Immunization procedure

No medical or therapeutic procedure comes without some risk to the patient. All possible steps are taken to ensure safely, quahty and efficacy of vaccines and immunological products (Chapter 15). The risks associated with immunization procedures must be constantly reviewed and balanced against the risks of, and associated with, contracting the disease, hi this respect, smallpox vaccination in the UK was abandoned in the mid 1970s as the risks associated with vaccination then exceeded the predicted number of deaths that would follow importation of the disease. Shortly after this, in 1980, The World Health Assembly pronounced the world to be free of smallpox. Similarly, the incidence of paralytic poliomyelitis in the USA and UK in 1996 was low but the majority of cases related to vaccine use. As the worldwide elimination of poliomyelitis approaches, there is much debate as to the value of the vaccine outside of an endemic area. 

Public confidence in the safely of vaccines and immunization procedures is essential if compliance is to match the needs the community. In this respect public concern and anxiety, in the mid 1970s, over the peroeived safety of pertussis vaccine led to a reduction in coverage of the target group from ca. 80% to ca. 30%. Major epidemics of whooping-cough, with over 100000 notified cases, followed in 1977/1979 and 1981/83. By 1992, public confidence had returned, coverage had increased to 92% and there were only 4091 reported cases. 

Immunization procedures and schedules vary depending on the laboratory. Usually an initial series of injections is followed by booster injections some weeks later. Animals are generally bled 7-14 days after each booster injection and the characteristics of the serum determined. Serum may be collected or pooled following numerous booster injections and(or) the animal may be exsanguinated. 

A variety of lipid adjuvants and protein mediators have also been shown to influence the immune response to antigens encapsulated in liposomes. The most widely used examples of such adjuvants for practical immunization procedures are endotoxin (including lipid A and lipopolysaccharide) and numerous types of lipophilic derivatives of muramyl dipeptide. 

Furthmayr, H. (1982). Immunization procedures, isolation by affinity chromatography and seriological and immunochemical characterization of collagen-specific antibodies. In Furthmayr, H., ed. immunochemistry of the Extracellular Matrix, Vol. 1. CRC Press, Boca Raton, FL. 

Apart from the spleen, other lymphoid tissues, such as tonsils and the mesenteric or popliteal lymph nodes, can be used as a source of lymphocytes. In the preparation of MABs of human or veterinary origins it is often not possible to obtain lymphoid tissue, and there have been many reports of the successful use of lymphocytes separated from peripheral blood. In some cases, for ethical or practical reasons, it is not possible to immunize the lymphocyte donor, as when human MABs are required, or acutely toxic antigens are used. Also, antigen is not always available in sufficient quantities to perform a successful immunization in vivo. In these circumstances, it may be possible to perform the boosting stage or, indeed, the entire immunization procedure on the lymphocytes in vitro. 

The generation of polyclonal antibodies to an antigen of interest is an important technique applicable to many areas of biological research. In this chapter, we describe a basic immunization procedure designed to generate polyclonal antisera in rabbits and two methods that are commonly employed in the subsequent preliminary characterization of antipeptide antibodies raised in this way. 

Two immunization procedures designed to enhance the immune response to multiple antigen mixtures have been reported recently. The cascade immunization technique (20) utilized in vitro depletion of E. coli proteins (ECPs) which had previously elicited an antibody response. The removal of these dominant immunogens from the mixture was accomplished by immunoabsorption with antibodies obtained from an earlier antiserum. The passive immunization procedure (21) relied on in vivo blocking of strong immunogens by the concurrent administration of early antiserum obtained previously. This latter report demonstrated the presence of an apparently poorly immunogenic ECP to which a humoral response could only be elicited by this passive procedure. 

Given the potential of these methods to improve antibody production to multiple antigen mixtures, we performed a comparison of these two methods to a conventional immunization procedure reported previously (6). In addition, we selected two dimensional electrophoresis and immunoblotting as a tool to examine the production of antibodies to minor components of the ECP mixture. 

The first group underwent a conventional immunization procedure (6) in which one half mg of ECPs were administered subcutaneously in Complete Freunds Adjuvant (CFA) on day one and in Incomplete Freunds Adjuvant (ICFA) on day 7. The rabbits were boosted every 14 days for 160 days and serum collected 7 days after each boost. 

