Application of an in vitro endopeptidase assay for detection of residual toxin activity in tetanus toxoids.

Tetanus vaccine is composed of chemically denatured tetanus toxin (TeNT), thus safety testing requires confirmation of freedom from residual and reversible toxicity. Currently, TeNT activity is estimated using in vivo assay models. Information that TeNT acts by selectively inactivating protein leading to the blocking of release of neurotransmitters has provided the opportunity to develop in vitro biochemical assay for toxin activity. In this study we describe development and use of an in vitro endopeptidase assay for detection of TeNT activity in toxoid vaccine formulations.

Therapeutic botulinum type A toxin: factors affecting potency.

The type A neurotoxin produced by Clostridium botulinum is a potent neuromuscular blocking agent which causes paralysis by preventing the release of neurotransmitter from motor neurons. This property has resulted in the use of the toxin in the treatment of a number of neuromuscular diseases involving muscle spasms. At present, the only recognised assay to estimate accurately the potency of botulinum toxin in clinical preparations is bioassay, in which lethality is used as the endpoint. Such bioassay is inherently variable and large interlaboratory variability has been reported, highlighting problems for standardisation of activity in the absence of any commonly used reference preparation. In the present study, we have confirmed that many different assay conditions can affect potency estimates of clinical formulations of type A botulinum toxin. Further, our studies indicate that different preparations, because of their unique formulation and stability, are differentially affected by some of these assay conditions and that these differences might well contribute to the differences observed in their clinical use.

Refinement and validation of an alternative bioassay for potency testing of therapeutic botulinum type A toxin.

The type A neurotoxin produced by Clostridium botulinum is a potent neuromuscular blocking agent which causes paralysis by preventing the release of neurotransmitter from motor neurones. This property has led to the use of the toxin in the treatment of a number of neuromuscular diseases involving muscle spasms. At present, the only recognised assay with the specificity and sensitivity to estimate accurately the potency of botulinum toxin in clinical preparations is bioassay, in which lethality is used as the end point. Refinement of this assay, with respect to the end point, was explored on the basis of the development of flaccid paralysis of muscles following subcutaneous injection of the toxin at the inguinocrural region. Potency estimates, relative to in house reference preparations, for different therapeutic preparations obtained using flaccid paralysis as a scored response gave excellent agreement with estimates obtained in independent assay using the currently required control method. This study demonstrates that an alternative, more humane bioassay for potency testing of clostridia neurotoxins gives valid estimates equivalent to those currently in use.

Immunological detection of Clostridium botulinum toxin type A in therapeutic preparations.

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 F(ab')2 fragment as second antibody. The limit of detection was 4-8 pg of purified toxin/ml (gcv < 13%), equivalent to 1-2 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.

Low affinity antibodies against collagen type II are associated with pathology in collagen-induced arthritis in mice.

The relationship between the functional affinity of antibodies against type II collagen (CII) and the development of arthritis was studied in mice with collagen-induced arthritis. The responses of DBA/1 strain mice were compared with those of mice selectively bred to produce antibodies of high functional affinity (HA mice) and low functional affinity (LA mice). HA and LA mice did not develop arthritis in response to immunization with CII whereas 86% of DBA/1 mice did, with 33% showing severe and 53% mild disease. Anti-CII antibodies of the highest titre, the lowest functional affinity, and the greatest affinity heterogeneity were associated with the development of the severest arthritis in DBA/1 mice: even in DBA/1 mice with moderate or no disease the amount of antibody and heterogeneity were higher and functional affinity lower than in either HA or LA mice. Antibodies of the G1, 2a, 2b and 3 subclasses were produced in all mice, and none of these alone accounted for the overall difference in IgG antibody titres or affinity in the groups of mice. Antibodies of the IgG2a subclass showed the closest association with the development of arthritis in the different groups. It is concluded that anti-CII antibodies of low functional affinity, and presumably also of the IgG2a subclass, influence the disease process in collagen arthritis.