Development of a highly quantitative, reproducible assay for determination of chicken T cell growth factor biological activity.

This report examines optimal culture conditions necessary for accurate and sensitive quantification of chicken T Cell Growth Factor (TCGF) activity. With this bioassay, TCGF is quantified by measuring its ability to cause proliferation of splenocytes prestimulated with mitogen. Proliferation is quantified by determining the optical density (OD) or "signal" of test samples in microtiter wells by measuring the incorporation of tetrazolium salt by live cells. To optimize assay conditions, systematic evaluation of the effects of cell culture variables was carried out with the constant aim of increasing signal to noise ratio in the assay. Higher signal to noise ratios were found when using Dulbecco's Modified Eagle's Medium (DMEM) rather than Roswell Park Memorial Institute Medium (RPMI) for basal tissue culture media containing the same supplements. The addition of lipid supplement to the assay system not only increased the proliferation signal, but also decreased the background OD. Incubation temperatures of 41 C rather than 37 C for both the mitogen prestimulation and proliferation phases of the assay also resulted in a higher signal to noise ratio. While incorporating the optimal experimental conditions, a finalized assay procedure employing test sample normalization with an internal assay standard was tested for accuracy. The assay can accurately detect 2 to 15 U/mL of TCGF activity. The within-assay variation ranged from 2 to 13% and the between-assay variation ranged from 11 to 22% depending upon the TCGF preparation being tested. The excellent reproducibility of this assay has facilitated investigations of TCGF production, processing, and purification.

Evidence for covalent attachment of phospholipid to the capsular polysaccharide of Haemophilus influenzae type b.

Cells of Haemophilus influenzae type b were grown in a liquid medium containing [3H]palmitate or [14C]ribose or both for two generations of exponential growth. Radiolabeled type-specific capsular polysaccharide, polyribosyl ribitol phosphate (PRP), was purified from the culture supernatant by Cetavlon precipitation, ethanol fractionation, and hydroxylapatite and Sepharose 4B chromatography. The doubly labeled ( [3H]palmitate and [14C]ribose) PRP preparation was found to coelute in a single peak from a Sepharose 4B column, suggesting that both precursors were incorporated into the purified PRP. A singly labeled ( [3H]palmitate) purified PRP preparation was found to be quantitatively immune precipitated by human serum containing antibody against PRP. The radioactivity of this preparation could not be dissociated from PRP by treatment with chloroform-methanol, 6 M urea, sodium dodecyl sulfate, or Zwittergent. Only after acid, alkaline, or phospholipase A2 treatment of PRP labeled with [3H]palmitate or [3H]palmitate and [14C]ribose followed by chloroform-methanol extraction could most of the 3H-radioactivity be recovered in the organic phase. The chloroform-soluble acid-hydrolyzed or phospholipase A2-treated product was identified as palmitic acid after thin-layer chromatography. These results strongly suggest that a phospholipid moiety is covalently associated with the H. influenzae type b polysaccharide PRP.