Immunological detection of Clostridium botulinum toxin type A in therapeutic preparations

ELSEVIER

Journal of Immunological Methods 180 (1995) 181-191

Immunological

detection of Clostridium botulinurn toxin type A in therapeutic preparations

Theresa A.N. Ekong Dikion

*,

Kay McLellan, Dorothea Sesardic

of Bacteriology, National Institute for Biological Standards and Control, South Mimms, Potters Bar, Hertjordshire EN6 3QG, UK

Received 4 October 1994; revised 4 November 1994; accepted 21 November 1994

Abstract The potent neurotoxins produced by strains of Clostridium botulinum act by blocking the release of acetylcholine from peripheral nerve junctions. This specific action of the botulinum neurotoxins is now being exploited therapeutically to treat a variety of conditions involving involuntary muscle spasms. We aimed to develop a sensitive and specific enzyme-linked immunosorbent assay (ELISA) which may be used in parallel with the currently accepted mouse bioassay test for the determination of botulinum neurotoxin type A in therapeutic preparations. High titre polyclonal antitoxins and their biotin derivatives, highly labelled horseradish peroxidase (HRP)-antibody conjugates, and streptavidin-biotin-HRP complexes were prepared and used in a sandwich ELISA for the detection of pure neurotoxin and neurotoxin in therapeutic material. The ELISA utilized either a monoclonal or polyclonal antibody as capture agent and HRP-labelled IgG or Flab’), fragment as second antibody. The limit of detection was 4-8 pg of purified toxin/ml (gcv < 13%), equivalent to l-2 mouse bioassay units/ml. The assay was used to evaluate therapeutic preparations and the results compared with the mouse bioassay. The lower limit of detection for a therapeutic preparation of BoTxA was 2-5 mouse bioassay units/ml. Although across different manufacturers and bulk products there was no correlation between immunologically detected neurotoxin and its biological activity in different therapeutic preparations (r = - 0.44, p = 0.34, n = 8), the assay could be used to quantify neurotoxin in therapeutic preparations derived from the same bulk concentrate and manufacturer. The assay is relatively simple, and may be readily adapted to routine monitoring of BoTxA content in therapeutic preparations. Keywords:

Botulinum

neurotoxin

type A, Therapeutic

use; ELISA; Monoclonal

antibody; F(ab’),-HRP

conjugate

Abbreviations: ABTS, 2,2’-azino-bis(3-ethylbenzthiazoline-6-suifonic acid); BoTxA, botulinum neurotoxin type A, ELISA, enzyme-linked immunosorbent assay: HRP, horseradish peroxidase; IgG, immunoglobulin G; GAR-HRP, goat anti-rabbit IgG conjugated to HRP; F(ab’),(GAR)-HRP, goat anti-rabbit F(ab’& conjugated to HRP; SDS-PAGE, sodium dodecyl sulphate-polyacrylamide gel electrophoresis; DAB, diaminobenzidine; FCA, Freund’s complete adjuvant; PBS, phosphate buffered saline; KLH. keyhole limpet haemocyanin. * Corresponding author. Tel.: ( + 44) 0707-654753; Fax: ( + 44107076-46730. 0022-1759/95/$09.50 0 1995 Elsevier Science B.V. All rights reserved SSDI 0022-1759(94)00313-O

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of Immunological Methods 180 (1995) 181-191

1. Introduction

Clostridium botulinum neurotoxin type A (BoTxA) is one of seven antigenically different but structurally and functionally similar types (AG) of neurotoxins produced by various strains of C. botulinurn and C. barati. It has long been known that these extremely potent toxins act by blocking the release of acetylcholine at the neuromuscular junction, thereby causing the flaccid paralysis of botulism in both humans and animals (Simpson, 1989). H owever, their exact mode of action has only recently been elucidated (Schiavo et al., 1992; Link et al., 1992; Blasi et al., 19931. These studies show that botulinum neurotoxins are zinc-dependent endopeptidases whose toxic action depends on the specific cleavage of a selective group of proteins involved in the docking and fusion of synaptic vesicles during the release of neurotransmitters. For BoTxA, the target protein, SNAP-25, is located at the pre-synaptic membrane and is cleaved at position 198-199 (Schiavo et al., 1993). Recently, BoTxA has found use in the symptomatic relief of a growing number of spasmodic neuromuscular disorders. Therapeutic formulations of BoTxA contain minute quantities of partially purified neurotoxin complexed to haemagglutinin, together with relatively large amounts of other proteins, such as human serum albumin, added to stabilize the preparation. Consequently, the need for sensitive and specific detection of BoTxA has become increasingly important, not just for investigations of suspected food-borne outbreaks of botulism, but also for monitoring this extremely potent toxin in therapeutic preparations. The mouse bioassay (Schantz and Kautter, 19781 is currently the most sensitive and indeed the only available method for the testing of toxin activity. This method, however is cumbersome, takes 3-4 days, requires laboratory animals, and is only made specific by the use of specific antitoxin in a parallel assay. It is therefore highly desirable to have a sensitive in vitro assay which is simple and which may be readily adapted for routine laboratory use. The development of immunoassays for the detection of BoTxA was pioneered by Notermans et al. (1982). Since

