A solution to the problems of cytolysis assays with additional applications to other immunological and biochemical assays.

After cellular immunoassays are compared with classical bioassays, conventional methods and consequent problems of data analysis for cytolysis assays are reviewed and a new solution is proposed. This solution incorporates new methods, called dose-response surface assays and analysis (DRSA), which estimate cytolytic activity coefficients on a surface in a three-dimensional space with two dose variables (killers and targets) and one response variable (counts). These new methods based on dose-response surfaces are demonstrated to be more informative and reliable than classical methods based on dose-response curves. In a test of the methods' robustness (sensitivity of parameter estimates to changes in the dose levels of the assay design), cytolytic activity coefficients estimated by DRSA varied by less than or equal to 30% over a reduction of three to four orders of magnitude in the dose levels. This remarkable robustness should be compared with the corresponding figures of as much as 500% over less than 1 order of magnitude for previously published results of coefficients estimated by conventional methods. DRSA is distinguished from replot-of-plots methods such as those used for enzyme inhibition assays in biochemistry, and is recommended as a more efficient method that should replace replot-of-plot methods now antiquated by the advent of microcomputers. DRSA can be applied to any experimental system that requires an activity coefficient to be estimated on a dose-response surface in a space of greater than or equal to 3 dimensions (greater than or equal to 2 dose variables and one response variable), regardless of the mathematical model and statistical estimators used to analyze the dose-response interaction. Finally, DRSA is compared with the methods known as response surface methodology (RSM), and is described as a new class of methods to be added to those that constitute RSM.

Limiting dilution assays for the determination of immunocompetent cell frequencies. III. Validity tests for the single-hit Poisson model.

A statistical method was developed to test the validity of the single-hit Poisson model in limiting dilution assays used to determine immunocompetent cell frequencies. Principles of bioassay, validity tests, and the distinction between model-discrimination experiments and parameter-estimation assays are reviewed in the Introduction. The new test derived and then demonstrated with previously published data is intended to be used for parameter-estimation assays based upon the single-hit Poisson model. It is a family of related chi 2, t, and F tests for deviations from zero of the slopes of weighted least squares regression plots. These plots regress the logarithms of single-dose estimates fi of the frequency phi on the total cell doses lambda i and fi on the total cell dose reciprocals 1/lambda i, that is, Yi = ln fi on Xi = lambda i and Yi = fi on Xi = 1/lambda i. The test discriminates against alternative models with multiple-hit/target response-generation processes, a variable number (dose-dependent) of false negatives, and a constant number (dose-independent) of false positives. Its purpose as a test for parameter-estimation assays, though, is to detect deviations from the single-hit Poisson model and not to select one of these alternative models. Tests for model-discrimination experiments to select or 'prove' an unknown alternative model are considered in light of relevant literature reviewed in the Discussion.

Limiting dilution assays for the determination of immunocompetent cell frequencies. I. Data analysis.

A statistical method was developed for the analysis of experimental data from limiting dilution assays. Formulas for the estimation of the frequency of immunocompetent cells within a test population were derived by the statistical methods of weighted averaging, likelihood maximization, and X2 minimization. Equations for the latter 2 were solved by Newton's method of iterative approximation. Estimates obtained by these methods were found to be more valid than those obtained by least squares (LS) fitting as judged by the X2 test and as established by Monte Carlo experiments. X2 minimization was chosen as the preferable estimation method with maximum accuracy and precision (minimum bias and variance) for the standard determination of frequencies; likelihood maximization was used only for the confirmation of results. When data from previously published experiments were reanalyzed, both results and conclusions were found to differ significantly from those originally obtained by LS fitting, thus demonstrating the importance of using proper data analysis methods. In conjunction with the use of available calculators or microcomputers, the method presented here provides a simple and rapid procedure for the valid determination of immunocompetent cell frequencies.

Clonal analysis of cytolytic T lymphocyte specificity. I. Phenotypically distinct sets of clones as the cellular basis of cross-reactivity to alloantigens.

The cellular basis of the cytolytic cross-reactivity observed in primary allogeneic (C56BL/6 anti-DBA/2 and C57BL/6 anti-C3H/He) mixed-leukocyte cultures (MLC) was investigated by analysis of the specificity of clonal progeny derived from individual cytolytic T lymphocyte (CTL) precursor cells (CTL-P) contained within these populations. A sensitive mixed-leukocyte microculture (micro-MLC) technique was used with limiting dilution analysis by Poisson statistics to determine the frequency of CTL-P reactive against both specific and third-party (P815 and AKRA) target cells, to calculate the probability that each micro-MLC was a clone derived from a single CTL-P, and to examine the specificity of each micro-MLC assayed separately against both target cells. A total of 287 phenotypically specific, heteroclitic, and cross-reactive micro-MLC from the 2 different strain combinations were observed with a relative frequency of 81, 11, and 8%, respectively, and were calculated to have mean clone probability of 90 and 99% when based, respectively, upon the frequencies of CTL-P reactive against the specific and third-party target cells. These clones were estimated to have an approximate size of 6 X 10(4) cells, which corresponded to roughly 16 cell doublings during the 7 d of culture. 22 clones were successfully subcloned and in virtually every case, the subclones retained the specificity phenotype of the original clone from which they were derived. These results provide direct evidence for three phenotypically distinct sets of CTL as the cellular basis of cross-reactivity in MLC populations assayed against two different target cells.

Limiting dilution analysis of alloantigen-reactive T lymphocytes. II. Effect of cortisone and cyclophosphamide on cytolytic T lymphocyte precursor frequencies in the thymus.

A minimal estimate of the frequency of cytolytic T lymphocyte precursors (CTL-P) in the thymus was determined by application of Poisson statistics to limiting dilution analysis. A mean CTL-P frequency of 1/1467 was obtained for C57BL/6 (H-2b) thymus cells activated by DBA/2 (H-2d) irradiated spleen cells and assayed against P-815 mastocytoma (H-2d) target cells. CTL-P frequencies were also obtained for spleen, nylon wool column purified spleen, peripheral blood, and lymph node cell populations. The effect of in vivo drug treatments on CTL-P frequencies was then examined. Cortisone at 100 mg/kg dramatically increased the CTL-P frequency in thymus by more than 20-fold despite a drastic reduction in the number of total thymus cells. The same cortisone treatment did not affect the CTL-P frequency in spleen. In contrast, cyclophosphamide at 300 mg/kg decreased the CTL-P frequency in spleen by more than 10-fold without affecting that in thymus. Cyclophosphamide at 100 mg/kg did not produce any significant change. A detailed explanation of the calculation of CTL-P frequencies is provided and their validity is discussed.