Purification of antibodies to O antigen of Salmonella Typhimurium from human serum by affinity chromatography.

Nontyphoidal Salmonellae (NTS) are a common cause of bacteraemia in children and HIV-infected adults in Sub-Saharan Africa. We have previously shown that antibodies play a key role in both bactericidal and cellular mechanisms of immunity to NTS, but found that high concentrations of antibody to Salmonella Typhimurium O antigen (OAg) in the serum of some HIV-infected African adults is associated with impaired killing of NTS. To further investigate the function of antibodies to the OAg of NTS, we developed a method to purify these antibodies from human serum by affinity chromatography. Purified Salmonella Typhimurium OAg was activated with adipic acid dihydrazide (ADH) via two different chemistries before linking to N-hydroxysuccinamide-Sepharose resin: one ADH molecule was introduced per OAg chain on its terminal 3-deoxy-D-manno-octulosonic acid sugar (OAg-ADH), or multiple ADH molecules were attached along the OAg chain after oxidation with sodium periodate (OAgoxADH). Both resulting columns worked well when tested with commercial polyclonal anti-O:4,5 antibodies from rabbit serum. Over 90% of the applied antibodies bound to the resin and 89% of these antibodies were then eluted as detected by ELISA. OAg-ADH was preferred as the method for OAg derivatisation as it does not modify the saccharide chain and can be applied to OAg from different bacteria. Both columns were able to bind OAg-specific antibodies in human serum, but antibody recovery was initially low. Different elution buffers were tested and different amounts of OAg-ADH were linked to the resin to improve the yield. Optimal recovery (51%) was obtained by loading 1mg of activated OAg per ml of resin and eluting with 0.1M glycine, 0.1M NaCl pH2.4. The column matrix could be regenerated following elution with no detectable loss in performance for over ten uses. This method offers the potential to purify antibodies to Salmonella OAg from polyclonal serum following vaccination or natural exposure to Salmonella and so investigate the functionality and diversity of the antibody response to OAg.

The influence of spacer-containing enzyme conjugate on the sensitivity and specificity of enzyme immunoassays for hapten.

BACKGROUND: In hapten enzyme immunoassays (EIA), there is an increase or decrease of labeled hapten recognition by antibody that affects sensitivity of the assay. We incorporated a spacer between a hapten derivative and enzyme to test its influence on the sensitivity and specificity of enzyme immunoassays. METHOD: Antibodies were generated against cortisol-3-O-carboxymethyl-oxime-bovine serum albumin (cortisol-3-O-CMO-BSA) and cortisol-21-hemisuccinate-bovine serum albumin (cortisol-21-HS-BSA) as an immunogen. Four cortisol horseradish peroxidase (HRP) enzyme conjugates were prepared using 2 cortisol derivatives (cortisol-3-O-CMO and cortisol-21-HS) with and without adipic acid dihydrazide (ADH) as a spacer. Eight combinations of homologous and heterologous assays were evaluated. RESULT: The incorporation of ADH spacer in cortisol-enzyme conjugate improved the sensitivity in heterologous (bridge and site plus bridge) EIA systems. In heterologous assays (site plus bridge), the presence of spacer in enzyme conjugate reduced the cross-reactivity with cross-reacting steroids. CONCLUSION: Spacer in the enzyme conjugate for hapten ELISA can improve the sensitivity of heterologous assay of hapten-like steroids. It may also reduce the cross-reactivity for some assays.

Immunogenicity of meningococcal B polysaccharide conjugated to tetanus toxoid or CRM197 via adipic acid dihydrazide.

Vaccine development against Group B Neisseria meningitidis is complicated by the nature of the capsular polysaccharide, which is alpha 2-8-linked poly-sialic acid, identical in structure to the poly-sialic acid found in many mammalian tissues during development. To test the feasibility of a vaccine based on this polysaccharide, we synthesized several conjugates of meningococcal B polysaccharide linked to a carrier protein (tetanus toxoid or diphtheria CRM197), via an adipic acid dihydrazide (ADH) spacer. All conjugates induced a strong immune response. However, most of the antibodies were not directed against the Meningococcus B polysaccharide and could not be inhibited by the purified polysaccharide alone. Further investigations showed that the antibodies recognized an epitope composed by the junction between the spacer and the polysaccharide and protein, that is not present in the native polysaccharide and is generated during the coupling reaction. This epitope becomes immunodominant with respect to the poorly immunogenic polysaccharide. While the majority of the immune response is directed against the above epitope, the conjugates induced also an immune response against the Meningococcus B polysaccharide. The anti-Meningococcus B antibodies elicited are of the IgM and IgG class and are inhibitable by the polysaccharide. Moreover, they are bactericidal, thus suggesting that they would induce protection against disease.