Cascade immunization procedure

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. 

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. 

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.

Figure 3. Selection of ECP subpopulations forjprogressive iterations of the cascade procedure by silver stained SDS-PAGE. Lane 2 in each panel shows the entire ECP mixture used as the column load and lane 3 shows the column flowthrough fraction used for the next injection. Panel A demonstrates the affinity chromatography performed with day 14 antisera, Panel B with day 28 antisera and Panel C with day 42 antisera. The arrow shows ECPs depleted by the early antibodies. The progression of the immune response is clearly apparent although it is clear not all of these proteins are equally immunogenic. A 50 Kd protein has saturated its respective antibody and begun to flow through the column (Panel C, lane 4). Reproduced with permission from Ref. 24. Copyright 1989 The Humana Press Inc.

These data suggested that a mechanism of early priming of the immune response though the cascade procedure resulted in a broader spectrum of antibody reactivity. This improvement also required additional time (56 days) and/or subsequent injections of the total antigen mixture because similar experiments with day 56 antisera demonstrated equivalent antisera reactivity (24). 

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