crude anti-OVA-DA serum diluted 1/2000 into UB with I% skim milk powder. This concentration of antibody was used consistently throughout the study. In the first experiment, samples of all three fluids were spiked with 10#g domoic acid/ml. Samples were diluted 1/50, 1/100, 1/500 and 1/1000. In the second experiment, biological fluids were spiked with domoic acid at concentrations rang- ing between 0.2 and 1011g/ml (urine), 0.25 and

100

75 ~ ~ J

, . 54.2 II|/ml ~ / " 117 IIII/ml

0 ~ tO0 200

D o m o i c A c i d ( n g / m l )

Fig. 2. Performance of ( • ) antiserum to ovalbumin domoic acid conjugate (anti-OVA DA serum) (I/2000) (r = 0.997), and (A) antiserum to keyhole limpet haemocyanin domoic acid conjugate (anti-KLH-DA serum) (1/25003 (r = 0.996) in competitive ELISA for domoic acid. Arrows indicate the concentration of domoic acid required (anti- OVA DA =54.2ngmkanti-KLH DA=217ngml) to re- duce the absorbance generated by the sera alone by 50%. Curves represent the least squares regression of y on log x, and error bars represent the standard deviation around the

mean of triplicate measurements.

Table 2. Cross-reacti,,ity of the antiserum to o~,albumin-domoic acid conjugate (anti-OVA-DA ,,erum) with '~arlous compounds m com-

petitive enzyme-linked immunosorbent assay IELISA)

Reduction Test Concentration absorbance (405 nm) °

compound (pg ml) (mean _+ SD) P'~f

Domoic acid 0.005 0.038 _+ 0.004 <0.005~ Kainic acid 50.0 0.000 _ 0004 >0.5§ Aspartic acid 50.0 <0.001 ± 0.02,1 >0.5~ Glutamic acid 500 <0.003 _+ 0.011 >0.5~ Saxitoxin 1.00 0.004 -+ 0.009 >0.5§

*Reduction in absorbances (405nm) generated in competitive ELISA by the test compound from the control (anti-OVA--DA alone). Values are means _+ standard des'iation (n = 31.

fProbability (P). (as determined by Student's t-test for paired observations) that the observed reduction in absorbance (405 nm) is due to random variation.

~Significant reduction in absorbance from control. §No significant reduction in absorbance from control.

1011g/ml (plasma), and I and 100/lg/ml (milk) to evaluate the accuracy of the competitive ELISA. Spiked urine was prepared for the competitive ELISA by diluting samples 1/100. Plasma samples were diluted 1/250 and 1/500. Milk samples spiked with domoic acid were cooled on ice and centrifuged at 12,000g for 15min at 2'C. The aqueous layer between the pellet and the fat plug was collected from each sample and diluted 1/500 and I/I000 in preparation for the competitive ELISA.

R ES L' I .TS

Domoic acid was coupled to OVA, KLH and BSA at molar ratios of 47:1, 44:1 and 30:1, respectively (Table I). Mice injected with the conjugates displayed no ill-effects or symptoms typical of ASP. The titre of the anti-KLH-DA against the OVA-DA conjugate was 1/35,000, whereas the titre of the anti-OVA-DA serum against the KLH-DA conjugate was a little higher, approximately 1/50,000 (Table I). Neither the anti-OVA-DA nor the anti-KLH-DA sera displayed any reaction with their heterologous unconjugated carrier proteins, KLH and OVA respectively (Table I). This indicated a high concentration of antibody in the anti-OVA-DA and anti-KLH-DA sera specific for domoic acid. Although domoic acid was success- fully coupled to BSA using HSAB, the BSA-DA conjugate failed to produce titres against either KLH-DA or OVA-DA (Table I). Cross-absorbing the anti-BSA-DA serum with BSA neutralized the anti-BSA activity in the serum (Table 1). The cross-

