A 51Cr-Release Assay, Essay Example

The principle experiment used in this paper was a 51Cr-release assay to measure changes in cytotoxic T-lymphocyte activity. Explain in detail how this assay was performed, beginning with the subcutaneous injection of adjuvant and antigen into mice and ending with the assay’s readout of “% lysis”.

When dying animal cells are co-injected with antigen, they afford an adjuvant effect for preparing T-cell responses. This endogenous adjuvant movement is existent in the cytosol of cells and significantly intensifies when cells are injured, as in, by ultraviolet irradiation. In this experiment, to identify this endogenous adjuvant, cytosol was fractionated from ultraviolet-irradiated BALB/c 3T3 cells by high-performance liquid chromatography (HPLC) on a sizing column scrutinized with a diode array ultraviolet spectrum detector.  Groups of four to five successive segments were examined for their ability to enhance the priming of CD8+ T-cell reactions when co-injected with particulate HIV gp120 antigen. Subsequently 14 d splenocytes from the prepared mice were stimulated ex vivo with antigen and then analyzed for their ability to kill antigen-bearing objective cells in a 51Cr-release test. A group of low molecular weight (LMW) segments that were beneath the optimum outcome array of the sizing supports had adjuvant activity that noticeably boosted the production of cytotoxic T lymphocyte (CTL) responses. At all four levels, the LMW adjuvant from the liver presented the same chromatography profile as the 3T3 cells, which indicate that both cells had the same adjuvant molecule, but these details were not expounded upon within the report. The dynamic segments from all four stakes had inimitable ultraviolet peak absorptions at 235 and 292nm, which faded at low pHand these details have also been excluded.

Since the possibility exists for large amounts of uric acid to be produced from tissue injury in vivo, 3T3 cells were given allopurinol, which is a uric acid analogue that reduces uric acid production, and then injured by ultraviolet irradiation and freezing to test this hypothesis. These cells were then injected, along with gp120 beads, into mice treated with allopurinol and uricase to terminate any uric acid that was released in vivo. Allopurinol and uricase treatment of mice significantly diminished their plasma uric acid concentrations and the priming of gp120-specific CTLs by 104 injured cells was extensively decreased by excluding the uric acid. Cultures of CTLs from control mice contained 11.8 lytic units compared with 2.0 lytic units in CTLs from mice treated with allopurinol plus uricase, which is an 83% inhibition, and is representative of several experiments. To omit the possibility of an enantiomeric or stereo-isomeric structure, the sanitized LMW molecule was incubated with uricase, which is a highly specific enzyme that breaks down uric acid to allantoin. Uricase quickly deteriorated the LMW molecule. Congruently, this information indicates that the main component of the exceedingly refined LMW division is uric acid. Further analysis attempted to ascertain whether uric acid was accountable for the organic activity seen in vivo and acquired highly purified uric acid to test it for adjuvant activity. This substance heightened CTL priming to a similar extent achieved by the purified LMW fraction and comparable results were obtained when C57BL/6 mice were immunized with pure uric acid and particulate ovalbumin antigen.  It can therefore be inferred that LPS is not accountable for the adjuvant activity in the examined preparations and the indications that uricase incapacitated the LMW fraction also indicates that LPS or other microbial contaminants do not excuse the adjuvant activity because both commercially attained uric acid and uric acid purified to homogeneity from the liver have adjuvant activity and uricase destroys this activity.  This supports the conclusion that uric acid is one of the endogenous adjuvants in cells