Immunoassays for Residue Analysis - American Chemical Society

Chapter 31

Immunochemical Approaches to the Analysis of Paralytic Shellfish Poisoning Toxins

Downloaded by PEPPERDINE UNIV on September 28, 2017 | http://pubs.acs.org Publication Date: May 5, 1996 | doi: 10.1021/bk-1996-0621.ch031

Richard Dietrich, Ewald Usleber, C. Bürk, and Erwin Märtlbauer Institute for Hygiene and Technology of Food of Animal Origin, Veterinary Faculty, University of Munich, Schellingstrasse 10, 80799 Munich, Germany

Using different paralytic shellfish poisoning (PSP)-protein conjugates several monoclonal antibodies against saxitoxin (STX) as well as polyclonal antibodies against STX and neosaxitoxin have been produced. The sensitivity and particularly the specificity of the antibodies will be discussed in detail, their use in microtiter plate EIA's and in immunoaffinity columns for the analysis of contaminated mussels is described.

Paralytic shellfish poisoning (PSP) toxins are produced by certain marine algae known as dinoflagellates. Contamination of shellfish has been associated with harmful algae blooms throughout the world. The first PSP component to be chemically characterized was saxitoxin (STX). Three groups of PSP toxins, iV-sulfocarbamoyl, carbamate and decarbamoyl toxins are known (Figure 1). A l l 18 toxins are naturally occurring; thus, meaning that a mixture of different toxins is usually present in a contaminated sample (7). PSP toxins act through a potent, reversible blockage of the sodium conductance in nerve and muscle membranes. The lethal dose for humans is 1-4 mg expressed as saxitoxin equivalents. Each of the PSP toxins has its characteristic toxicity expressed as mouse units (MU) per /xmole. The toxicity of iV-sulfocarbamoyl derivatives is relatively low (18-430 M U ) , that of the carbamate toxins is significantly higher (673-2045 MU) and the decarbamoyl toxins exhibit intermediate toxicity (530-1220 MU). The classical method for the analysis of PSP toxins is the mouse-bioassay, based on the lethal effect of intraperitoneally administered PSP toxins. In most countries this method is still used for monitoring purposes. But in addition to objections to the use of experimental animals, sensitivity of the mouse bioassay is too close to the regulatory limit, which is in the range of 40-80 /zg per 100 g shellfish (2). The assay also lacks specificity. 0097-6156/96/0621-0395$15.00/0 © 1996 American Chemical Society Beier and Stanker; Immunoassays for Residue Analysis ACS Symposium Series; American Chemical Society: Washington, DC, 1996.


396

IMMUNOASSAYS FOR RESIDUE ANALYSIS

R4:

R4:

Ο

R1

R2

R3

H OH OH H H OH

H H H H oso3 oso-

H H OSO" 3 OSO" 3 H H

R4:

Ο

Carbamate Toxins

N-Sulfocarbamoyl Toxins

Decarbamoyl Toxins

STX neoSTX GTX 1 GTX2 GTX 3 GTX 4

B1 B2 C3 C1 C2 C4

dc-STX dc-neoSTX dc-GTX 1 dc-GTX 2 dc-GTX 3 dc-GTX 4

Figure 1. Structures of paralytic shellfish poisoning (PSP) toxins.

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

31. DIETRICH ET AL.

Analysis of Paralytic Shellfish Poisoning Toxins

397

The following is a brief overview of the characteristics of antibodies against PSP toxins produced in our laboratory with some examples for the applicability of immunochemical methods used in analyzing for these algal toxins.


