Development of a new method for the quantitative analysis of the extracellular polysaccharide of Neisseria meningitidis serogroup A by use of high-performance anion-exchange chromatography with pulsed-amperometric detection.

A new method for the quantitative determination of Neisseria meningitidis group A (MenA) capsular polysaccharide (CPS) has been developed. The method is based on trifluoracetic acid (TFA) hydrolysis of the CPS (2 M at 80 degrees C for 3 h), followed by chromatographic separation and quantification of the liberated mannosamine-6-phosphate from the area of the peak obtained using an IonPac AS11 column coupled to the sensitive pulsed amperometric detector ED40. The highly selective nature of this method circumvents the interference problems associated with the classical method based on a colorimetric assay for phosphorus. Provided that suitable hydrolysis conditions can be found, this chromatographic approach might be applicable to the quantification of other bacterial antigens containing phosphorylated sugars such as meningococcal groups H, L, X and Z, and pneumococcal serotypes 6, 10A and 19.

Quantitative determination of saccharide in Haemophilus influenzae type b glycoconjugate vaccines, alone and in combination with DPT, by use of high-performance anion-exchange chromatography with pulsed amperometric detection.

The stability and integrity of glycoconjugate vaccines requires determination of the total saccharide and quantification of the unbound or free saccharide present. The traditional assay for Hib conjugates, based on colorimetric determination of ribose, has been much improved by the use of base hydrolysis and analysis of the Hib subunit generated using high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD). The production of this subunit was confirmed by NMR analysis. However, quantification of free Hib saccharide using this method was not possible in the combination vaccines evaluated due to interferences emanating from DPT. Thus a method based on TFA hydrolysis followed by the chromatographic separation and quantification of ribitol on a CarboPac MA1 column was developed. The method is selective, and with the use of ED40 electrode, requires only nanomole amounts for the chromatographic step, thereby ensuring that free saccharide can be monitored accurately in the formulated Hib-CRM vaccine alone and when in combination with other vaccines.

Unresponsiveness to human leukocytes in immunosuppressed mice by combined donor-derived human transferrin and antigens.

Previous work on the facilitation of xenogeneic and allogeneic bone marrow engraftment in irradiated mice and dogs with transferrins allowed the development of a model for induction of an apparently durable state of immunological unresponsiveness or 'tolerance' in chemically immunosuppressed mice. The system is based on the simultaneous and combined administration of donor-derived cell antigens, namely human leukocytes, and specific donor-derived or plasma pool human transferrin into BALB/c or C57BL/6 mice previously treated with prednisolone and cyclophosphamide on day 0 and day 1 of the experiment. A properly timed presentation of both donor-specific or plasma pool transferrin and leukocyte antigens into the mice on day 3 and day 16 of the experiment, in the course of initial restoration of their lymphohaemopoietic tissues and cells after severe immunosuppression, results 1-3 months later, in their inability to 'recognize' human donor lymphocytes and to mount an immediate or a delayed-type immune response against human antigens. This durable state of unresponsiveness was evaluated by a complement-mediated cytotoxicity assay, with a mixed lymphocyte culture method and confirmed by the abrogation of the humoral (antibody response to human erythrocytes) and of the cell-mediated (popliteal lymph node test) immune responses in vivo. Our findings demonstrate the capacity of human plasma-derived transferrins to induce a state of durable unresponsiveness (xenogeneic tolerance?) in mice when administered with human antigens in the course of regeneration of stem cells in the bone marrow and lymphatic organs.