Search

The memory response of BALB/c mice to phosphocholine-keyhole limpet haemocyanin (PC-KLH) consists of two antibody populations, designated Group I and Group II, that differ in their fine specificity, as determined by hapten inhibition using phosphocholine (PC) and p-nitrophenylphos-phocholine (NPPC). It is known that Group I response is dominated by T15 idiotype-positive antibodies that utilize the VH I heavy-chain gene in combination with VK22 light-chain gene. In this paper we show that Group II serum antibodies of BALE/c mice are also highly restricted in their heterogeneity, as determined by N-terminal amino acid sequence analysis. Furthermore, we demonstrate that the Group II response is not affected by neonatally induced T15 suppression, whereas the Group I response in these mice consists of T1 5-negative antibodies. This suggests that the expression of the two antibody populations is regulated independently. Finally, we show that the isotype distributions within a fine specificity are the same in normal and T15-suppressed mice. Interestingly this is true not only for the Group II but also for the Group I antibodies. Because the isolated Group I antibodies from normal mice differ in structure from those of T15-suppressed mice, i.e. different light chains, our data indicate that the isotype distribution of these two populations is associated with their fine specificity in addition to their clonal origin. [ABSTRACT FROM AUTHOR]

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 serological cross reactions between Mycoplasma conjunctivae, the etiological agent of infectious keratoconjunctivitis (IKC), and the antigenetically and phylogenetically closely related Mycoplasma ovipneumoniae, which is often found in sheep, were analysed. Cross reacting antigens were identified using sera from sheep with IKC and from sheep of herds known to be free of IKC, as well as rabbit hyperimmune serum specific to the two Mycoplasma species. Cross reactions were predominantly due to the strongly antigenic proteins of 42 kDa and 83 kDa. Serospecific antigens of M. conjunctivae could be separated from cross-reacting antigens by the extraction of Tween 20-soluble membrane proteins. The Tween 20-extracted proteins of the M. conjunctivae strain HRC/581T were used for the development of an indirect ELISA test. This ELISA test was shown to be a useful serological method for the diagnosis of M. conjunctivae infections and to identify infected sheep herds.

Sporadic cases of infection with Mycoplasma bovis have been observed in Switzerland since 1983. However, five severe outbreaks of endemic mastitis in a geographically distinct, small region (Canton du Jura) during the period 1995 to 1997 prompted the present investigation on the seroprevalence of infection with M. bovis among milking cows in Switzerland. A commercially prepared indirect enzyme immunoassay was used. Among a stratified random sample of 118 herds of milking cows in Switzerland, at least one positive animal was detected in 56 (47%) of the herds, whereas 6.1% of the 1816 individual animals tested positive. An epidemiological study was performed in the Canton du Jura region among 51 herds in order to assess the importance of management factors in the spread of M. bovis infection. The herd-level prevalence was 78%, and the seroprevalence at the level of the 1354 individual animals tested was 13.4%. A multivariate analysis of possible risk factors showed purchase of animals to be the only variable significantly associated with serological status of the herd with an "odds ratio" of 10.8.

Despite advances in the in vitro immunization of human B cells (Borrebaeck et al. 1988) and the development of immunodeficient mice (McCune et al. 1988) for the reconstitution of the human immune system ex vivo, immortalization of antigen-specific human B cells remains the limiting step in the generation of human monoclonal antibodies. Typically this is performed with the aid of Epstein-Barr virus transformation followed by subcloning, confirmation of antigen binding and hybridization of the B lymphoblasts to a suitable fusion partner such as GLI-H7. This general approach is effective and widely used; however, it is time-consuming with erratic results. These were the immediate reasons we and others devised methods to directly obtain the variable regions from small numbers of human B cells (Larrick et al. 1987). The success of the PCR-based approach is illustrated above. In the present studies we successfully captured and stably produced antibodies from the V regions of two potent human anti-tetanus antibodies secreted by heteromyelomas that were too unstable for scale-up production. Although further preclinical evaluation of these antibodies is in progress, results to date indicate that the recombinant antibodies produced in myeloma-based cell lines or CHO cells are equivalent in binding specificity and activity to the native heteromyeloma-derived antibodies. Recent studies from this laboratory indicate that effective anti-tetanus protection will require a cocktail of anti-tetanus antibodies. Details of this work will be the subject of a future communication (Lang et al., in preparation).

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.