Antibodies Against PSP Toxins Although the first antibodies against STX were described in 1966 by Johnson and Mulberry (3), immunochemical methods still play only a minor part in the field of PSP toxin analysis. This is primarily due to the multiplicity of STX analogs that makes it unlikely or even impossible, that a single antibody reactive with all PSP toxins could be produced. For screening purposes, however, it would be sufficient, if the immunochemical methods used in a monitoring program were able to detect only the highly toxic compounds, namely the decarbamoyl and carbamate toxins. Polyclonal Antibodies. Using a previously described formaldehyde condensation procedure (4,5) for the preparation of the immunogen, STX was coupled to keyhole limpet hemocyanin (KLH). After immunization of rabbits, highly sensitive antibodies were obtained and a competitive indirect enzyme immunoassay (EIA) was established, in which STX-bovine serum albumin (BSA) served as coating antigen (6). A 10-fold increase of the test sensitivity was observed after development of a direct EIA using STX coupled to horseradish peroxidase via a modified periodate reaction as the enzyme conjugate (7). The detection limit of this test system was 7 pg STX/mL. Taking into account that the tolerance level for PSP toxins is set at 40 to 80 μ% per 100 g shellfish, the sensitivity of both EIA's is more than sufficient for all analytical purposes. But, as mentioned above, the usefulness of immunochemical test systems in the field of PSP analysis is a question of the specificity of the antibodies, particularly, recognition of the highly toxic carbamate and decarbamoyl toxins. Until recently, the determination of cross-reactivity was nearly impossible as a result of the lack of commercially available pure standards. During the last few years, however, a Canadian group has isolated several PSP toxins, in particular STX, neosaxitoxin (neoSTX), a mixture of the gonyautoxins-2/3 (GTX-2/3) and GTX-1/4, decarbamoylSTX (dc-STX) and a mixture of the N-sulfocarbamoyl toxins C - l / 2 (8). Using these well characterized standards, the polyclonal antibodies against STX showed crossreactivities of 27.8% and 12.1% with dc-STX and GTX-2/3, respectively; whereas, their reactivity towards neoSTX and particularly GTX-1/4 and C l / 2 was very low (Table I). But, as a result of the extremely high sensitivity of the test system, the detection limit for C l / 2 is still less than 1 ng/mL. Studying the effect of using a heterologous PSP toxin-enzyme conjugate on the cross-reactivity, we observed that mainly the detection limit for neoSTX could be improved (9). In a direct EIA with dc-STX-HRP, the detection limits (25% inhibition dose) for STX, neoSTX, dc-STX, and GTX-2/3 were 6.2, 68.0, 24.3, and 96.1 pg/mL, respectively. Compared with the homologous assays, the recognition of the toxins was more uniform, thus reducing the risk of an over- or underestimate of the total toxin content in an unknown mixture. Low cross-reactivities with neoSTX in assays using polyclonal antibodies against STX also were described by other authors (5,10).



398


In order to facilitate the detection of the #-l-hydroxy derivatives (neoSTX and the corresponding structural analogues), rabbits were immunized with neoSTX conjugated to the glycoprotein glucose oxidase (GlcOx) using the periodate method. Competitive indirect E I A , using STX-BSA as coating antigen, revealed that the antibodies are specific for neoSTX, but that they also have substantial cross-reactivity with GTX-1/4 (Table I). The detection limits are 18 pg neoSTX/mL and 26 pg G T X 1/4/mL, respectively. Considering the complementary cross-reactivities of the polyclonal antibodies against STX and neoSTX it is obvious, that a combination of these two test systems could be very useful for monitoring purposes. The only other polyclonal antiserum against neoSTX, desribed by Chu and Huang (77), mainly crossreacts with STX (11.4%), but no data were given concerning the reactivity of these antibodies with gonyautoxins.

Table I. Relative Cross-Reactivities (RCR) of the Polyclonal Antibodies against Saxitoxin and Neosaxitoxin. Antibodies against Saxitoxin 50% dose (pg/mL)

Toxin Saxitoxin

15.0

dc-Saxitoxin

47.5 163.5

Gonyautoxin-2/3 C-l/2

5,053

Neosaxitoxin Gonyautoxin-1/4

510.0 6,242

Neosaxitoxin

%RCR°

50% dose (pg/mL)

a

%RCR

2,420

3.0

27.8

42,054

0.2

12.1

2,886

3.3

0.5

202,350

0.1

100

3.1

76.1

0.3

111.2

100 88.5

Cross-reactivity was calculated on a molar basis.

Monoclonal Antibodies. A l l monoclonal antibodies described so far show very low affinities for STX and therefore are not suitable for the detection of PSP toxins in food (72). In our first attempts to produce monoclonal antibodies against STX we used the S T X - K L H and other STX-protein conjugates all produced by the Mannich reaction (4,5). Unfortunately, all of these conjugates proved to be poor immunogens in mice. Much better results could be achieved by using another coupling procedure, namely the modified periodate reaction. Coupling STX to the glycoprotein glucose oxidase yielded a highly immunogenic antigen-carrier preparation. After only a single immunization, 10 out of 12 mice showed high serum antibody titers against STX.