then, a number of radioimmunoassays (Ashton et al., 1985) and enzyme-based immunoassays (ELISAS) (Dezfulian et al., 1984; Shone et al., 198.5; Michalik et al., 1986; Gibson et al., 1987) for this toxin have been described. The majority of these have lacked the sensitivity required for the detection of very low levels of toxin. Some, however, have been able to achieve sensitivities approaching that of the mouse bioassay, but only by the use of fairly complex signal amplification systems (Shone et al., 1985; Doellgast et al., 1993,1994X This has limited their general applicability. In the present work, we describe the development of a relatively simple enzyme immunoassay which would be of sufficient sensitivity for detecting and monitoring the consistency of botulinum toxin type A in therapeutic preparations. 2. Materials and methods 2.1. General reagents

Goat anti-species antisera, F(ab’), fragment of goat anti-rabbit IgG, horseradish peroxidase type IV, pepsin, HRP-labelled goat anti-species IgG (H + L), ABTS tablets, Sigma fast OPD and DAB tablets, and urea-H,O, buffer tablets were purchased from Sigma (Poole, UK). Streptavidin, biotin-labelled HRP, sulpho-NHS-biotin and streptavidin HRP were purchased from Pierce (IL, USA). Molecular weight markers for SDSPAGE and FPLC were purchased from Amersham (Buckinghamshire, UK) and Pharmacia (Uppsala, Sweden) respectively. All other reagents were of analytical grade and were purchased from BDH or Sigma (Poole, UK), unless otherwise stated. The microtitre plates were NUNC-immunoplate Maxisorb and were obtained from Gibco (Paisley, UK). Desalting columns were purchased from Pierce (IL, USA). An Anthos microtitre plate reader, model 2001 (Anthos Labtec Instruments, Austria) connected to an Olivetti PCS286 running an Arcom 2.4 software package was used to measure optical density and analyze results. Water was doubledistilled and deionized using a Milli Q system (Millipore, UK).

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of Immunological Methods 180 (1995) 181-191

Toxin and toxoid

A highly purified preparation of BoTxA, with specific activity of 1.5 x lo8 mouse LD,,/mg was prepared as described elsewhere (Shone et al., 1985) from culture supernatant of C. botulinurn type A (A 7272). The intact toxin was chemically detoxified by incubation with 0.2% formaldehyde in 0.05 M Hepes buffer, pH 7.4 for 21 days at 37°C. 2.2. Antibody reagents

183

lowed by affinity chromatography on protein GSepharose (Pharmacia MAb Trap G kit) according to the manufacturer’s instructions. In some cases, antibodies were affinity-purified using antibody-specific affinity columns prepared by coupling the specific antigen to CNBr-activated CM Sepharose 6B (Sigma, UK) according to the manufacturer’s instructions. The purity of antibody reagents was determined by SDS-polyacrylamide gel electrophoresis according to the method of Laemmli (1970). Protein was determined by the BCA assay system (Pierce, IL, USA).

