Immunization of Laboratory Animals

Dilute the protein solution to a concentration corresponding to the 1.5-fold of the optimal dilution. Pour 1 vol. of the diluted protein into a centrifuge tube and quickly add 5 vol. of dialyzed gold colloid. Vortex and incubate at RT for 15 min. Add 0.1 vol. Soln. C per 10 rrd protein gold mixture and vortex again. 

Spin with 11 000 x g at RT for 30 min and resuspend the pellet into a volume of Soln. D corresponding to the original volume of the gold sol. Spin again and carefully resuspend the pellet in 1/10 volume of Soln. D. 

The red solution is stable at 4 °C for 2-3 weeks. If sediment occurs, spin with 500 x g for 5 min and discard the pellet. 

Geoghegan WD, Ackerman GA (1977) J Histochem Cytochem 25 1187 Goodman SL, Hodges GM, Livingston DC (1980) Scanning electron microscopy 1980 11 133 

As conjugates, complexes from biotinylated hapten and strep-tavidin maybe used. In that case perform the detection with avidin-biotinylated hapten because avidin and streptavidin do not cross-react. 

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Benzylpenicillenic acid, which forms readily from benzylpeniciUin in aqueous solution, is unstable and is thought to be allergenic, particularly in contact skin allergy, after direct reaction with disulfides and cysteine sulfhydryl groups (Fig. 5.6). Immunization of laboratory animals with penicil-lenate-protein and penicilloyl-protein conjugates... 

The field of DNA vaccination started when eukaryotic expression vectors were injected into the muscle of laboratory animals [2]. The authors observed protein expression for more than 2 months after injection and noted that no special delivery system was required to obtain this expression. Subsequently, it was demonstrated that antibodies can be induced simply by injecting plasmid DNA into the muscle of mice [3]. Subsequent studies found that the injection of expression plasmids also leads to the induction of a cytotoxic T-cell response. After injection, the DNA enters cells of the vaccinated host and the encoded gene becomes expressed. This eventually leads to the induction of a cellular cytotoxic T-cell, T-helper, and/or humoral (antibody) immune response. 

Dioxins are of concern because they accumulate in the biosphere, where they have highly deleterious effects. Tests have shown that when the concentration of dioxins in the blood of laboratory animals reaches a critical level, reproductive and immune system defects result. Moreover, recent data indicate that the concentration of dioxins in the blood of the average U.S. resident has nearly reached that level. A major reason is that dioxins are not veiy water-soluble, so they accumulate in the body rather than being readily processed and excreted. Consequently, several groups, including the American Public Health Association, have issued calls for phasing out the use of industrial chlorine. 

Vos JG, Moore JA, Zinkl JG. 1973. Effect of 2,3,7,8-tetrachlorodibenzo-p-dioxin on the immune system of laboratory animals. Environ Health Perspect 5 149-162. 

There are, however, in vivo methods of obtaining mAbs, and these produce much larger quantities. One of these is intraperitoneal administration of the hybridomas into histocompatible animals (of the same cell line as the parents of the hybridoma) or immunodeficient animals (individuals with no functional immune system). These receptor animals will develop ascitic tumors containing from 1 to 40 mg/ml of the mAb secreted by the hybridoma (Kretzmer, 2002). However, despite the fact that this method is well documented and has been used widely in the past, the procedure is presently being rejected because of ethical concerns over the use of laboratory animals. 

Embryonic exposure to vinclozolin, an antiandrogenic endocrine disruptor, has been shown to promote prostate disease, kidney disease, immune system abnormalities, testicular abnormalities, breast and other tumor development, and a number of blood abnormalities in the F1-F4 generations of laboratory animals. I8,91 The effects observed were noted in the adults of the four ensuing generations that followed the exposure. 

Purification processes require several steps in order to obtain a commercially pure product. Affinity chromatography is therefore extremely useful, but until a few years ago, was limited to antibodies produced by the immune systems of laboratory animals. However, antibodies often cannot discriminate between closely related impurities. In addition, the drastic sanitation conditions used in the production of therapeutic products may denature the antibodies. Phage display technology allows the isolation of affinity ligands with the required physical and chemical properties. This technique can also discriminate between the target and closely related impurities. Small peptides bound to resins are well suited for use in purification of proteins that are normally used as drugs. Once a peptide with... 

Sensitization and Irritation ISO 10993-10 are two more test that are required under ISO 10993-1. Sensitization or hypersensitivity reactions usually occur as a result of repeated or prolonged contact with a chemical substance that interacts with the body s immune system. Because most such reactions to biomaterials have been of the dermal cell-mediated type, rather than the humoral or antigen-antibody type, the skin of laboratory animals is used in sensitivity testing. Dermal sensitization reactions are marked by redness and swelling. The guinea pig, a species known to be nearly as responsive to dermal sensitizers as human beings are used in the sensitization test. 

Other investigators have been unable to demonstrate lead-induced effects on various components of the immune system in laboratory animals. The effects of lead exposure of varying duration on natural killer cell and T-lymphocytc function were investigated in rats. Male Alderly Park rats received lead as lead acetate in the drinking water at lead concentrations equivalent to 14.3 and 143 mg lead/kg/day for... 

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