31. DIETRICH ET AL.


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Using the STX-glucose oxidase immunogen, 9 hybridoma cell lines, all secreting antibodies of the IgGl subclass, were established. Table Π shows the characteristics of the monoclonal antibodies (Mab) designated 5F7, 7H11, 1E8, each representing a group of antibodies different in terms of sensitivity and particularly in their specificity relative to STX. The monoclonal antibodies were less sensitive than the corresponding polyclonal antibodies, but all showed considerable cross-reactivity to GTX-2/3. Only weak cross-reactivity to the corresponding N-sulfocarbamoyl toxins C - l / 2 was observed indicating that the R4 side chain had great influence on the affinity of the monoclonal antibodies. Additionally, Mab 1E8 showed very low reactivity with dc-STX, whereas Mabs 7H11 and 5F7 exhibited cross-reactivities of 11.4 and 18.2%, respectively. Also, each change in the side chains reduces the reactivity of Mab 5F7 by a factor of 10, whereas, a change at side chain R2/3 had little influence (86.3% cross-reactivity with GTX-2/3) on the affinity of Mab 7H11. Additionally, this Mab showed remarkable cross-reactivity with all tested carbamate and decarbamoyl toxins; e.g., neoSTX, GTX-1/4, GTX-2/3 and dc-STX. Considering the cross-reactivity of both the polyclonal antibodies against neoSTX and the monoclonal antibodies against STX, there is strong evidence that using the periodate method for the preparation of PSP toxin-carrier conjugates, resulted in antibodies with improved cross-reactivities with the respective, structural related gonyautoxin. Application of Immunochemical Methods for the Analysis of PSP Toxins Using highly sensitive and well characterized antibodies, immunochemical methods could be very useful at different stages of an integrated analytical system for the analysis of PSP toxins. Depending on the required result, immunochemical test systems can be designed as rapid qualitative (7,75) or quantitative assays. Additionally, immunochemical methods may be combined with physico-chemical methods, thus improving both sensitivity and specificity of traditional chromatographic techniques. Quantitative Test. The microtiter plate assay has the potential to replace the mousebioassay as the screening assay. Currently, a comparison study between the two assays is being performed using the EIA based on the polyclonal antibodies against STX as an immunochemical method. So far 64 samples (mussels, King scallops and Queen scallops) were analyzed, and 44 were positive in the mouse-bioassay (Donald, M . , M A F F , Aberdeen, U K , personal communication, 1995). Generally, analysis by EIA resulted in lower values (28-370 μg STX equivalents per 100 g shellfish) than the bioassay (37-657 /zg STX equivalents per 100 g shellfish), but all samples which reacted positive in the mouse-bioassay were also positive in the enzyme immunoassay. Nearly all bioassay negative samples also yielded negative results or very low values (< 10 /xg/100 g) in the EIA. The differences between the two methods of analysis can be simply explained: lower values in the immunochemical assay are caused by the low cross-reactivity of the EIA with toxic analogues, and higher values occur, i f high concentrations of relatively non-toxic N-sulfocarbamoyl toxins are present in the sample material.



1.5

15.9

163.8

299

256

394

473

315

410

Saxitoxin

dc-Saxitoxin

Gonyautoxin-2/3

C-l/2

Neosaxitoxin

Gonyautoxin-1/4

1.3

10.0

500.0

14.0

11.8

50% dose (ng/mL)

mol wt

Toxin

5F7 a

13.2

17.9

76.5

2.2

7.2

1.5

50% dose (ng/mL)

15.1

8.5

3.0

86.3

18.2

100.0

% RCR

7H11 a

4.6

7.1

161.3

0.7

18.1

0.2

5.1

2.6

0.2

33.1

0.9

100.0

%RCR

1E8 50% dose (ng/mL)

Table IL Relative Cross-Reactivities (RCR) of the Monoclonal Antibodies Against Saxitoxin.


a

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401


In view of these first and preliminary results, it seems possible to replace the mouse-bioassay by an enzyme immunoassay or at least by a combination of immunoassays with complementary specificity like the S T X and neoSTX-EIA described above. Immunoaffinity Chromatography (IAC). As for every screening assay, positive samples should be reanalyzed by physico-chemical methods at least for legal or statutory purposes. In the analysis of PSP toxins the common method is H P L C with fluorescence detection after pre- or postchromatographic oxidation (14,15). The problems encountered with this method of analysis are mainly of a chromatographic nature. To separate the 18 structurally related compounds, sophisticated gradient elution programs and mobile phases were developed, but clean-up methods for the purification of shellfish extracts are barely described. To circumvent drawbacks of chromatographic analyses through sample interferences we developed immunoaffinity chromatographic columns using the broadly cross-reactive Mab 7H11 (16). The antibody was coupled to CNBr activated Sepharose 4B, mini-columns containing 200 uL of the immunosorbent were used in all of our studies. Each immunoaffinity-column had a capacity of approx. 3 μ% PSP toxin. In order to determine recovery rates for the available toxin standards, 2 μg of each toxin in a concentration of 100 ng per mL were passed through the column and subsequently the bound toxin was eluted with acetic acid. For STX, all G T X toxins and dc-STX the recoveryrateswere in the range of 70-90%. NeoSTX and C - l / 2 also were retained by the immunoaffinity columns, but to a minor extent. In order to check the applicability of the IAC columns for analysis of shellfish extracts, we passed unœntaminated, artificially contaminated and naturally contaminated mussel extracts through the column and analyzed the corresponding eluates by H P L C according to (14). The chromatograms of uncontaminated samples (Figure 2) showed the excellent clean-up provided by the IAC columns. No sample peaks could be observed. The recovery of PSP toxins from mussel extracts were in a similar range as observed applying the pure standards to the IAC columns. The analysis of naturally contaminated mussels gives evidence, that in addition to the known reactivity, the Mab 7H11 also binds dc-GTX-2/3 and the N-sulfocarbamoyl toxin B l (Figure 2). Conclusions The presented results strongly support the use of immunochemical methods for improved analysis of the PSP toxins. In our laboratory work is still in progress to obtain more data concerning the correlation between bioassay and enzyme immunoassay and to improve the clean-up of mussel samples using IAC columns. Acknowledgments We thank B. Minich and M . Straka for excellent technical assistance.