Site-specific and polyclonal antitoxins

Site-specific, anti-peptide antibodies to BoTxA were produced in female New Zealand White rabbits (2.5-3.0 kg) by the use of chemically synthesized peptides corresponding to surface loop regions of the toxin heavy chain (based on predictive algorithms) as described previously by Sesardic et al. (1992) for diphtheria toxin. Peptides (tagged with cysteine at the amino end) were coupled to maleimide-activated KLH using the Pierce Immunoject conjugation kit. Rabbits were immunized with 90-300 pg of the conjugates in Freund’s complete adjuvant (FCA). Booster injections were given at two week intervals using the same amount of the conjugates in PBS. Antitoxin was obtained from rabbits injected with toxoid (100 pg initially and again for subsequent booster). Antitoxins were also prepared by immunization of rabbits and guinea pigs (Duncan Hartley, female, 350-400 g), initially with toxoid, subsequently with toxoid and purified neurotoxin, and finally with purified neurotoxin alone. All injections were given subcutaneously and blood was collected for serum preparation 7 days after the final injection. Monoclonal antibody BA93

The monoclonal antibody BA93, specific for BoTxA, was obtained from C.C. Shone (CAMR, UK). 2.3. Purification of antibodies BoTxA-specific monoclonal and polyclonal antibodies and goat anti-species antibodies were purified by ammonium sulphate precipitation fol-

2.4. Screening of antibodies Antibodies were screened for reactivity (titrel against botulinurn type A toxin by ELISA and Western blotting. For screening ELISA, wells of microtitre plates were coated for 18 h at 4°C with 100 ~1 of purified neurotoxin (1 pg/ml) or peptide (2 lug/ml) solutions in coating buffer (0.05 M NaCO, pH 9.6). Non-specific sites were blocked for 1 h at 37°C with 150 pi/well of 5% skim milk in PBST (M-PBST). Plates were then incubated with 100 pi/well of serial dilutions of the antibodies to be tested in M-PBST (1 h at 37”Q followed by incubation with a 1 in 1000 dilution of the goat anti-species IgG-HRP conjugate (Sigma) in M-PBST (1 h at 37°C). Finally, the substrate solution containing 0.5 mg/ml 2,2’azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) and 0.04% H,O, in 0.05 M citric acid, pH 4.0 was added. Colour was allowed to develop for 30 min at room temperature and absorbance was read at 405 nm. Western blotting was performed according to the method of Towbin et al. (1979). Following SDS-PAGE, samples were blotted onto nitrocellulose membrane. Non-specific sites were blocked with 5% skim milk in PBST, stained with polyclonal antitoxin (10 pg/ml, 2 h, 37°C) and developed with 1 in 1000 dilution of the goat anti-species lgG-HRP conjugate from Sigma (2 h, 37°C). Colour was developed using the Sigma Fast DAB system. Antibodies were also screened for their ability to neutralize biologically active botulinurn toxin type A using an in vivo bioassay as described in the British Pharmacopoeia (BP 1993, p 1178). The neutralizing po-

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of Immunological Methods 180 (1995) 181-191

tency was expressed in terms of the first international standard for botulinum antitoxin type A.

HRP-antibody absorbance ratio at 403 nm and 280 nm, the absorption maxima of the two proteins.

2.5. Label&g and conjugation of antibodies 2.6. Development of ELISA for botulinurn toxin Biotin labelling

Affinity purified antibody (5 mg) was dissolved in 1 ml 0.1 M sodium borate buffer, pH 8.5. A 1Zfold molar excess of freshly prepared sulphoNHS-biotin (Pierce) in water was added. After 2 h at room temperature, excess biotin-ester was removed by desalting on a cross-linked dextran column (5 ml bed volume) (Presto, Pierce), equilibrated with PBS. The biotin-labelled antibody was stored frozen at -20°C. F(ab’),

fragments

F(ab’), fragment of IgG was prepared according to Johnstone and Thorpe (19871, with a slight modification; to reduce loss of antibody by precipitation during peptic digestion and so improve the yield of F(ab’), fragments, digestion of IgG by pepsin was carried out in the presence of 1 M NaCl in 20 mM sodium acetate, pH 4.0 for 18 h at 37°C (Rea and Ultee, 1993). F(ab’1, fragments were purified by FPLC gel filtration (Superose 6 column, 10 cm x 3 cm) (Pharmacia). Purity was determined by SDS-PAGE (Laemmli, 1970).

type A

A number of different reagent combinations were examined for their ability to amplify the signal and increase sensitivity in a single-site or sandwich ELISA for the detection of BoTxA in purified or therapeutic preparations. Single-site ELZSA