30o absorbed anti-BSA-DA serum also displayed no

Table 3. Recovery of domoic acid (DA) from human fluid samples spiked with 10p8 D A m l as determined by competitive enzyme-

linked immunosorbent assay (ELISA}

DA concentrations in biological fluids Lug ml) °

Dilution Urine Plasma Milk

I 50 1100 I 500 I 1000

8 4 *_ 0.8 (84) IZ.9 ± 0.6 (IZ9) 26 8 + 0.2 (Z6X) 10.8±1.1 (10g) 13.0_1.1 (130) 218 ~ 2.6 (218} I I . 0 ± 1 . 1 (110) 9.45____0.7(94.5) 1 5 8 + 2 . 6 ( t 5 8 ) 10.8+0.9(108) 9.58__+0.8{95.9) 169_+0.9(169)

°Values are means _+ SD. v, ith percentage recover) in parentheses.


| 0 I

60

0

2 O

J O ~

0 20 40 6 0 | 0 1 0 0

D o m o i c A c i d ( n l l / m l )

Fig. 3. Domoic acid determination in human urine diluted 1/100 (A) compared with a pure domoic acid standard (0 ) in competitive enzyme-linked immunosorbent assay. Error bars represent the standard deviation around the mean. Curves represent the least squares regression of) ' on log x.

significant activity against B S A - D A (Table I), thus indicating a lack of measurable anti-domoic acid activity in this serum.

The an t i -OVA-DA and the a n t i - K L H - D A sera were compared in the competit ive ELISA. Free domoic acid logarithmically inhibited absorbance generated with both the an t i -OVA-DA (r = 0.997) and the a n t i - K L H - D A (r = 0.996) sera (Fig. 2). The concentration of free domoic acid required for 50% inhibition with an t i -OVA-DA was 54.2 ng/ml, whereas 217 ng/ml domoic acid was required with the anti- K L i I - D A (Fig. 2). This indicated that the anti- O V A - D A serum had higher affinity for free domoic acid than the a n t i - K L H - D A serum, and it was concluded that the an t i -OVA-DA serum would therefore provide the more sensitive assay for domoic acid.

The specificity and cross-reactivity of the anti- O V A - D A serum was then determined by measuring the reduction in absorbance generated by free domoic acid and other compounds in the competitive ELISA.

Domoic acid (5.0 ng/ml) significantly (P < 0.005) re- duced the absorbance from that of the control, which contained only an t i -OVA-DA serum diluted 12000 in UB with I% skim milk powder (Table 2). Kainic acid (Fig. Ib; 50pg, ml), a compound structurally related to domoic acid (Fig. la: Debonnel e t al . ,

1989), had no significant effect (P > 0.5) on absorbance, even at a concentration 10,000 times greater than the level of domoie acid tested (Table 2). Similarly, the addition of aspartie acid (50,ug/ml), glutamic acid (50/tg/ml) or the paralytic shellfish toxin, saxitoxin ( l .0pg /ml ) , displayed no significant (P > 0.5) cross-reactivity with the an t i -OVA-DA serum in competitive ELISA (Table 2).

In testing the ability of the competitive ELISA to measure domoic acid in human fluids, a preliminary experiment was performed in which human urine, plasma and milk were each spiked with [0 pg domoic acid/ml. These spiked samples were diluted 1/50, 1/100, 1/500 and 1/1000 and analysed for domoic acid in the competit ive EL[SA with pure domoic acid used as a standard. A dilution of 1/100 was necessary for an accurate determination of domoic acid in urine (Table 3). In plasma, domoic acid determinations were overestimated at dilutions of 1/50 and 1/100, but accurately determined at a dilution of 1/500 (Table 3). Accurate determinations of domoic acid concentration in milk could not be made at any of the dilutions without prior centrifugation (Table 3).

Subsequent experiments were designed to estimate the sensitivity of the competitive ELISA for accurately measuring less than 10 pg domoic acid ml in human urine, plasma and milk. The competitive ELISA of domoie acid in urine generated an inhibition curve (r -- 0.999) which corresponded closely to the pure domoic acid standard curve (r = 0.993) at concentrations between 2.0 and 100 ng/ml (Fig. 3). Accurate determinations of domoic acid were therefore achieved between 0.2 and 10pg/ml of spiked urine diluted 1/100.

