Complex nature of the human antisperm antibody response in SCID mice.

Human peripheral blood mononuclear (PBMs) cells were introduced into the peritoneal cavity of severely-combined immunodeficient (SCID) mice in concentrations of 2.5-4.0 x 10(7) cells per mouse. Whole mononuclear cell suspensions were used either unstimulated or following primary in vitro culture with human spermatozoa. In some experiments, immunodepletion of CD8(+) cells was carried out prior to grafting. Lymphocytes were obtained from nonsensitized (to antigen) human subjects or from individuals who were primed in vivo (vasectomized individuals in case of sperm antigens). An enzyme-linked immunosorbent assay was employed to assess total human immunoglobulin (G or M) levels as well as the specificity of the antibodies generated. We have been successful by generating primary and secondary immune responses with 'naïve' human lymphocytes, challenged with chlamydia or ovalbumin but without adjuvant or CD8(+) immunodepletion; however, we were unable to induce specific antibodies to spermatozoa under this regime in SCID male mice. We then employed female SCID mice, treated with sperm antigen extracts (glycosylated or deglycosylated) encapsulated in liposomes and human lymphocytes obtained from 'naïve' or pre-sensitized in vivo subjects. It was found that the most pronounced humoral response to sperm antigens was obtained with deglycosylated antigens and PBMs from vasectomized (in vivo pre-primed to spermatozoa) individuals. A presented SCID mice model can be helpful at understanding of antisperm antibody development and the molecular nature of generated antibodies to modified sperm antigenic entities.

Immunoneutralisation of GnRH-I, without cross-reactivity to GnRH-II, in the development of a highly specific anti-fertility vaccine for clinical and veterinary use.

In recent years, several forms of gonadotrophin releasing hormone (GnRH) molecules have been isolated from primate brain. These molecules are very similar in sequence and this raises the question of whether previously developed neutralisation vaccines based on GnRH (now termed GnRH-I) would remove other forms of GnRH (namely GnRH-II) as well. As the function of these other molecules has not yet been clearly defined, potential health risks could exist by their ablation. In view of the high sequence homology between the molecules, this paper describes the production of highly specific polyclonal antibodies against GnRH-I and GnRH-II, with negligible cross-reactivity. The ultimate aim of this is to develop an anti-fertility vaccine which does not present any inappropriate side-effects, caused by neutralisation of a GnRH molecule which may or may not be directly involved in reproduction. Several formulations were investigated, based on analogues of the following molecules, conjugated to tetanus toxoid: 1. GnRH-I pGlu-His-Trp-Ser-Try-Gly-Leu-Arg-Pro-Gly-NH2 and 2. GnRH-II pGlu-His-Trp-Ser-His-Gly-Trp-Tyr-Pro-Gly-NH2. The specificity of the antibodies produced was examined, together with effects on fertility and any inappropriate side-effects. Immunostaining of hypothalamic sections was carried out, using the generated antisera, to determine the regional distribution of GnRH-I and GnRH-II neurones, as well as to further evaluate the specificity of the antibodies.

Characterization of monoclonal antibodies with specificity for the core oligosaccharide of Shigella lipopolysaccharide.

Monoclonal antibodies (MAbs) were prepared against different strains of Shigella, following immunization of BALB/c mice with a heat-killed preparation of Shigella. Antibody-producing hybridomas were screened in an indirect enzyme-linked immunoadsorbent assay (ELISA) and epitope specificity determined using chemically defined lipopolysaccharide, lipid, and KDO fragments. Five MAbs were characterized and the following specificities identified: 2C32E6 and 4D64B9 (reactive to S. flexneri and S. boydii), 5E45D8 (reactive with S. flexneri), 4B33D10 and 1B52F10 (all species of Shigella). The properties of 1B52F10 revealed its potential importance in immunological detection of Shigella from unknown samples, as it was able to bind to all strains of Shigella.

Cardiac myofibrillar proteins: biochemical markers to estimate myocardial injury.