The wells of microtitre plates were coated for 18 h at 4°C with serial dilutions of samples to be tested. After blocking and washing three times with PBST, plates were incubated for 2 h at 37°C with 100 pi/well of 10 pg/ml of antibody in M-PBST (antipeptide, rabbit or guinea pig polyclonal antitoxins). Plates were washed three times with PBST before incubation with the appropriate dilutions of anti-species IgG-HRP conjugates in M-PBST (2 h at 37°C). After a final wash with PBST, substrate solution was added and the colour developed was measured as described above. Two-site (sandwich) ELISA

Preparation of HRP conjugates

Horseradish peroxidase (type IV) was conjugated to IgG or F(ab’), fragments according to a modification of the periodate method of Wilson and Nakane (1978) described by Madersbacher et al. (1992); to increase sensitivity while still retaining low non-specific background, highly purified antibody preparations and a 4-fold molar excess of the enzyme was employed rather than the usual 2-fold excess. Conjugates were purified by ultracentrifugation (to remove any insoluble material) followed by FPLC gel filtration (Superose 6 column, 10 cm x 3 cm) (Pharmacia). Purified conjugates were stored in PBS containing 0.1% thiomersal, 1% BSA and 50% glycerol. Properties of conjugates were evaluated by ELISA using antigen-coated microtitre plates (purified rabbit or guinea pig IgG). The amount of enzyme conjugated to IgG was estimated by determining the

A sandwich ELISA was developed using either a monoclonal antibody or polyclonal antitoxin as capture antibody. Wells of microtitre plates were coated with 100 ~1 of 1 pg/ml of the capture antibody in coating buffer for 18 h at 4°C. After blocking and washing three times in PBST, serial dilutions of samples to be tested in PBST were added and incubated for either 2 h at 37°C or 18 h at 4°C. Captured toxin was then detected as described for the single-site assay. For some assays, biotin-labelled polyclonal antitoxin (10 pg/ml) and streptavidin-biotin-HRP (SBHRP) complexes were used to detect bound neurotoxin. SBHRP complexes were prepared by incubating 2.5 pg each of streptavidin and biotin-labelled HRP in M-PBST for 30 min at room temperature. 100 ~1 of the complex were then added to wells of microtitre plates for 30 min at room temperature.

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18.5

of Immunological Methods 180 (1995) 181-191

In order to amplify signal and improve sensitivity, ABTS was replaced by the Sigma OPD Fast substrate system in some assays, since this substrate is known to increase sensitivity (Sigma product literature). In this case absorbance was measured at 492 nm after stopping the reaction with 50 pi/well of 3 M H,SO,. All assays were performed in duplicate. The mean OD of wells incubated with non-immune IgG provided background (negative control) OD values. The cut-off point/sensitivity was calculated as the mean OD plus two standard deviations of negative control wells. Values above this were taken to be positive for botulinum toxin type A. 2.7. Intra- and inter-assay variation lntra- and inter assay variation in OD was estimated by testing several replicates of a toxin dilution series (1000 ng/ml-0.01 ng/ml) on a single plate using 10 pg/ml of the polyclonal rabbit antitoxin and F(ab’),-HRP diluted 1 in 10000. Differences between plates were estimated by testing the same toxin dilution series on six different plates analyzed on three different days (two per day). The assays were analyzed as parallel line bioassays, with relative potencies and percent geometric coefficient of variation (% gcv) being calculated for individual and paired well data (Finney, 1978). 2.8. Bioassay The potency estimates of the therapeutic preparations of botulinum toxin type A were performed using the currently accepted bioassay. The methods used were as previously reported (Schantz and Kautter, 1978), with minor modifications. 3. Results 3.1. Generation and characterization of antipeptide and polyclonal antitoxins

Four antibodies (B8, C2, C4 and C6) to peptides corresponding to surface loop regions of

