Detection and quantification of Pneumocystis carinii using a sandwich ELISA.

Antigenic sites on Pneumocystis carinii, the basis for organism enumeration by an enzyme-linked immunosorbent assay (ELISA) were adversely affected by incubation in detergents. However, stronger detergent concentrations were needed to eliminate high levels of non-specific background. Good P. carinii quantification was obtained with low non-specific background when the detergent was used only in the washing steps and not in cell suspension solutions. Formalin fixation of the cells resulted in good ELISA quantification of organism numbers with low non-specific background. No adverse effects were observed using a detergent on fixed cells. Although the system's range of accuracy needs to be expanded, a reduction in the number of organisms in response to the effects of pentamidine in vitro could be demonstrated by ELISA.

Production, characterisation and use of monoclonal antibodies to human interleukin-5 in an enzyme-linked immunosorbent assay.

Two mouse monoclonal antibodies (Mabs) against recombinant human interleukin-5(rhIL-5) have been produced, characterised and purified. Both are IgG1 antibodies and neutralised the activity of rhIL-5 in the B13 assay. Neither Mab cross-reacted with mouse IL-5. A two-site sandwich enzyme-linked immunosorbent assay (ELISA) was developed with different combinations of the mouse Mabs and also a rat anti-mouse IL-5 Mab, TRFK5, which also has activity against rhIL-5. The most sensitive assay, with a lower detection limit of 0.5 ng/ml IL-5, used TRFK5 as the capture antibody and the mouse anti-human IL-5 Mab as second antibody. The sensitivity of this assay was increased by an enhanced chemiluminescent reagent and resulted in a lower limit of detection around 40 pg/ml IL-5.

Functional and phenotypic analysis of human T-cell clones which stimulate IgE production in vitro.

Peripheral blood mononuclear cells (PBMC) from a patient suffering from the hyper IgE syndrome were used to generate phytohaemagglutinin (PHA)-expanded T-cell clones (all CD4+, CD8-, CD23-). A selection of the clones was tested for their ability to help IgE secretion by culturing with normal B cells in the presence of solid-phase antibody to CD3. Supernatants were harvested on Day 7 and assayed by ELISA for IgE, IgG and IgM. Lymphokine secretion by the clones was assessed by culturing clones for 24 hr with solid-phase antibody to CD3 followed by assay of the supernatants for IL-2, IL-4 and interferon-gamma (IFN-gamma) production. In addition, clones were analysed by flow cytometry for CDw29 and CD45R expression. Initial experiments with seven clones indicated that those clones that could help IgE secretion also stimulated optimal IgG and IgM responses. All clones appeared to secrete IL-2, IL-4 and IFN-gamma, although the amounts of each varied. These results confirm recent findings that human T-cell clones do not fall into Tinf (Th1) and Th (Th2) type subsets as described in the mouse. There was no clear correlation between the lymphokines secreted by the clones and their capacity to help IgE production. However, the helper function of the clones for all isotypes, including IgE, appeared to be related to the level of expression of the surface antigen CDw29.

Single-step monoclonal antibody affinity purification of human urogastrone from urine.

The use of a monoclonal antibody (MAb) affinity column to purify epidermal growth factor/urogastrone from human urine in a single-step process is described. The MAb was raised against purified recombinant human urogastrone derived from the expression of a cloned synthetic urogastrone gene. The MAb was characterised and shown to cross-react fully with recombinant and native human urogastrones. The material eluted from the column was shown to have retained biological activity. When the eluted material was run on SDS/PAGE under reducing conditions an apparently homogeneous species of 5400 Da apparent molecular weight was seen. When examined on acid PAGE, 2 molecular species corresponding to beta and gamma urogastrone were observed.