Functional and proteomic comparison of different techniques to produce equine anti-tetanus immunoglobulin F(ab')2 fragments.

Tetanus is still a major cause of human deaths in several developing countries. In particular, the neonatal form remains a significant public health problem. According to the World Health Organization, administration of tetanus toxoid is recommended for neonatal tetanus patients. Furthermore, tetanus antitoxin or anti-tetanus immunoglobulin (Ig) are used for mild case or intensive care. This paper discusses a novel purification technique for improving equine anti-tetanus Ig production. First, equine plasma dealt with two steps salting out with ammonium sulfate; second, ultrafiltration concentration liquid purified by one successive protein G based affinity chromatography steps; finally, the purified F(ab')2 fragments was characterized using biochemical and proteomic methods and shown to be pure and homogeneous. Compared with the original technique product, specific activity increased by 80% (about 90,000 IU/g) and recovery of F(ab')2 is approximately equal 75%. Furthermore, Proteomic profiling of total technique process is demonstrated by nano-HPLC-MS and bioinformatics analysis. New technique to produce equine anti-tetanus immunoglobulin F(ab')2 fragments from crude plasma in high quality and yield. And it also could be used for industrial amplification.

[Sex and age-dependent gaps in tetanus immunization]. / Geschlechts- und altersabhängige Lücken im Tetanusimmunschutz.

To test tetanus immunity, tetanus antitoxin titres were measured in the serum of 692 subjects (354 males, 338 females), aged one day (newborns) to 92 years (mean age 29 years). Those aged 18 to 65 years were first-time blood donors, the remainder were healthy newborns, while the children and those over 65 years were patients without immune-compromising disease. An inadequate protection (titre < 0.1 IU/ml) was found in 107 (15.5%), of whom 75 (70%) were females. Women aged 20 years and above also had significantly lower average antitoxin titres than men (1.7 vs. 3.5 IU/ml); P < 0.0001). The inadequate immunization protection of many young women is reflected in the lack of protective antibodies in 10 of the 49 examined newborns. In addition, 18% of children aged between 1 and 15 years had inadequate immunity against tetanus. In the whole group the titre level decreased with age, while the proportion of unprotected persons increased. Apart from the obvious age and sex dependency of the demonstrated inadequacy of immunological protection against tetanus, attention should also be paid to the lack of protective antibodies in newborns and the marked gaps of immunity among children.

[A gel filtration study of the fractional composition of tetanus antitoxic sera]. / Izuchenie fraktsionnogo sostava stolbniachnykh antitoksicheskikh syvorotok metodom gel'-fil'tratsii.

The fraction composition of tetanus antitoxic sera has been studied by the method of gel filtration in columns packed with Sephadex G-200. Differences in the fraction composition of preparations manufactured by different enterprises have been revealed. The specific activity of antitoxic sera has been found to depend on the degree of their fragmentation.

Establishment of stable mouse/human-human hybrid cell lines producing large amounts of anti-tetanus human monoclonal antibodies with high neutralizing activity.

To establish stable hybrid cell lines producing human anti-tetanus antibody with high toxin-neutralizing activity, peripheral lymphocytes from humans hyperimmunized with tetanus toxoid were, after in vitro antigen stimulation, fused with a mouse/human heteromyeloma or human lymphoblastoid cell line and cloned. Unlike the IgM secretors (six clones), the IgG secretors we obtained (six clones) produced anti-tetanus human monoclonal antibodies with high neutralizing activity (the highest one, cell line G2, 4.3 IU/100 micrograms IgG). Appropriate combinations of three or four kinds of monoclonal antibodies of the IgG type resulted in markedly increased neutralizing activity comparable with that of anti-tetanus human polyclonal immunoglobulin preparations currently used clinically on the basis of toxin-specific IgG content. Five of these cell lines produced 10-20 micrograms of antibody per ml for more than 3 months. The cell line G2 produced 6 mg of antibody per day in serum-free medium in a 500-ml bioreactor in perfusion culture and 13-104 mg in a nude mouse. These cell lines satisfied, for the first time, the minimal requirements for applying human monoclonal antibodies to clinical use.

Use of chromatography for the preparation of homologous tetanus antitoxin.

To obtain immunoglobulin of selected action with a higher titre, techniques for isolating antibodies from a donor's normal immunoglobulin by means of affinity chromatography were used. To obtain an affinity sorbent, tetanus toxoid was used as a ligand. Using an immunosorbent based on the purified ligand, tetanus antibodies were prepared with a specific activity of 65 IU/mg. According to gel-permeation chromatography, the tetanus antibodies obtained were represented mainly by IgG monomer (greater than 90%). The anticomplementary activity of the preparations obtained, being at least 11 mg of protein per 2CH50, does not rule out the possibility of intravenous administration of tetanus antibodies.

Immune response and reactions to various dose regimens for raising hyperimmune antisera in sheep.