Table 1 Titres of anti-BoTxA antibodies reagents [antibody concentration

used as primary antibody at OD,os of 1.0 in pg of

IsG/mll Antibody

Species

Titre

Antipeptide c2 c4 C6 B8

Rabbit Rabbit Rabbit Rabbit

7.9 39.7 79.2 120.0

Polyclonal Al A2 A3

Rabbit Rabbit G. pig

0.41 0.22 0.07

BoTxA heavy chain region were raised in rabbits. Polyclonal antitoxins were also raised in rabbits (Al and A2) and guinea pigs (A3). Antipeptide antibodies recognized specifically the heavy chain, while polyclonal antibodies recognized both the heavy and light chains of the toxin on Western blots; there was no cross-reactivity with other clostridial neurotoxins, or with tetanus toxin (data not shown). Anti-BoTxA activity of antipeptide and polyclonal antitoxins was determined by ELISA. Antipeptide antibody titres were generally low, with 50% activity being detected at IgG concentrations of 7.9-120 pg/ml (Table 1). Polyclonal antitoxins were of relatively higher titre, with 50% activity being detected at IgG concentrations of 0.07-0.41 pg/ml (Table 1). Polyclonal antitoxins were neutralizing with potencies of 20 (Al), 160 (A2) and 300 (A3) IU/ml, as determined by the in vivo toxin neutralization assay, indicating reactivity with biologically active neurotoxin. Antipeptide antibodies however, were non-neutralizing. 3.2. HRP conjugates A number of HRP-antibody conjugates were prepared and their properties evaluated by ELISA: Al-HRP and A2-HRP (rabbit antitoxins), A3-HRP (guinea pig antitoxin), * GAR-HRP (goat anti-rabbit IgG prepared in-house), GAG-HRP (goat anti-guinea pig IgG), F(ab’),(GAR)-HRP, (F(ab’), fragment of GAR) and F(ab’),(RAG)HRP, (F(ab’), fragment of rabbit anti-guinea pig

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T.A.N. Ekong et al. /Journal

Table 2 Titres of conjugates used for toxin detection conjugate at OD, of 1.01 Conjugate

Titre

Al-HRP a A2-HRP a Sigma GAR-HRP b Sigma GAG-HRP b * GAR-HRP ’ F(ab’),(GAR)-HRP d A3-HRP e GAG-HRP ’ F(ab’),(RAG)-HRP a

1000 1000 20000 43 000 126000 500 000 800 65 000 63 000

of Immunological

[dilution of

IgG). Direct conjugation of HRP to antitoxin (Al, A2 and A3) resulted in products of low titre (range 1:800-1:lOOO) (Table 2) and avidity. However, conjugation to the second antibody resulted in a significant increase in sensitivity, with titres

Reagents

(n)

C2 + * GAR-HRP C4 + * GAR-HRP Al-HRP Al + S GAR-HRP a Al + * GAR-HRP Al-B ’ + SBHRP ’ A2-HRP A2 + SGAR-HRP A2 + * GAR-HRP A2 + SBHRP ’ A2 + F(ab’),(G.AR)-HRP A3 + GAG-HRP A3 + F(ab’),(RAG)-HRP

2 1 3 3 7 3 3 2 8 2 5 6 2

ranging from 1:65,000 for GAG-HRP to 1:126000 for * GAR-HRP (titres for the equivalent Sigma products were 1:20 000 for GAR-HRP and 1:43 000 for GAG-HRP). HRP-antibody absorbance ratios as defined in the methods ranged from 0.42 to 0.65 for these conjugates. The highest sensitivity was achieved using HRP conjugated to purified F(ab’), fragment of goat antirabbit IgG. This reagent had a titre 1:500000 (Table 2) and a HRP-antibody ratio of 1.15. 3.3. ELISA for botulinum type A toxin

a Rabbit polyclonal antitoxin Al or A2 conjugated to HRP. b Goat anti-rabbit (GAR) or goat anti-G. pig (GAG) HRP conjugate from Sigma. ’ * Goat anti-rabbit-HRP conjugate prepared in-house. ’ F(ab’), fragment of affinity purified goat anti-rabbit IgG conjugated to HRP. e Guinea pig polyclonal antitoxin conjugated to HRP. f Goat anti-guinea pig IgG conjugated to HRP. g F(ab’), fragment of goat anti-guinea pig IgG conjugated to HRP.

