[ANTITOXIC ANTISTAPHYLOCOCCAL IMMUNITY IN PUERPERANTS AND NEWBORN INFANTS IN CONNECTION WITH THEIR IMMUNIZATION WITH STAPHYLOCOCCAL ANATOXIN].
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The need for a reference cholera antitoxin to serve as a standard for the calibration of cholera enterotoxin and toxoid as well as for measurement of the antitoxin response in animals and patients was recognized by the NIH Cholera Advisory Committee, NIAID, DHEW, USA. Two cholera antitoxins have been used for several years as provisional references, but neither was considered to embody all of the properties of an ideal standard. Accordingly, a lot of cholera antitoxin was prepared by immunization of goats with a formalinized, highly purified cholera toxin adsorbed on aluminum phosphate adjuvant. Booster injections were given at 8, 16 and 24 weeks. Plasma samples obtained from the 25 to 27 week bleedings were converted to serum, pooled, and freeze-dried. This serum possessed both high and constant toxin neutralizing activity in rabbit skin, rabbit ileal segment, mice, Y-1 adrenal cells and Chinese hamster ovary cells, and had high avidity by the rabbit skin assay. Hemagglutination tests gave identical values. It was shown to be highly specific by gel diffusion, but flocculation was relatively poor. On the basis of specificity, high avidity and toxin-neutralizing capacity this goat antitoxin (NIH Lot 1) was considered superior to previous provisional standards and is proposed as a satisfactory standard reference reagent.
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Antibody neutralisation and toxin A elution experiments showed that toxin A uptake from rabbit intestinal lumen was a continuous process. The kinetics of the ileal and colonic responses were significantly different; a much longer incubation (4 h) with toxin was required for colon, compared with 45 min for the ileum, to induce fluid accumulation at 12 h. Fluid secretion was induced only when toxin had gained access to deeper tissues, probably achieved by several toxin uptake-tissue damage cycles. Toxin A induced haemorrhage in both ileal and colonic tissues. In ileum, the villus architecture was severely damaged and this gave rise to protein-rich bloody luminal fluid. In the colon, although colonocytes were removed, the basement membrane remained intact; this resulted in a tissue-localised haemorrhage and a protein-low watery ultrafiltered luminal fluid. Toxin A is thus a novel type of histotoxic enterotoxin.
A column chromatography method was employed to obtain a monospecific botulinus type A toxin. The latter was found to be a homogeneous product via column chromatographic and electrophoretic studies. The immunodiffusion test after Ouchterlony with a serum obtained with the use of a native toxin yielded one precipitation line. No such lines were produced with a serum obtained from animals immunized with a type B toxin. The purified toxin could be detoxicated by routinely applied methods. When injected to rabbits it induced the production of monospecific antibodies.
Serum samples from 101 individuals were titrated for Corynebacterium diphtheriae antitoxin by an IgG-specific enzyme-linked immunosorbent assay (ELISA) and a neutralisation test in tissue culture (TC). In some of the sera, the concentrations of antitoxin determined by the two assays were different; antitoxin values in these sera were titrated again by neutralisation tests in guinea pigs (GNT). Antitoxin concentrations of greater than 0.01 IU/ml by GNT partly corresponded to values obtained in both ELISA and TC. Only the values from TC agreed with lower GNT results. Heat inactivation of sera was investigated and rejected as a possible reason for the discrepancy in the results. ELISA can be used to detect levels of less than 0.1 IU/ml, although the accuracy below 0.01 IU/ml, often considered a protective level, is questionable. At higher levels ELISA was reproducible for the titration of diphtheria antitoxin in human sera and offers a useful alternative to both in-vivo assays and TC.
Hybridomas producing monoclonal antibodies against type A Hall strain Clostridium botulinum toxin were generated by fusing mouse myeloma cell line P3-X63-Ag8.653 with spleen cells of Balb/c mice immunized with C. botulinum type A toxoid. The monoclonal antibody from one hybridoma, identified as No. 424, was selected from 61 others for its high antibody titre. This monoclonal antibody was used in a double-sandwich enzyme-linked immunosorbent assay (ELISA) system to detect type A toxin in culture fluids and in foods. The monoclonal antibody did not react with either C. botulinum toxin types B, C, D, E and F or with other clostridial species tested. This particular monoclonal antibody (No. 424) did not neutralize type A toxin in the mouse bioassay procedure but detected approximately 10 mouse lethal doses of type A toxin/ml culture fluid. Monoclonal antibody and rabbit antitoxin to type A C. botulinum toxin were useful in a double-sandwich ELISA for the rapid and specific detection of type A toxic fluids in culture and in food samples.
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Schaeffer, W. I. (Massachusetts Institute of Technology, Cambridge), J. Gabliks, and R. Calitis. Interaction of staphylococcal enterotoxin B with cell cultures of human embryonic intestine. J. Bacteriol. 91:21-26. 1966.-The cytotoxic effect of staphylococcal enterotoxin B upon human embryonic intestine cell cultures is characterized by retraction of cells from the monolayer. This is followed by clumping of the retracted cells to form clear areas in the monolayer and finally by sloughing of the clumps from the glass surface. The 50% effective dose of the toxin, determined by protein analysis of the cultures used in titration studies, was found to be between 40 and 60 mug/ml. The cytotoxic property of the enterotoxin was completely neutralized by 3.9 x 10(-5) ml of specific antitoxin per mug of toxin. The cytotoxicity was found to be slightly enhanced by 2.2 g of bicarbonate per liter of Eagle's basal medium (Earle's salt solution level), the absence of serum, the absence of penicillin and streptomycin, and the presence of 2.8 mmoles of calcium in the medium. The cytotoxicity was profoundly influenced by the age of the culture. No cytotoxicity was evident until after 2 days of growth had taken place, when the cell number was approximately 4.0 x 10(5) cells per culture.
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