8 0

7, 4o

2O

0 I i L A i

0 I o 20 l O 4 0 $0

Domotc Acid (ng/ml)

Fig. 4. Domoic acid determination in human plasma diluted 1250 (&) and I 500 (A) compared with a pure domoic acid standard ( e ) in competitive enzyme-linked immunosorbent assay. Error bars represent the standard deviation around the mean. Curves repre~nt the least squares regression ofy

on log x.

3 0

o

,ID

40

2 O

0 ' i

20 40 60 | 0 100

Domoic A c i d ( n g / m l )

Fig. 5. Domoic acid determinations in human milk diluted 1:500 (&) and 1/1000 (A) compared with a pure domoic acid standard (Q) in competitive enzyme-linked immunosorbent assay. Error bars represent the standard deviation around the mean. Curves represent the least squares re-

gression of y on log x.

152 D. S S'qlrl-t and D. D. KJtTS

Diluting domoic acid-spiked plasma I 500 resulted m accurate determinations between 2.5 and 2011g ml (r = 0.998). which compared closely with the domoic acid standard {r =0.999) (Fig. 4). Although a dilution of I 250 resulted in an over-estimation of domoic acid concentration m the competitive ELISA at le~,els higher than 5.0 ng ml (1.25 pg of the original plasma sample ml), greater accuracy was achieved in plasma diluted I,250 to a lower limit of 0.251,g domoic acid/ml spiked plasma (Fig. 4).

Milk samples spiked with I.O-100;tg domoic acid/ml required centrifugation to remove the fat. The dilution of these delipidated samples to I 500 resulted in an overestimation of domoic acid concentration (Fig. 5). Domoic acid determinations in samples diluted 1/1000, however, yielded accurate results in competitive ELISA (r = 0.990). which compared with the pure domoic acid standard curve (r =1).9891 (Fig. 5). The minimum concentration of domoic acid accurately determined in milk using the competitive ELISA was 11) l~g'ml.

I)ls(+l SSION

Since the ASP outbreak of 1987 there has been considerable interest in developing methods [\~r measuring domoic acid concentrations in biological tluids. Tracer kinetic studies in animals have used radiolabellcd domoic acid to fi~llow the excretion pattern of domoic acid m urine, (Newsome et al . ,

1991; Suzuki and Ilicrlihy. 1993), and plasma and brain tissue uptake (Preston and Ilynic, 1991; Suzuki and llicrlihy, 1993). hnmt, nological and chromatographic methods oiler a direct means of determining domoic acid concentrations in biological fluids. Ahhough radioimmunoassays (RIA) are sensitive and accurate, expenses are incurred in purchasing radiotracers vnd arranging I\~r the disposal of radioactive waste. Furthermore, RIA methods are not applicable in field situations because of the potential for radioactive contamination. EI.ISA procedures are usually the methods of choice, as they combine the advantages of RIA without the associated problems.

An ELISA previously developed with anti-domoic acid serum raised in rabbits u, as effective in determining domoic acid concentrations in rail urine. ~ith a reported lower qt,antilication limit of 41)ng ml (Ncv,'- some et al . , 1991). Dotnoic i, cid levels in rat and monke~ plasma, hm~ever, could not be determined a, ccurately t, sing this antiserum in ELISA {New, some et al . . 1991).

Although attempts to de',elop a simple chromatographic technique for domoic acid determinations in urine have been unsuccessful (l.awrencc, 1991)), such an analytical method has been developed l\)r accurately determining domoic acid levels in plasma (Blanchard and Tasker, 1990) v, ith a sensitivity com- parable to that of the ELISA described herein. Unlike the chronmtogr;,phic method, however, the ELISA method does not require a sol'~ent extraction step

prior to anal',sis, thus eliminating the need to estimate domoic acid reco,,cr,,.