Table 3 Average minimum levels of purified BoTxA detected (standard deviation in brackets)

Methods 180 (1995) 181-191

Different combinations of the reagents described above were compared in two ELISA systems for the detection of botulinum toxin type A. The first approach was a single site ELISA, based on the direct capture of toxin onto microtitre plates. Although some reagent combinations provided sufficient sensitivity to detect very low levels of purified neurotoxin (limits of 0.1 ng/ml for A2 + F(ab’),(GAR)-HRP and 1.1 ng/mi for A2 + *GAR-HRP) (Table 31, this system was not capable of detecting neurotoxin in therapeutic preparations. The second approach was a two-site, sandwich ELISA and was developed using the monoclonal antibody BA93 for the specific capture of toxin.

by single-site ELISA using different

reagents and ABTS as substrate

Toxin concentration [ng/mll

Equivalent [MLDSO/ml]

22.2, 26.0 730 562 (34.6) 178 (10.6) 6.53 (0.84) 5.50 (0.89) 126 (21.5) 15.6, 24.4 1.10 (0.11) 4.65, 5.35 0.10 (0.04) 4.1 (0.83) 14.4, 17.6

4 840 146 000 112400 35 600 1306 1100 25 200 4 000 220 1000 20 824 3 200

a Goat-HRP conjugate purchased from Sigma. b Biotin-labelled polyclonal antitoxin Al. ’ Complexes of streptavidin, biotin and HRP prepared as described in the text.

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of Immunological Methods 180 (19951 181-191

This assay was 8-22 fold more sensitive than the single-site assay (Table 4); the most sensitive assay was achieved using either biotin-labelled polyclonal antitoxin (Al-B) + SBHRP complexes or A2 + F(ab’),(GAR)-HRP to detect captured toxin, with detection limits of ranging from 35-45 pg/ml or 20-40 pg/ml respectively. The polyclonal antitoxin A3 was also used to capture toxin, followed by detection of toxin with biotinlabelled guinea pig polyclonal antitoxin (A3-B) + SBHRP complexes, or A2 + F(ab’),(GAR)-HRP (data not shown). The detection limit of 14-26 pg/ml was achieved, similar to limits obtained using monoclonal antibody coated plates. The guinea pig antitoxin/anti-guinea pig conjugate gave relatively poor sensitivity and so did not correspond to in vivo toxin neutralization data which had indicated that the guinea pig antitoxin had greater anti-BoTxA activity than the rabbit antitoxin. Antipeptide antibodies failed to recognize toxin captured by monoclonal or polyclonal antibody. The sensitivity of the assay could be further increased up to 2-5-fold by replacing ABTS with the Sigma OPD Fast system as enzyme substrate. Consequently, detection limits of 0.1-l ng/ml for direct assays and 4-12 pg/ml for capture assays could be achieved (not shown).

187

3.4. Inter- and intra-assay variation The gcv was similar for within-plate and between-plate variation (same-day and differentdays), with values ranging from 4.8 to 12.5% (gcv). 3.5, Measurement of toxin in therapeutic preparations A single preparation of therapeutic toxin (the internal standard) was used to estimate the sensitivity of the ELISA for measurement of toxin therapeutic preparations. Fig. 1. shows the titration curves for therapeutic and purified neurotoxins. The minimum level of therapeutic toxin that 1.6-5.0 could be detected ranged from MLD,,/ml, equivalent to 8-25 pg/ml (n = 151, and a good dose-response was obtained with 5.0500 MLD,, of toxin per ml. The calculated toxin protein content per vial was 1.9 * 0.37 ng (n = 111. In general, the detection of toxin by different reagent combinations in therapeutic and purified preparations correlated closely. Intra- and interassay variation for therapeutic material ranged from 8.6 to 14.5% (gcv). Four different therapeutic preparations of BoTxA from different manufacturers and of dif-

Table 4 Average minimum levels of purified BoTxA detected by BA93 capture ELISA using different reagents and ABTS as substrate (standard deviation in brackets) Reagents

(n)

C2 + * GAR-HRP C4 + *GAR-HRP AI-HRP Al + S GAR-HRP Al + * GAR-HRP A2-HRP A2 + * GAR-HRP A2 + F(ab’),(GAR)-HRP Al-B + S-B-HRP A3-HRP A3 + GAG-HRP A3 + F(ab’)JGAG)-HRP

-

Toxin concentration

h0-d

a Antipeptide

3 4 7 2 8 6 2

1 3 4

_a -a 40 (8.6) 8.0 (2.1) 0.31 (0.11) 6.4, 13.6 0.08 (0.01) 0.030 (0.005) 0.035, 0.55 251 1.23 (0.24) 1.6 (0.14)

antibodies failed to recognize toxin captured by antibody.