402



A uncontaminated mussel sample

Β mussel s a m p l e artificially contaminated with d c - S T X a n d S T X STX

dc-STX

C naturally contaminated mussel sample dc-STX

B1

dc-GTX-2/3

GTX-2/3

STX

Figure 2. H P L C chromatograms of extracts of (A) uncontaminated, (B) artificially contaminated (100 ng/g S T X and 25 ng/g dc-STX) and (C) naturally contaminated mussels. A l l extracts were purified by I A C columns. H P L C conditions were exactly as described by Lawrence et al. (14). The retention time of STX was 13.8 min.


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Literature cited 1. Hall, S.; Strichartz, G.; Moczydlowski, E . ; Ravindran, Α.; Reichhardt, P. B. In Marine Toxins: Origin, Structure and MolecularPharmacology;Hall, S.; Strichartz, G., eds.; American Chemical Society: Washington, DC, 1990, ACS Symposium Series No. 418; pp 29-65. 2. Van Egmond, H. P.; Aune, T.; Lassus, P.; Speijers, G. J. Α.; Waldcock, M . J. Nat. Toxins 1993, 2, 41-83. 3. Johnson, Η. M . ; Mulberry, G. Nature 1966, 211, 747. 4. Johnson, Η. M . ; Frey, P. Α.; Angelotti, R.; Campell, J. E . ; Lewis, Κ. H. Proc. Soc. Exp. Biol. Med. 1964, 117, 425-430. 5. Chu, F. S.; Fan, T. S. L. J. Assoc. Off. Anal. Chem. 1985, 68, 13-16. 6. Renz, V.; Terplan, G. Arch. Lebensmittelhyg. 1988, 39, 30-33. 7. Usleber, E . ; Schneider, E . ; Terplan, G. Lett. Appl. Microbiol. 1991, 13, 275-277. 8. Laycock, M . V.; Thibault, P.; Ayer, W.; Walter, J. A. Nat. Toxins 1994, 2, 175-183. 9. Usleber, E.; Dietrich, R.; Märtlbauer, E.; Terplan, G. Lett. Appl. Microbiol. 1994, 18, 337-339. 10. Cembella, Α.; Parent, Y.; Jones, D.; Lamourex, G. In Toxic Marine Phytoplankton; Graneli, E.; Sundstrom, B.; Edler, L . ; Anderson, D. M . , eds.; Elsevier Science Publishing: New York, NY, 1990, pp 339-344. 11. Chu, F. S.; Huang, X. J. AOAC Int. 1992, 75, 341-345. 12. Huot, R. I.; Armstrong, D. L . ; Chanh, T. C. J. Toxicol. Environ. Health 1989, 27, 381-393. 13. Usleber, E.; Schneider, E.; Terplan, G.; Laycock, M. V. Food Addit. Contam. 1995, 12, 405-413. 14. Lawrence, J. F.; Menard, C. J. Assoc. Off. Anal. Chem. 1991, 74, 1006-1012. 15. Sullivan, J. J. In Marine Toxins: Origin, Structure and Molecular Pharmacology; Hall, S.; Strichartz, G., eds.; American Chemical Society: Washington, DC, 1990, ACS Symposium Series No. 418; pp 66-77. 16. Dietrich, R.; Usleber, E . ; Terplan, G. 107th Annual AOAC Meeting, Washington, DC, 1993; p 143. RECEIVED

October 3, 1995


Immunoassays for Residue Analysis - American Chemical Society

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