Ischaemic heart disease represents the most common of the serious health problems in the contemporary society and acute myocardial infarction (AMI) is the major cause of cardiovascular morbidity and death. The accurate localization and determination of the infarct size and the volume of myocardium at risk at the time of insult is crucial and vital for the choice of treatment. Initially the ischaemic cells are reversibly injured. However, if these changes are not reverted at the earliest, it results in the death of the myocyte. This irreversible myocyte necrosis travels transmurally towards epicardium in the form of a wavefront. A timely intervention during evolving infarct could reduce and delimit the infarct and preserve the left ventricular function. Enzyme analysis and electrocardiography (ECG) along with the clinical history of the patient is still considered to constitute a reliable triad in the diagnosis of myocardial infarction (MI). Efforts have been made to relate infarct size with the serum enzyme level changes without much success. In addition, a number of specialist techniques such as planar radioisotope imaging, single photon emission computed tomography (SPECT), positron emission tomography (PET), Echocardiography, Ventriculography and nuclear magnetic resonance (NMR) imaging have been devised to support diagnosis in the patients who show ambiguous symptoms and ECG findings. However most of these procedures are unavailable to the patients due to economic reasons while others have suffered due to non-availability of ideal radiopharmaceuticals. Major advances have been made in the methods based on immunological techniques to improve the detection and estimation of infarct. These methods are exclusively based upon the production and availability of specific antibodies against intracellular, cardiac specific components.

Anti-gonadotropin releasing hormone vaccines and their potential use in the treatment of hormone-responsive cancers.

Gonadotropin-releasing hormone (GnRH) and its analogues have been used clinically to treat a range of hormone-dependent conditions. It is often necessary for large, toxic and expensive drug doses to be administered. Improvements in drug delivery have necessitated new developments in formulation, but these in turn can induce new adverse effects. Immunological neutralisation of GnRH has been examined as a less toxic and cheaper replacement therapy, and has been studied closely in different animal species. However, only a few clinical trials have been carried out with respect to hormone-dependent cancers. Based on clinical trials of the free peptide drug in cancer patients, it would appear that there is an increasing trend towards using GnRH and its analogues in adjuvant therapy and that antibody-based GnRH neutralisation will have a role in this treatment regimen.

Fertility-disrupting potential of synthetic peptides derived from the beta-subunit of follicle-stimulating hormone.

PROBLEM: Hormone immunoneutralization is hampered by immunologic cross-reactivity caused by close-sequence homology between related molecules. One solution is to use smaller fragments to induce antibodies of greater specificity. METHOD OF STUDY: A number of peptides selected from beta-follicle-stimulating hormone (FSH) were conjugated to tetanus toxoid and were used to immunize female rats. The antisera were examined for FSH cross-reactivity by immunoassays and in an in vitro bioassay. RESULTS: In the immunoassays, the antisera did not react with FSH but did react with their respective peptides. In the bioassay, sera from VYKDPARPC- and CDSLYTYP-immunized animals inhibited FSH-receptor interaction by 73% and 68%, respectively. These animals also showed reduced estradiol levels. Sequences were synthesized around VYKDPARPC and were tested on a FSH-receptor-bearing Chinese hamster ovary cell line. LVYKDPARPC, VYKDPARPC, YKDPARPIC, CLVYKDPARP, and LVYKDPARP inhibited FSH-receptor interaction by greater than 50%. In female mice, TRDLVYKDPARPKI and LVYKDPARP disrupted estrous cycling in all animals; LVYKDPARPC and CLVYKDPARP disrupted cycling in three of five animals, whereas VYKDPARPC disrupted cycling in one of four animals. CONCLUSIONS: Peptides from two areas of beta-FSH (VYKDPARP and DSLYTYP) were shown to raise FSH-neutralizing antibodies, which were able to suppress estradiol levels. An additional leucine residue to VYKDPARP greatly enhanced the peptide's ability to inhibit FSH-receptor binding and caused fertility disruption in vivo.

Investigation into suitable carrier molecules for use in an anti-gonadotrophin releasing hormone vaccine.

Gonadal function can be controlled through immunoneutralisation of gonadotrophin releasing hormone (GnRH), with an analogue, GnRH-glycys, linked to a carrier molecule and an appropriate adjuvant. In this study, four different types of carrier molecule were investigated: (a) single and branched amino acid polymers--[poly-(D-glu, D-lys) and poly-(phe, glu)-poly(DL-ala)-poly(lys)]; (b) bacterial toxoids--diphtheria (DT) and tetanus (TT); (c) synthetic T-helper epitopes--derived from malarial circumsporozite protein (CS) and measles virus fusion protein (MVF); and (d) thyroglobulin (Thy)--a large protein. The effect of non-ionic surfactant vesicles (NISV) and an aluminum hydroxide based adjuvant (alum), was also examined. Although good antibody responses were achieved with GnRH-glycys-DT, GnRH-glycys-TT and GnRH-glycys-Thy, adsorbed onto alum and the dimerised synthetic T-helper epitope constructs, incorporated into NISV, a critical antibody titre was necessary to result in morphological changes in the gonads and complete suppression of spermatogenesis. This was only achieved with tetanus toxoid and the dimerised T-helper epitopes.

