Paralytic shellfish poisoning (PSP) toxin binders for optical biosensor technology: problems and possibilities for the future: a review

Figures & data

Figure 1. Structure, relative molecular mass (RMM) and toxicity factor of paralytic shellfish poisoning (PSP) toxins.

Figure 2. SPR optical biosensor sensorgram of the molecular interaction between the binder and immobilised surface. Source: Abery (2001). Courtesy: Biacore International AB, GE Healthcare.

Figure 3. SPR optical biosensor inhibition assay format.

Figure 4. Typical response versus PSP toxin concentration calibration curve using a polyclonal antibody binder.

Figure 5. Trends in carbamate toxin response at a fixed concentration of 10 ng ml⁻¹ with increasing percentage of NEO antibody in STX/NEO antibody binder mix. Source: Campbell et al., unpublished data.

Table 1. General advantages and disadvantages of binders previously assessed for SPR analysis of PSP toxins.

Binder	Advantages	Disadvantages
Receptors	Have a binding affinity that generally correlates with the biological response or toxicity of the toxins Can be isolated rapidly compared with antibody production Good sensitivity	Wider scope of binding to all compounds that act on the same receptor Have to be isolated form biological tissues and in some cases the species of origin is difficult to obtain May display poor stability May be temperature sensitive for analytical purposes
Monoclonal antibodies	Constant renewable source of binder once produced with minimal batch to batch variation During production less immunizing agent is required compared with polyclonal production and it is possible to select for specific epitope specificities and generate antibodies against a wider range of antigens React with a single epitope on an antigen with high specificity Binding affinity for the immunizing antigen is generally better than a receptor Less background signal than polyclonal antibodies	For production, time, effort and commitment is high and use animals On average the binding affinity of a monoclonal antibody is lower than a polyclonal antibody As monoclonal antibodies are highly specific to a single epitope they may lose affinity to other antigens within a group with minor molecular modifications The binding affinity of an antibody does not correlate with biological response for a toxin group Antibodies sometimes display unexpected cross-reactivity with unrelated antigens in biological matrices
Polyclonal antibodies	For production relatively quick and inexpensive to produce compared with monoclonal antibodies Polyclonals will recognize multiple epitopes on any one antigen and are therefore more tolerant of minor changes in the antigen than monoclonal antibodies providing better cross-reactivity to structural antigens within a group The recognition of multiple epitopes generally provides more robust detection Binding affinity for the immunizing antigen is generally better than a monoclonal antibody or receptor	Animals are required and are prone to batch to batch variability They produce large amounts of non-specific antibodies which can sometimes give background signal in some applications Multiple epitopes make it important to check for any cross-reactivity in biological matrixes The binding affinity of an antibody does not correlate with biological response for a toxin group

Table 2. Sensitivity and specificity data for both STX and NEO antibody on a NEO chip surface (Campbell et al., unpublished data).

Chip surface	Neosaxitoxin chip surface
Antibody	STX			NEO
Antibody dilution in HBS-EP	1/250			1/50
Antibody ratio to standard	1:1			1:1
Flow rate (µl min^−1)	25			25
Contact time (min)	2			2
PSP toxin analogue	IC50 (ng ml^−1)	Percentage cross-reactivity	Dynamic range, IC20 to IC80 (ng ml^−1)	IC50 (ng ml^−1)	Percentage cross-reactivity	Dynamic range, IC20 to IC80 (ng ml^−1)
Saxitoxin dihydrochloride	4.8	100	2.7–8.1	41.8	100	6.6–203
Neosaxitoxin	13.4	35.8	3.7–84.8	2.4	1742	1.1–5.5
Gonyautoxin 1/4	474^b	1.0^b	77–2780^b	4.1	1032	1.6–14.9
Gonyautoxin 2/3	7.0	68.7	2.1–22.0	19.6	213	4.1–91.6
Decarbamoyl saxitoxin	2.9	165.1	1.3–6.3	>1000	<4.2	n.d.^a
Decarbamoyl neosaxitoxin	100	4.8	14.4–200	192.1	21.8	41.9–512
Decarbamoyl gonyautoxin 2/3	18.7	25.8	4.4–62.3	>1000	<4.2	n.d.
C1/C2	14.8	32.6	3.3–53.6	>1000	<4.2	n.d.
Gonyautoxin 5	4.1	117	2.7–7.7	>1000	<4.2	n.d.
C3/C4	206^b	2.3^b	27–590^b	100	41.3	n.d.
Notes: ^an.d., Not determined due to lack of the standard.
^bValues calculated based on extrapolation of the four-parameter fit applied to the standard curve due to availability of the standard.