Using this ELISA method, the urine or plasma samples need only be diluted (a mmimunl of I 100 and I 500. rcspectivc[,,) to a final domoic acid concentration bctv, een 2 and 25 ng ml. This range was found to yield the steepest slopes (Figs 3 and 4), and therefore represents the most accurate range of the assay. Mi lk samples would require centrifugation to remove the flit plug and subsequent dilution of no less than 1/1000, to a final domoic acid concentration of no less than 10ngml. Because the domoic acid concentration in human fluid samples would not be known prior to analysis, a series of sample dilutions would probably require analysis.

Domoic acid can inflict excitotoxic damage to the central nervous system as evidenced by numerous clinical signs when given at an oral dose of 0.5~10mgkg in monkeys and 60 80mgkg in rats (Tryphonas et al., 1990a). Although it has been estimated that human intoxication occurs at an oral dose of I 5 mg domoic acid:kg body weight in sus- ceptible individuals (Iverson et al. , 1989). the levels of domoic acid present in urine, plasma or milk from humans displaying the symptoms of ASP are not known. In the absence of such hurnan data, animal studies have shown that the kidneys play an import- ant role in clearing domoic acid from the system (l 'reston and tlynie, 1991; Suzuki and tlicrlihy, 1993). in rats, an ip domoic acid injection of 2 mg/kg body weight produced only equivocal, transient symptoms (Tryphonas et al . , 1990b), but domoic acid was excreted in the urine at concentrations exceeding 141) pg/ml when sampled more than I hr after injection, and 40pg /ml when sampled 2 hr after injection (Newsome et al. , 1991). All the domoic acid injected into rats at this dose (61)1) 801)itg) was recovered m the urine within 160 rain [Suzuki and Hierlihy, 1993). With the assay reported in this study, a lower quantification limit of 0 .2pg domoic acid ml t, rinc was achieved. Assuming a similar clearance of domoic acid in humans, the sensitivity of this ELISA would be adequate [\)r determining domoic acid concentrations in human urine during the early stages of ASP.

Because impaired renal function has been identilied as a predisposing l\tctor in human ASP (Pcrl ct al.,

1991)), accurate domoic acid determinations in plasma ma) be more import;,nt than those in urine, especially in patients displaying more severe symptoms of ASP. Ahhough the le~,el,, of domoic acid reached in pk, sma of animals displa)ing ASP symptoms has never been directly measured, Blanchard and Tasker 11991)) have estimated that a concentration of 2 p g domoic acid ml plasnw approaches the threshold o fdomoic acid toxicit', in rodents. With a Io~er quantitation limit of 1').25l~g ml. it is likeb that the competiti ' ,e ELISA described here is ade- quately sensitive for determining domoic acid concentrations in the plasma of humans ~ith ASP.


Quanti t ies of domoic acid secreted in the milk of lactating subjects previously exposed to various amounts of the amnesic shellfish toxin arc unknov, n. Other mar ine toxins such as tc t rodotoxin have been s h o ~ n to produce neurotoxic effects in the foetus (Shatz and Stryker, 1988) and there is one report of a severe episode of ciguatra poisoning (Senecal and Osterloh, 1991). Moreover . offspring delivered from mice given a single subsymptomat ic dose of domoic acid dur ing pregnancy displayed histological and e lect roencephalographic evidence of brain damage (Daksh inamur t i et al., 1993). It is conceivable that an asymptomat ic mothe r could intoxicate her infant with domoic acid th rough her milk after ingesting domoic ac id-contamina ted shellfish. An assay for measuring domoic acid in milk could be used to identify and confirm the source of ASP-like symptoms in nursing infants. Al though thcrc have bccn no such published assay methods, this issue is of particular conccrn, since infant mice arc three or I\~ur timcs more sensitive to the toxic effects of domoic acid than adult mice (Bose et al., 1989). and neonates arc likely to be even more vulnerable.

