Preparation of antisera

Until now, R-specific determinants or carrier linked artificial antigens of the Rc to Re types have not been synthesized, although their availability might prove most useful. In contrast to killed bacteria which are presently used for the preparation of antisera, synthetic immunogens would be non-toxic. Further, one would be able to investigate mono-specific sera and to identify those R determinants which are most effective in protection. 

There are few instances in which categorical evidence has shown one species of common laboratory animal to give consistently better responses than another to any particular immunogen. Some fairly well know exceptions are the superiority of guinea pigs for production of antiinsulin sera (presumably because the endogenous hormone in this species is most unlike the other mammalian insulins) and of horses for preparation of antisera for immunoelectrophoresis. The latter preference is due to the... 

Various preparations of antisera to atropine have been reported. A racemic hemisuccinate ester was prepared and conjugated to bovine serum albumin by the carbodiimide technique. Antisera formed to the original immunogen selectively bound the R isomer (34), but a later antiserum prepared by this approach was reported to bind both R and S forms with "equal efficiency" (35). R, S-atropine was treated with diazotized p-amino-benzoic acid, and the resulting compound (which was not further characterized) was used for conjugation to bovine serum albumin by means of a carbodiimide-mediated reaction. Antisera resulting from use of this material were quite selective for the R isomer, with a cross-reaction of only about 2% for the S isomer (36). Virtanen et al. followed this procedure with S-atropine. Their antiserum bound equally to S- and R,S-atropine, as measured by displacement of tritium-labeled R,S-atropine (37). In another study (31), both racemic atropine and the S isomer were coupled to human serum albumin by the technique of Wurtzburger et al. (36), Antisera were obtained that were selective for both the R and S isomers (33). 

Native calmodulin has in general proven to be a poor antigen for the reproducible preparation of antisera of useful titer and of high affinity, although there have been some reported successes (43). Most of the antibodies which have been prepared against either native or derivitized calmodulin have not differentiated between the Ca -free and Ca +-saturated conformation. 

Preparation of Antisera Bleed at day 10 after final immunization. Keep the blood at room temperature for a while and then centrifuge at 1500g for 20 min twice. Collect the supernatants (antisera) and keep at - 20°C. 

L. Chen, N. Zhang, B. Adler, J. Browne, N. Freleen, M. Pierce, Preparation of antisera to recombinant, soluble N-acetylglucosaminyltransferase V and its visualization in situ, Glyco-conjugate J, 1995, 12, 813-823. 

The simplicity of this assay makes it highly attractive for further development. Some of the limitations include the need for the preparation of relatively large amounts of appropriately modified DNA for the initial immunization. This may be reduced with the development of in vitro immunization techniques in which as little as 5ng of antigen can be effective. The antibodies must also be characterized once prepared. For example, a variety of monoclonal antibodies against B[a]PDE-modified DNA have been prepared (42) which show varying specificity. At one extreme, some require the full structure of the adduct bound to DNA, at the other, BtalP tetraol will effectively compete. What is not clear at the moment is how specific such antisera are for a particular PAH. Will these antisera recognize only B[a]P tetraol structures or those of any diol epoxide modified DNA ... 

Antibody preparations used to induce passive immunity may be obtained from either animal or human sources. Preparations of animal origin are generally termed antisera , and those sourced from humans are called immunoglobulin preparations . In both cases, the predominant antibody type present is IgG. 

The Western blot method is often used in the analysis of host cell impurities. It can be used to identify a recurring impurity. O Keefe et al. used a Western blot to identify an E. coli protein impurity in the preparation of the recombinant fibroblast growth factor (aFGF).29 By using specific antisera to the E. coli host cell proteins, they were able to isolate the impurity and determine its N-terminus amino acid sequence to confirm its identity. Antibodies could be used to determine the concentration of this impurity in sample preparations. 

Aqueous extracts of cotton dust and cotton bract induced the formation of specific precipitating antibodies in rabbits. The antisera cross-reacted with both extracts as well as with extracts of cotton stem, leaf, and burr, baled cotton and gin trash. Cross-reactivity was also demonstrated with extracts of flax, soft hemp, sisal, and jute. No antigen-antibody reaction was obtained with extracts of cottonseed hulls, cottonseed proteins, noncontami-nated cotton lint, or house dust. No reaction was obtained between the antisera to dust and several connmercial preparations of bacterial lipopolysac-charides believed to be present in cotton dust. 

Application of the RIA. The RIA was initially used to evaluate the cross-reaction of the STXOL antisera to STX. The antibody was found to have excellent STX cross-reactivity (93%). Subsequent preparation of a logit/log (37) standard curve for STX (Figure 4) demonstrated that the chosen assay format would give good reproducibility and a desirable order of magnitude linear sensitivity range. 

Cooper HM, Paterson Y (2000) Preparation of polyclonal antisera. Current protocols in molecular biology. Wiley, Unit 11.12 Green JA, Manson MM (1992) Production of polyclonal antisera. In Man-son MM (ed.) Immimochemical protocols. Methods in molecular biology, vol. 10. Humana Press, Totowa, N.J., p 1 Harlow E, Lane D (1988) Antibodies. A laboratory manual. Cold Spring Harbor Laboratory, p 92... 

Serological Properties. Antisera prepared against purified preparation of Acineto-bactor pyrocatechase cross-react and inhibit the enzyme activity in the crude extracts prepared from other strains of Acinetobactor calcoaceticus, but fail to cross-react and inhibit isofunctional enzymes prepared from strains of Pseudomonas, Nocardia, and Alcaligenes114. ... 

Butler, J. E. and Maxwell, C. F. 1972. Preparation of bovine immunoglobulins and free secretory component and their specific antisera. J. Dairy Sci. 55, 151-164. 

The development of an O serotyping scheme is complex. Heated O antigen preparations of unknown cultures are tested first for agglutination on slides with droplets of pooled, polyvalent O antisera. If agglutination occurs with one of the pooled antisera, the heated suspensions are then tested for agglutination using the individual O antisera contained in that pool. Next, for positive reactions, the heated... 

Complement-mediated binding may occasionally be a cause of background in frozen tissue when whole antisera are used. However by the time large pools of antisera have been prepared for use, several of the complement factors are usually inactivated. 

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