Table 3. Sensitivity and specificity of the STX/NEO antibody mix for each PSP toxin (Campbell et al., unpublished data).

	STX			1/500
Antibody dilution in HBS-EP	NEO			1/50
Antibody mix	20% STX/80% NEO
Antibody ratio to standard	1:1
Flow rate (µl min^−1)	12
Injection volume (µl)	60
Contact time (min)	5
	Toxin concentration (ng ml^−1)			Toxin concentration as STXdiHCl equivalents (ng ml^−1)
PSP toxin analogue	IC50 (ng ml^−1)	Percentage cross-reactivity	Dynamic range, IC20 to IC80 (ng ml^−1)	IC50 (ng ml^−1)	Percentage cross-reactivity	Dynamic range, IC20 to IC80 (ng ml^−1)
Saxitoxin dihydrochloride	2.8	100	0.2–68.8	2.8	100	0.20–68.8
Neosaxitoxin	2.8	100	0.8–9.9	3.1	90.3	0.87–10.8
Gonyautoxin 1/4	4.4	63.6	0.8–25.3	4.0	70	0.72–22.8
Gonyautoxin 2/3	4.9	57.1	0.9–28.0	2.9	96.6	0.54–16.8
Decarbamoyl saxitoxin	0.4	700	0.1–1.2	0.3	933	0.07–0.9
Decarbamoyl neosaxitoxin	9.1	30.7	1.7–48.7	6.4	43.8	1.19–34.2
Decarbamoyl gonyautoxin 2/3	17.1	16.4	3.1–94.7	6.8	41.1	1.23–37.7
C1/C2	14.5	19.3	2.0–102.7	1.1	255	0.15–7.7
Gonyautoxin 5	0.4	700	0.1–1.5	0.03	9333	0.01–0.1
C3/C4	91.8	3.0	24.0–198	4.0	70	1.03–8.5

Table 4. Advantages and disadvantages of potential binders for SPR analysis of PSP toxins.

Binder	Advantages	Disadvantages
Chemosensors	Non-animal-based binders Can be incorporated into self-assembly monolayers for sensing on a surface	Lower binding affinity than antibodies No correlation between the binding intensity and biological response or toxicity
Molecular imprinted polymers (MIPS)	Non-animal-based binder Target analyte defines its own recognition site Stable at extreme temperature and pH Show specificity in natural systems Adaptable and flexible in their use Low cost and ease of manufacture May be used in non-aqueous media or aggressive environments	May be sensitive to small alterations in target analyte structure and properties Show non-specific binding Diversity of binding sites Template bleeding requires suitable template analogue for imprinting and affects quantitative applications Generally lower binding affinities than antibodies
Aptamers	Nucleic acid aptamers are non-animal-based binders produced by chemical synthesis resulting in limited batch to batch variation Selectively bind to low molecular weight compounds Binding affinities of aptamers are in the µM to nM range and kinetic parameters can be altered on demand Toxins that do not illicit a good immune response can be used to generate high-affinity aptamers Aptamers are stable for long-term storage and can be transported at ambient temperature Selection conditions can be manipulated to obtain aptamers with properties desirable for in vitro assays, e.g. non-physiological buffers, solvents, etc. Reporter molecules can be attached to aptamers at precise locations so as not to affect binding	Aptamers are costly to generate and long aptamer sequences are difficult to achieve DNA aptamers have a smaller range of three-dimensional structures obtainable compared with RNA aptamers but they can bind their target to the entire sequence DNA and RNA aptamers are susceptible to enzymatic degradation by nucleases, thus requiring highly pure environments. This can be overcome Peptide aptamers are constrained to the scaffold protein so they are less flexible, which may affect their effectiveness. Only the variable range is used for binding Peptide aptamers require biological systems for selection purposes

Abery , J . 2001 . Detecting the molecular ties that bind . Modern Drug Disc , 4 : 34 – 36 .

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