Equivalent [MLD,,/ml] _a _a 8 000 1600 62 2000 16 6 9 50 200 272 320

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T.A.N. Ekong et al. /Journal 3.0

~

1

n

:I:

1

g

1.5-

9” %

l.O-

z!

Purified

of Immunological

Methods 180 (1995) 181-191

BoTxA

Therapeutic

Preparation,,,/

Preparation

L

0.5 -

-#Ll

B

/

.-----A

!

0.0

6

L

I

/

/

I

1

5

4

3

2

1

0

Log10

BoTxA

dilution

Fig. 1. Determination of botulinum neurotoxin type A in therapeutic preparations. Mab-coated microtitre plates were used to capture toxin from serial dilutions of purified neurotoxin (initial concentration 1 pg/ml) or a therapeutic preparation (reconstituted with 200 ~1 PBST per vial). Bound toxin was detected using the rabbit polyclonal antitoxin, A2 and F(ab’),- HRP as described in the text.

ferent purities (different bulk concentrates) were compared for relative neurotoxin content and activity by ELISA and bioassay respectively. There was no correlation between the amount of neurotoxin detected by ELISA and its biological activity as determined by the bioassay (Fig. 21, indicat3.0

ELISA

2.5 w. .= ,z z =l F ‘G $

Bioassay

2.0 i 1.5 1.0

a 0.5

$ ol

!

0.25

0

-.25 Log10

relative

activity

0.50

1

0.75

(bioassay1

Fig. 3. Comparison of the relative content and biological activity of different filling lots of therapeutic botulinurn neurotoxin type A derived from the same preparation by ELISA and bioassay. ELISA was performed by the capture assay described in the text, while bioassay was performed by a modification of Schantz and Kautter (1978).

ing that the ratio of the amount of biologically active and immunodetected toxin varies between different preparations. Different filling lots of therapeutic material derived from the same manufacturer and bulk concentrate were also compared. The mean relative activity and geometric coefficient of variation were very similar for the two assays (0.96 * 0.11 and 11.4% respectively for the ELISA, and 0.92 + 0.10 and 11.6% respectively for the bioassay), indicating that the ratio of the amount biologically active and immunodetected toxin was comparable in these samples. Four filling lots of therapeutic material from two different bulk preparations each were further compared for neurotoxin protein content and biological activity. Fig. 3 shows that biological activity was positively correlated with immunodetected toxin when filling lots from the same preparations were compared.

Preparation Fig. 2. Comparison of the relative content and biological activity of therapeutic botulinum neurotoxin type A in four different preparations by ELISA and bioassay. ELISA was performed by capture assay as described in the text, and bioassay was performed using a modification of the method of Schantz and Kautter (1978).

4. Discussion The recent application of BoTxA, a powerful neurotoxin, to the therapeutic management of a growing number of reactive muscle conditions

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of Immunological Methods 180 (1995) 181-191

presents a number of technical challenges for the monitoring of the biological material in therapeutic use. These include the need for a specific, sensitive, and accurate method for measuring the minute quantities of toxin present in the therapeutic material. The most sensitive and only accepted method currently available for the measurement of this toxin is the bioassay in vivo (Schantz and Kautter, 1978; Tsuzuki et al., 1988). The method however is expensive and time-consuming and it is desirable to develop methods avoiding the use of laboratory animals. This has resulted in a widespread search for alternative in vitro assays of similar sensitivity. To achieve this sensitivity has proved difficult; enzyme immunoassays have been reported which were capable of detecting as little as 10-100 MLD,,/ml for type A toxin (Dezfulian and Bartlett, 1984; Shone et al., 19851, compared to 5 MLD,,/ml for the in vivo assay. However, some of these assays employed elaborate enzyme amplification systems which has limited their general applicability. Ogert et al. (1992), utilizing a sophisticated fiberoptic based biosensor, were only able to achieve a detection limit of 5 ng/ml, some 200-fold less sensitive than the in vivo assay. Doellgast et al. (1993,1994) have recently developed a modified ELISA for the measurement of toxins A, B and E from C. botulinurn which relies on the detection of complexes by a solid-phase coagulation assay. The method is very sensitive, with detection limits similar to that of the mouse bioassay. However, the complicated nature of the system is likely to limit its general applicability. In this study, we aimed to develop a simple enzyme-based immunoassay, the sensitivity of which would at least equal that of the LD,,, and which would be adequate in the first instance for monitoring the toxin content of therapeutic preparations of BoTxA. The most important factor determining the detection limit in an immunoassay is the binding affinity of antibody for its specific antigen (Avrameas, 1992). Initially site-specific antipeptide antibodies to the toxin were prepared, in the hope that these would possess the desired specificity and sensitivity for the detection of very low levels of toxin, thereby establishing a relatively