The evolution of immunoassay technology.

Since they were first utilized, immunoassays have witnessed phenomenal growth in the range and scope of their applications. A vast array of different labels and assay strategies has been developed to meet the requirements of sensitivity, accuracy and convenience. The development of increasingly sensitive labels and detection equipment has seen a drastic improvement in the sensitivity of immunoassay systems, allowing an ever-increasing range of analytes to be measured accurately. At the same time, simple to use, inexpensive assay systems have been developed with the necessary reliability, accuracy and sensitivity to bring immunoassay technology to much more diverse areas such as home testing, near-patient monitoring, and large screening programmes in developing countries. Recent developments in molecular biology techniques have made possible the production of fusion antibody conjugates, which can lead to further improvements in sensitivity and cost of reagents, as well as possibly revolutionizing the production of monoclonal antibodies. However, dissatisfaction with various aspects of existing immunoassay technology will necessarily lead to the continued development of this already widely diverse subject.

Radiolabelled monoclonal antibodies (McAb): an alternate approach to the conventional methods for the assessment of cardiomyocyte damage in an experimental brain-death pig model.

The present study was carried out to determine the possible use of cTn-I in the cardiac myofibrillar architecture, as a potential target for in vivo radioimmunodetection of cardiac damage in a brain death pig model. Radioiodination of the anti-cTn-I 5F4 McAb was carried out by lactoperoxidase method. The percentage iodine incorporation achieved was 70-75%. The radioiodinated McAbs were purified on Sephadex G-25 column and characterised by Paper chromatography, Phast Gel electrophoresis and electroimmunoblotting. Radioiodinated anti-cTn-I 5F4 McAbs were employed alongside Pyrophosphate (Tc99m-PPi) and Thallium201 chloride (Tl201) in 24 landrace pigs (brain-dead = 18 & sham-operated = 6). The percentage cardiac uptake of the radiolabelled antibody injected dose was significantly higher in the brain dead animals (0.196%) as compared to that of sham-operated animals (0.11%). Specific in vivo localization of radiolabelled McAbs in the infarcted cardiac tissue was confirmed by computer-aided reconstruction of 3-D images of the isolated heart. The preliminary results of the study revealed preferential uptake of radiolabelled antibody at the site of myocyte damage resulting from artificially induced brain death.

Immunoneutralisation of gonadotrophin releasing hormone: a potential treatment for oestrogen-dependent breast cancer.

The aim of this study was to assess the therapeutic potential of active immunisation with GnRH-glycys-PPD in a hormone-dependent experimental model. Mammary tumours were induced in female rats using dimethylbenzanthracene (DMBA) and the effects of GnRH immunoneutralisation on tumour development were evaluated. High titres of anti-GnRH IgG correlated with a decrease in oestrogen levels and subsequent tumour suppression. A comparison of immunised and non-immunised animals showed that when GnRH-specific IgG levels were at a maximum titre (80-100 micrograms/ml), nearly 10% of the GnRH-glycys-PPD treated animals showed mammary masses, compared with all the non-treated animals at the same stage in the study. When the antibody levels fell, tumour regrowth was observed, but to a level below that observed in the non-treated animals. Following further treatment with the analogue, the tumours regressed again, showing their retention of hormone dependency. This is consistent with other endocrine manipulations in the treatment of breast cancer; the advantages of immunisation with GnRH-glycys lies in its non-toxicity and reduction in side-effects, which were mainly adjuvant-induced.

Effects of adjuvant, dose and carrier pre-sensitisation on the immunisation efficacy of a GnRH analogue.

A GnRH-neutralising vaccine, with potential applications in the treatment of human sex hormone-dependent disorders, was developed by conjugating GnRH-glycys to tetanus toxoid. An evaluation of adjuvant, dose and carrier pre-sensitisation was made. Male rats immunised with the conjugate, adsorbed onto alum, showed higher anti-GnRH antibody levels and suppressed testosterone concentrations, compared with animals immunised without adjuvant. Conjugate administration in a four injection regime proved to be the most effective in disrupting fertility, as assessed by the degree of lowered testosterone levels and gonadal atrophy. Pre-sensitisation with tetanus toxoid had an initial marked effect on immunisation, observed following 2 drug doses; the pre-sensitised animals showed a lower antibody response to the conjugate than did the non-primed animals. However, as the number of drug doses increased to 4, there was no significant difference between the primed and non-primed animals.