To determine the optimum procedure for raising hyperimmune sera to tetanus toxin, three adjuvants, four antigen preparations and two routes of administration in various combinations were investigated in sheep. Oil-in-water adjuvants alone or in combination with aluminum gels were superior to aluminium gels on their own. This disadvantage of aluminium gels was partially but not completely abrogated when the frequency of doses was increased to three per week. Intensity of local reaction was strongly correlated with immune response; the more immunogenic a dose, the more reactive. Reactivity of oily adjuvants could be lessened by use of a more suitable route of administration, thus oily adjuvants appeared suitable for use when administered by the intraperitoneal route even though moderate to severe reactions resulted from subcutaneous injections. Of other variables investigated, toxin did not confer any advantage over toxoid as an immunogen, purified toxoid was a significantly better immunogen than unpurified toxoid and two large bleeds (30% of total blood volume each) every six weeks rather than 20 ml test bleeds did not affect the titre of the hyperimmune serum produced.

A new method for the large scale preparation of antitoxic antibodies exhibiting high specific protective activities.

A method using polyethylene-glycol and immobilized pepsin for purifying heterologous antitoxic antibodies is described. Using horse antitetanus plasma or sera, F(ab')2 fragments exhibiting specific activities in the range of 150 IU per mg protein were repeatedly isolated with a yield around 80%. The procedure was scaled up from 200 ml up to 201.

[Biological properties of immunochemically pure tetanus antitoxin]. / Biologicheskie svoistva immunokhimicheski chistogo stolbniachnogo antitoksina.

Immunochemically pure tetanus antitoxin obtained from enzyme-treated horse serum is less reactogenic and anaphylactogenic and possesses higher therapeutic properties than antitoxin purified by nonspecific physico-chemical methods and containing ballast antigens. Due to its increased persistence in the recipient's body, the immunochemically pure antitoxin induces passive immunity in considerably lower doses than the preparations purified by the method "Diaferm-3".

[Therapeutic effectiveness of tetanus antitoxins purified by nonspecific and immunochemical methods]. / Terapevticheskaia éffektivnost' stolbniachnykh antitoksinov, ochishchennykh nespetsificheskimi i immunokhimicheskimi metodami.

The treatment of experimental tetanus intoxication in 120 rabbits by the intrathecal injection of antitoxins revealed that the therapeutic effect was directly proportional to the dose of antitoxins, expressed in antitoxic units. The maximum effect (the survival rate 78.6%) was obtained by the injection of antitoxins purified on immunoadsorbents and containing no admixtures. The use of such antitoxins ensured the absence of postinjection complications, while commercial antitetanus serum "Diaferm-3" gave only 16.7% survival rate and produced the equal percentage of postinjection complications.

[Preparation of highly potent lyophilized tetanus antitoxin (author's transl)].

The neutralizing antibody titre of tetanus antitoxin could be elevated 3.19 times by the purification of the antitoxin with pepsin digestion method, which is superior to ammonium sulfate salting out method by 1.5 times. But the antitoxin recovery rate by the pepsin digestion method was about 65.37% of the salting out method. Addition of 2% glycine significantly improved the solubility of lyophilized antitoxin. The effective period of liquid antitoxin was 1 year, while that of lyophilized products could be as long as 5 years.

Employment of toxins bound to porous silica beads for isolation of pure antibodies by immunoadsorption.

A simple technique for the isolation of pure antibodies by use of bacterial toxins or toxoids bound to porous silica beads "Spherosil" is described. Employment of toxins instead of toxoids has the advantage of resulting higher yields of purified antibodies.

[Admixtures present in antitoxic horse sera and pepsin preparations]. / Izuchenie primesei, prisutstvuiushchikh v antitoksicheskikh loshadinykh syvorotkakh i preparatakh pepsina.

Antitoxic therapeutic sera produced by the method "Diaferm-3" (devised at the Institute of Epidemiology and Microbiology of the USSR Academy of Medical Sciences) are contaminated in the process of production with group-specific substances and extraneous enzymes contained in crude preparations of swine pepsin. These ballast substances have a negative influence on the process of purification and the stability of antitoxin during the storage of the sera and constitute one of the reasons of their high reactogenicity.

Immunity to tetanus and diphtheria in various age groups of the Polish population.

The concentration of tetanus and diphtheria antitoxins was determined in 279 sera of 18 to 81-year-old women and 509 sera of 22 to 46-year-old men. Tetanus antitoxin content was determined also in 246 samples of fluid from human placentas and 428 lots of commercial human normal immunoglobulins. Immunity to tetanus was clearly age-dependent: in younger age groups the percentage of immunized persons amounted to 90%-100%, in middle-age groups to about 80%, and at age above 60 to about 25%. The findings are in agreement with age-dependent incidence of tetanus, which has become now in Poland a disease primarily of older people. Two groups of the population are now protected against diphtheria: a younger group under 20, and an older one above 40. Immunity in younger age groups was induced by artificial immunization, and among older persons by natural immunization through contact with diphtheria bacilli during severe diphtheria epidemics in the past. Between these groups, gaps exist including 20-30 years of age, who are sensitive to diphtheria.

Passive transfer of antibodies of maternal origin from blood to cerebrospinal fluid in infants.

The demonstration that specific IgM antibodies are present in the serum of infants is useful in the diagnosis of several congenital infections. However, it is less certain whether the detection of antibodies in cerebrospinal fluid (c.s.f.) of infants indicates congenital infection of the central nervous system, because the origins of such antibodies have not been established. In the present study diphtheria and tetanus antitoxins of maternal origin have been detected both in the serum and in the c.s.f. of infants. These observations suggest that an important source of immunoglobulins in c.s.f. is passive transfer of antibodies from serum which should be considered in interpreting serological studies with c.s.f.