189

safe and inexpensive means of preparing toxinspecific antibodies. This approach had proved successful in the mapping of epitopes of diphtheria toxin (Sesardic et al., 1992). However, although the antipeptide antibodies obtained were specific, they were of relatively poor titre in direct assays, and did not recognize antibody-captured toxin in capture assays, possibly indicating the presence of shared epitopes between capture and antipeptide antibody and/or conformational changes on the captured toxin. Subsequently, high affinity polyclonal antibodies were prepared by immunizing with a combination of highly purified toxoid and toxin. These were labelled with HRP and biotin in order to increase the detection limit. Although the monoclonal antibody first described by Shone et al. (1985) was used as capture antibody in initial studies, subsequent work showed that the polyclonal antitoxin (A31 was at least as effective for this purpose (data not shown). In ELISA techniques using HRP conjugates, a crucial step is the conjugation of HRP to antibodies (first described by Nakane and Kawaio, 1974) such that reagents of high specific activity and low background noise are produced. Commercially prepared conjugates gave relatively low signal amplification and were therefore not sufficiently sensitive for the desired purpose. Affinity-purified IgG or their F(ab’), fragments were therefore used to prepare HRP conjugates. The anti-rabbit HRP conjugates prepared in this manner had very high titres compared to their commercial equivalents and gave backgrounds which were acceptable. However, the equivalent anti-guinea pig products were of comparatively low titre. The most sensitive ELISA was the capture assay in which toxin-specific antibody was used to capture toxin, with detection by affinity-purified antitoxin and high titre conjugates of HRP and IgG or its F(ab’), fragment. This assay was sufficiently sensitive to detect toxin at the limits of the mouse bioassay, with both purified and therapeutic neurotoxin preparations. The useful working range of the assay was 0.06-1.60 ng/ml of toxin protein for purified preparations, and 5-500 LD,,/ml for the therapeutic preparation. This is

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at least comparable to the most sensitive ELISAs previously reported for BoTxA (Shone et al., 1985;Doellgast et al., 1993,1994). The assay could be completed in 6h using pre-coated and blocked plates. It requires minimum instrumentation and is technically simple to perform. Furthermore, the assay shows good reproducibility/precision, with intra- and inter-assay variations of 4.8%12.5% (gcv) for toxin concentrations of 0.01-1000 ng/ml. The single-site ELISA developed here could not be used for the assay of therapeutic toxin because of the presence of relatively large amounts of other proteins in the therapeutic preparations. However, it is very sensitive (minimum level of toxin detected, 0.10 ng or 20 MLD,,/ml) and should prove useful for the measurement of toxin from other sources, such as in culture supernatants. A major drawback of this and other immunoassays is that they do not provide a measure of the biologically active material, only of total antigenicity. This was confirmed by results showing that relative potencies of different preparations of the therapeutic material determined by in vivo assay and ELISA showed no correlation. This is due presumably to the presence of varying amounts of immunoreactive, non-functional toxin. Notwithstanding, there was excellent correlation between immunoreactive toxin and biologically active toxin in different fills derived from the same neurotoxin preparation, suggesting that there is negligible loss of activity as a consequence of the filling process and subsequent storage. Hence, from the data presented here, it would appear that the sandwich immunoassay developed provides the sensitivity and specificity required for monitoring consistency of toxin content in therapeutic preparations derived from the same bulk source.

Acknowledgements

We are grateful to Dr. Cliff C. Shone, Porton Down, Wiltshire, for his kind gift of monoclonal antibody. We would also like to thank Dr Alan Heath and Dr. Rose Gaines-Das of the Division

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of Informatics, NIBSC, for their invaluable help with statistical analyses, and Dr. Mike J. Corbel of the Division of Bacteriology for critical evaluation of the manuscript. This work was funded by the Home Office under the Animal Procedures Research Scheme Grant.

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