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Serological differentiation between infection and vaccination depends on the detection of pathogen specific antibodies for an epitope that is modified or lacking in a vaccine. Here we describe a new assay principle that is based on differences in the binding properties of epitope specific antibodies. C-DIVA is a potent Classical swine fever vaccine candidate that differs from the parental C-strain life attenuated vaccine in the highly immunogenic TAVSPTTLR epitope by the deletion of two and the mutation of one amino acid (TAGSΔΔTLR). We show that C-DIVA vaccination elicits antibodies with high affinity for both the TAGSΔΔTLR and TAVSPTTLR epitope, whereas infection elicits only TAVSPTTLR specific antibodies. Differentiation is achieved with a double competition assay with negative selection for antibodies with affinity for the TAGSΔΔTLR epitope followed by positive selection for antibodies with affinity for the TAVSPTTLR epitope. Our findings add a new strategy for the development of marker vaccines and their accompanying discrimination assays and offer an alternative to the devastating stamping out policy for Classical swine fever., (Copyright © 2015 Elsevier B.V. All rights reserved.)

The direct propagation of newly formed human hybridomas in serum-free medium selects for hybrids with a metabolism best suited to growth in this environment. Under optimal culture conditions, this procedure results in the generation of antigen-specific human hybridomas comparable in frequency, stability, and antibody secretion rate to that obtained with murine hybridomas. After a transient phase of a few days in the appropriate selection medium supplemented with 1% serum, hybridomas grow in serum-free medium in stationary cultures with a cell doubling time of 15-25 h and an antibody production rate averaging 12 micrograms/10(6) cells/day. Clones propagated in bioreactors exhibited a cell doubling time of 29-35 h and an antibody secretion rate of 10-21 micrograms/10(6) cells/day.

The potential of an ELISA based detection of affinity distributions within polyclonal populations of antigen-specific serum antibodies was assessed by analyzing defined probes composed of monoclonal antibodies (MAb). In a competitive binding ELISA in which the concentration of antigen in the liquid phase and the solid phase was varied, we analyzed mixtures containing defined percentage compositions of MAb exhibiting apparent affinity constants (aK) between 3 x 10(6) and 2 x 10(9) M-1 for Pseudomonas aeruginosa exotoxin A. Our results indicate that the detectability of antibody populations depends on the antigen concentrations in the solid phase and on the affinity distribution of the probe to be analyzed. In wells coated with high antigen concentrations, antibody titers reflected antibody concentrations, whereas at low antigen concentrations antibody titers primarily reflect antibody affinities. Independent of their affinities, subpopulations less than 10% could not be detected. Low affinity antibodies were preferentially underestimated. The degree of distortion depended on the composition of the probe to be analyzed. In general, the higher the absolute and the relative affinity of a population, the stronger was its capacity to interfere with the detection of other populations. As a consequence, the heterogeneity of affinity distributions in polyclonal samples may be substantially underestimated. The experiments reported provide guidelines for an optimal design and an adequate interpretation of ELISA based qualitative analyses of polyclonal antibody samples.

We describe a rapid in vitro assay for the evaluation of in vivo properties of conjugate vaccines. Using human and murine monoclonal antibodies (mAb) specific for lipopolysaccharides (LPS), isolated from Pseudomonas aeruginosa, we determined in a competitive binding assay the amount of LPS or conjugate vaccine which was required to inhibit the antibody binding to LPS by 50% (I50 values). Furthermore, utilizing a murine burn wound sepsis model, we determined the potential of the same conjugates to induce protection in vivo against infection with the corresponding bacteria. Protective mAb have approximately 100-fold lower I50 values for preparations which are highly effective in inducing protection than for preparations which are ineffective. Furthermore, in the case of potent preparations it was noted that protective mAb exhibit similar I50 values for the conjugates and for the corresponding LPS. These results suggest that the fast and easily interpretable in vitro assay described may significantly facilitate the development and optimization of vaccines.