An investigation into the immunogenicity of a GnRH analogue in male rats: a comparison of the toxicity of various adjuvants used in conjunction with GnRH-glycys.

Immunization of male Copenhagen Fischer rats with a gonadotrophin releasing hormone (GnRH) analogue, conjugated to PPD resulted in high levels of antibody being produced which disrupted gonadal function in male rats. The antibody reduced serum testosterone levels and subsequently suppressed spermatogenesis. Alternatives to Freund's adjuvant were tested, namely, aluminium hydroxide and non-ionic surfactant vesicles (NISV). The study showed that aluminium hydroxide was as effective as Freund's adjuvant and less toxic, in both BCG and non-BCG primed animals. However, NISV were completely non-toxic and most effective in conjunction with BCG priming. The data obtained showed that NISV have the potential to be used as an alternative to FCA and aluminium hydroxide-based adjuvants.

Production and characterisation of anti-cardiac troponin-I monoclonal antibodies.

Cardiac troponin-I (cTn-I) was isolated from bovine left ventricular tissue and used as immunogen. Sixteen murine hybridoma lines were produced with two of them, 1D12 and 5F4, showing a high specificity for cTn-I; both of these monoclonal antibodies (McAbs) were isotyped as IgG1 with kappa-light chains. The specificity of the McAbs for cTn-I was confirmed by ELISA, western blotting and by the ability of the antibodies to block actomyosin ATPase inhibition by cTn-I. The McAbs may be useful for human in vivo imaging of myocardial infarcts and other pathological conditions related to cardiac myocyte damage.

A novel method for boronating antibodies without loss of immunoreactivity, for use in neutron capture therapy.

The search for suitable boron containing compounds for 10B neutron capture therapy (BNCT) is based on the principle that boron atoms must be delivered specifically to tumour cells at a concentration high enough to be effective without being toxic to normal cells. Specificity may be achieved through monoclonal antibodies. However, it has been difficult to conjugate large numbers of boron atoms to the antibody molecules without inactivating them. We have devised a strategy to do this indirectly through the use of a boronated glutamate-lysine polymer in conjunction with biotin and streptavidin.

Development of a hormone neutralizing vaccine, using GnRH-glycys-PPD, for use in the treatment of oestrogen-dependent disorders.

The aim of this study was to develop an effective and nontoxic vaccine, suitable for use in humans, which was capable of effectively controlling oestrogen levels. Female Sprague-Dawley rats were immunized with a conjugated analogue of gonadotrophin releasing hormone, GnRH-glycys-PPD. This resulted in high levels of neutralizing antibody which disrupted GnRH function and consequently caused a reduction in serum oestrogen. The effect of oestrogen deprivation correlated well with ovarian failure and gonadal atrophy. An examination was made of various adjuvants in conjunction with the analogue to determine the suitability of the combinations in the formulation of an effective human vaccine. This investigation included a novel adjuvant, non-ionic surfactant vesicles (NISV); the results showed that NISV are completely nontoxic and in terms of potentiating and sustaining an immune response, compare favourably with Freund's adjuvant and alum. In addition the long term effects of immunization were investigated and the data showed that immunoneutralization of GnRH effectively suppresses fertility on a long-term basis.

Accurate determination of adjuvant-associated protein or peptide by ninhydrin assay.

Modern peptide and subunit vaccines are increasingly having to rely on the use of immunological adjuvants to achieve effective immunity. However, the only adjuvant currently approved for use in humans is aluminium hydroxide, although many adjuvants are currently under preclinical development. Determining immunogen concentration in the presence of adjuvants such as aluminium hydroxide gel, liposomes or NISV has proved to be problematic. One approach has been to use radiolabelled antigens to extrapolate concentration to a preparation using native immunogen. However, the use of a colorimetric assay would allow greater flexibility in terms of immunogen used and would reduce costs and remove safety problems. Of the colorimetric methods we have examined thus far, only the manual ninhydrin assay has produced consistent results with detection of microgram quantities of protein or peptide in the presence of NISV or Alhydrogel, but not liposomes. As the assay relies on the detection of free amino groups after protein hydrolysis, peptides as well as proteins may be effectively determined irrespective of amino acid composition, a considerable advantage over other colorimetric assay systems.