We have analyzed the developmental patterns of two groups of B cell precursors in nonimmunized BALB/c mice with respect to their relative proportions, absolute frequencies, V gene usage, fine specificity, and avidity for antigen. One group of B cells (group I) secretes antibodies specific for PC and PC-containing bacteria, whereas the other group (group II) produces antibodies recognizing only nonenvironmental PC-protein conjugates. A marked shift in the proportions of group I and group II occurs during ontogeny: while the group I B cells dominate (greater than 85%) the adult antibody repertoire, the group II B cells have equal representation in neonatal mice from Days 1 to 7, and remain as a significant portion until 2 weeks of age. Examination of the absolute frequencies of group I and group II B cells revealed that the frequency of group II B cells remained relatively stable throughout ontogeny, whereas group I B cells expanded rapidly after 7 days of age to predominate in the adult. Genetic analysis indicated that early group I antibodies were encoded by VH and VL genes different from adult group I antibodies which are mostly encoded by a single VH (S107) and VL (V kappa 22) gene combination (the T15 idiotype). On the other hand, early group II antibodies used VH genes comparable to their adult counterparts. The majority of early group I antibodies have lower avidity for PC than adult T15+ antibodies, whereas the avidity of neonatal group II antibodies varies considerably and is comparable with that of the adult group II antibodies. Our results suggest that the ontogeny of phosphocholine-specific B cells may be regulated according to their fine specificity rather than to their avidity or V gene usage.

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 an approach that allows the generation of stable hybridomas secreting antigen specific human IgG antibodies with an efficiency comparable to that of the generation of IgM and IgA secreting hybridomas. This was achieved by evaluating means to increase the frequency of human hybridoma formation and the stability of the generated hybridoma cells when subjected to conditions for large scale growth. To this end, we generated new fusion lines with an increased human DNA content and modified the culture system. However, the application of these new fusion lines primarily resulted in unstable giant cells. As a consequence, we evaluated whether the viability of newly formed hybrids between existing fusion lines and lymphoblastoid cell lines might be improved. In an attempt to provide as many components necessary for the growth of antibody secreting hybridomas as possible, we propagated fused cells in medium supplemented with human serum. Our results show that with this approach the frequency of initially growing hybrids was significantly increased. Furthermore, only in culture medium supplemented with human serum was it possible to obtain stable IgG secreting clones.

We have shown previously that anti-phenylphosphocholine antibodies elicited by phosphocholine-keyhole limpet hemocyanin can be divided into two populations according to their ability to recognize the two hapten analogues p-nitrophenylphosphocholine (NPPC) and p-nitrophenyl 3,3-dimethylbutyl phosphate (NPDBP). These analogues differ from each other in that NPPC has a positively charged nitrogen in the choline moiety, whereas NPDBP lacks the positively charged nitrogen. Group II-A antibodies bind only NPPC, whereas group II-B antibodies bind both ligands. Here, by infrared and nuclear magnetic resonance spectroscopic investigations, we find that when free in solution NPPC has a predominantly fixed structure in which the termini approach each other, probably due to electrostatic interactions within the molecule; this "bent" structural feature is retained when the ligand is bound by antibody. In contrast, the structure of unbound NPDBP is less fixed, being characterized by rapidly interchanging conformations corresponding to an open chain structure with less overall proximity of the termini compared to NPPC. The overall shape of NPPC is essentially unaltered by binding, whereas in the case of NPDBP what was a minor conformation in the unbound state becomes the predominate conformation of the bound ligand. Thus, our results are consistent with these antibodies providing a molecular template for stabilizing the conformation of the bound ligand.

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.

The memory response to PC-KLH is dominated by two antibody populations differing in fine specificity. Group I antibodies show affinity for both phosphocholine (PC) and p-nitrophenyl phosphocholine (NPPC). Group II antibodies exhibit significant affinity only for NPPC. Here, we describe the binding site characteristics of Group II antibodies and show that in recognizing NPPC these antibodies have a common requirement for the phenyl moiety, a negatively charged phosphate, and the trimethyl structure of the choline. However, Group II antibodies were found to differ in their requirement for the positively charged nitrogen of choline and thus could be divided into two subgroups. In contrast to Group II-A, Group II-B antibodies recognize not only NPPC but also its analog p-nitrophenyl-3,3-dimethyl butyl phosphate (NPDBP), which differs from NPPC by substituting a carbon for the positively charged nitrogen of the choline moiety. These results suggest that Group II-B antibodies do not require the positive charge in order to bind, although the binding constant, Ka, was increased when the nitrogen was present. Furthermore, heterogeneity within Group II antibodies was characterized by differences in binding to dinitrophenyl phosphocholine which has an additional phenyl ring and aminophenyl phosphocholine which has an amino group in place of the nitro group of NPPC. The results indicate that diversity in the memory response to PC-KLH is reflected in the Group II antigen-binding phenotype by antibodies which differ appreciably in their recognition of various structural aspects of the hapten.