Evaluat ion of this ELISA method sho~cd that domoic acid conccnt ra t ion in milk was overest imated at dilutions sufficient l\~r accurate de terminat ion m urine and plasma. This was probably duc to thc higher fat content of the milk, as shown by the improvement in accuracy obta ined by removing a large propor t ion of the milk fat by centrifugal(on, compared with the non-centr i fuged milk. No further a t tempt was made to cwdu:tte the etlicacy of a methanol or solid phase extract ion method commonly used for i i P L C analysis of domoic acid in shellfish materials (Quill(am et al., 1991). Rather, a greater di lut ion of milk was tested, and a l though accurate resuhs were obta ined compared with the domoic acid s tandard at a di lut ion of I / t000, the sensitivity of the assay was limited to I0 izg. ml. To improve the sensitivity of the ELISA, human milk samples may require extract ion prior to analysis. It is apparen t that domoic acid levels in milk excceding IOug ml could be accurately measured using the ELISA method presented herein.

In summary, the high spccilicity and sensitivity of this competi t ive ELISA, together with simple methods for hapten conjugat ion and sample prep- arat ion, make this assay at t ract ive for routine domoic acid analyses in various human biological fluids, particularly urine and plasma. Al though chromatographic analyses of shellfish material for domoic acid con tamina t ion would still bc required to identify thc source of an intoxication, a highly spccific ELISA for directly quant i fying domoic acid in human body fluids, such as the one dcscribcd here. could be uscd to confirm suspected cases of ASP in a more timely manner,

.4cknowh'd.~'mcnts--This ~.ork v,'as supported by a grant to DDK b~ the Science Council of British Columbm A

preliminary report of this v.ork ',,,as presented to the Cana- dian Federation of Biological Societies, Windsor, Ontario, 17 19 June 1993

REFERENCES

Blanchard J. and Tasker R. (1990) High-performance liquid chromatographic assay for domoie acid in serum of different species. Journal ~/" Chromatography 526, 546 549.

Bose R,, Glavin G. and Pinsky C. (1989) Neurotoxicity and lethality of toxic extracts from Atlantic coast shellfish. Progress in Neuro-Psychopharmacoh~,~y and Biological Psychiatry 13, 559-562.

Bradford M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochem- istry 72, 248 254.

Dakshinamurti K., Sharma S. K., Sundaram M. and Watanabc T. (1993) Hippocampal changes in developing postnatal mice follov, ing intrauterine exposure to domoic acid. Jourttal o f Neurascic'nce 13, 4486 4495.

Debonncl G., Bcauchcsne L. and De Montign~ C. (1989) Domoic acid, the alleged 'mussel toxin', might produce its neurotoxic effect through kainate receptor activation: an elcctrophysiological study in the rat dorsal hippocampus. ('anadtatt Journal 0 / Physiology and Pharmacob~g.v 67, 29 33.

Dcbonncl (i.. Weiss M. and De Montigny C. (1990) Ncuro- toxic effect of domoic acid: mediation by kainate receptor clcctrophysiological studies in the rat. ('ana~ht I)i.~ca.~t'.~ II'cck/l' Rcport 16 (Suppl IE), 59 68.

Dcshpandc S. and Sharma B. (It193) Diagnostics: im- munoassays, nucleic acid probes, and hioscnsors-two decades of development, current slatus, and future projec- tions in clinical, environmental, and agricultural appli- cations. In I)iagno,~tic~ hz l'hc }'car 2(X)O, Antibody, Bio.~'ensor, and Nuch'ic Acid l~'chmdogics. [!ditcd by P. Singh, B. Sharma and P. Tyle. pp. 459 526. Van Nos- trartd Reinhold. New' York.

Dickey R., Fryxcll (J., (iranadc II. and Roclkc D. (1992) Detection of the marine toxins okadaic acid and domoic acid in shclllish and phytoplankton in the Gulf of Mexico. 7"oxicmt ,~1, 355 359.

Impcllizzeria G., Mangiatlco S., Oricnte G., Piattclli M., Sciuto S., Fattorusso E., Magno S., Santacrocc C. and Sica D. (1975) Amino acids and low molecular weight carbohydrates of some marine red alg,tc, g/U'lochemi.~lry 14, 1549 1557.

Ivcrson |:., Truelove J., Vcra E., Tryphonas L., Campbell J. and Lok E. (1989) Domoic acid poisoning and musscl- associalcd intoxic:ttion: preliminary invcstig:ltions into tile response of mice and rats It) toxic mussel extract. Food and Chemical "Foxh'ol, Jgy 27, 377 384.