A second group underwent the passive immunization procedure. The protocol was similar to the conventional immunization procedure except injections of antigen also included a concurrent intravenous injection of 0.5 ml of serum obtained from the same individual rabbit seven days earlier. 

The third group underwent a modification of the cascade immunization procedure (24). After a primary injection of ECP in CFA and a subsequent injection in ICFA a serum sample was taken seven days later and the IgG fraction used to prepare an affinity column. The entire ECP mixture was passed over the column and fractions which were depleted of one or more ECPs (as determined by silver stain SDS-PAGE and compared to the starting preparation) were used for the subsequent immunization injection. This procedure of ECP adsorption was repeated with subsequent antisera obtained on days 28 and 42 and the resulting depleted ECP fractions used for injections on either days 35 or 49 and 62, respectively (Figure 3). Thereafter the rabbits received injections as described in the normal immunization procedure. 

Figure 4. Separation and detection of ECPs by two dimensional gel electrophoresis. A Silver stained. B Immunoblot with conventional procedure day 112 antisera. C Immuunoblot with cascade procedure day 112 antisera. D Immunoblot with passive immunization procedure day 112 antisera. Exposure time was 24 hours. Reproduced with permission from Ref. 24. Copyright 1989 The Humana Press Inc.

Although the assays using antibodies have reached a high state of sophistication, a definitive work on immunization procedures is still lacking. It is not generally possible to reproduce the exact titer and specificity of antibodies even in apparently identical animals. This lack of reproducibility in the raising of antibody titers may have led to the reluctance on the part of pesticide analytical chemists to embrace immunochemical techniques. However, the animal is only the tool used to obtain the antibody, and once the antibody is in hand, the assays are physical in contrast to biological assays. Most radioimmunoassays use serum dilutions of 1 5,000 to 1 100,000 so... 

Immunization procedures vary and are dependent on type of antigen to be used, duration of immunization process, and the amounts of immune product needed. The antigen suspension may be administered intravenously, intramuscularly, or subcutaneously. The amount of antigen injected can range from 1 to 200 mg. The quantity is determined by the availability of and the potency of the antigen. The time schedule also varies. Protocols for the three types of immunizations used to produce anti-carbohydrate antibodies are recorded in the following. 

Many published immunization procedures terminate with one or more intravenous injections of soluble immunogen given without adjuvant after a course of intramuscular Freund s emulsions. In the authors experience, this produces a less satisfactory response (about half the final titer of avid antibody) than can be obtained with a final injection of intramuscular emulsion. 

Polyclonal antibodies are widely used in clinical laboratories for the measurement of plasma protein concentrations. However, immunoassays are often sensitive to the nature of the antibody used. The development of polyclonal antibodies is affected by several factors, such as the purity and dose of the antigen used, the species of host animal, and the immunization procedure. Monoclonal antibodies are viewed as a viable alternative to alleviate these problems. However, the expression of particular epitopes varies with the hpoprotein particles and among individuals in addition, the apohpoproteins themselves are polymorphic in nature. Therefore the use of a single monoclonal antibody might not detect a particular variant. If a monoclonal antibody is used in the determination of an apohpoprotein, it should he directed to an epitope that is expressed on all polymorphic forms of that particular apoprotein. Furthermore, the epitope should be equally reactive to the antibodies regardless of which hpoprotein class contains it. Alternatively a mixture of monoclonal antibodies directed at different epitopes of the apohpoprotein may also be used. Such mixtures are referred to as panmonoclonal antibodies. 

Soluble factors, such as lymphokines and monokines, produced by T cells and monocytes, respectively, influence B-cell activation. Thymocytes also produce soluble factors which stimulate and support growth of B cells in vitro (Andersson et al., 1977). This forms the basis for the in vitro immunization and production of monoclonal antibodies (Reading, 1982). Soluble factors can be specific or nonspecific for the antigen (Volkman and Fauci, 1981). The antigen-specific factor may represent secreted T-cell receptors. Some monokines may also replace lymphokines in the immunization procedure in vitro (Jonak and Kennett, 1982). 

This chapter summarizes our recent efforts to establish two different protein/ enzyme dosing models in mice, using intratracheal and intranasal dosing. Both models have been used to measure antigen (enzyme) specific antibody responses as a function of the dose of antigen administered, the immunization procedure and regimen, and the isotype of antibody produced. 

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