Lav.rcnce J. 11991)) Determination of domoic acid in scal\~ods and in biological tissues and Iluids. Canada Di,wase~ Weekly Report 16 (Suppl IF,). 27 31.

Newsome I1., Truelove J., !licrlihy L. and Collins P. (1991) Determination of domoic acid in serum and urine by immunochcmicat analysis, lhdh,tm o/Enrironmcntal Con- rumination am1 ToxicohJ~y 47, 329 334.

Pcrl T.. Bcdard L., Kosalsky T., Ilockin J., Todd E. and Remis R (1990) An outbreak of toxic encephztlopathy caused by caring mussels contaminated with domoic acid. New L)zt.,hzm/Journal O/Medicine 322, 1775 17SO.

Preston E. and Ilynic I. (1991)Transfer constant fi~r blood brain barrier permeation of the neuroexcilalory shellfish toxin, domoic acid. ('dHtldttttt Journal n/ A,'currp- bJgical Scicnce.~ 18, 39 44.

Quill(am M. A.. Xic M. and |lardstaff W. R. {1991) ,4 Rapid Extraction and Ch'an-up Pr~t edure [br the Dt'lcrmi- tZ~lliotl o/ Domol( Acid ill ~7~ll~' Sclmph'~. l]'chnical

I 154 D.S. S~ITH and D. D. KITTS

Report 64: NRCC no. 33001. National Research Council of Canada. Institute for Marine Biosciences. Halifax. Nova Scotia.

Senecal P. E. and Osterloh J. D. (1991) Normal fetal outcome after maternal ciguateric toxin exposure m the second trimester. Clinical Toxicoh~¢y 29, 473 478.

Shatz C. J. and Stryker M. P. (1988) Prenatal tetrodotoxin infusion blocks segregation of retinogeniculate afferents. Science 242, 87-89.

Subba Rao D., Quilliam M. and Pocklington R. (1988) Domoic ac id- -a neurotoxic amino acid produced by the marine diatom Nitzsehia pungens in culture. Canadian Journal o[" Fisheries and Aquatic Sciem'e 45, 2076 2079.

Sutherland R., Hoesing J. and Whishav,' I. (1990) Domoic acid, an environmental toxin, produces hippocampal damage and severe memory impairment. Neuroscience Letters 120, 221 223.

Suzuki C. A. M. and Hierlihy S, L. (1993) Renal clearance of domoic acid in the rat. Food and Chemical Toxicoloey 3l, 701 -706.

Takemolo T. and Daigo K. (1958) Constituents of Chondria armata. Che,Uc'al and Pharmaceutical Bulletin 6, 578 -580.

Teitelbaum J., Zatorre R.. Carpenter S., Gendron D , E',ans A . Gjedde A. and Cashman N. (1990) Neurologic seque- lae of domoic acid intoxication due to the ingestion of contaminated mussels. New En,~land Journal of Medicine 322, 1781 1787.

Tryphonas L., Truelove J., Todd E., Nera E. and Iverson F. (1990a) Experimental oral toxicity of domoic acid in cynomolgus monkeys (Macaca fascicularis) and rats. Food and Chemical Toxieoh~g.v 28, 707 715.

Tryphonas L., Truelove J., Nera E. and Iverson F. (L990b) Acute neurotoxicity of domoic acid in the rat. Toxicoh~gic Pathology 18, I-9.

Wright J., Boyd R., DeFreitas A.. Falk M., Foxall R., Jamieson W,. Laycock M., McCulloch A.. Mclnnes A., Odense P.. Pathak V., Quilliam M., Ragan M.. Sim P., Thibault P. and Walter J. (1989) Identification ofdomoic acid, a neuroexcitatory amino acid, in toxic mussels from eastern Prince Edward Island. Canadian Journal ¢~fChem- istrv 67, 481 490.

a competitive enzyme-linked immunoassay for domoic acid determination in human body fluids

Documents