IMMUNE RESPONSE IN COLD EXPOSURE 



visable precipitation in double diffusion tests is dependent upon 

 three factors: (1) the diffusion co-effecient of each of the reactants, 

 (2) their absolute concentrations, and (3) the speed with which the 

 antigen- antibody front can form insoluble complexes. 



Assuming that each of the rabbit antibody preparations possesses 

 an equal diffusion co- efficient, and using the same antigen prepara- 

 tion for both systems, the time of appearance of each of the pre- 

 cipitating bands on the Ouchterlony gel diffusion plate should depend 

 upon the concentration of the antibody and/or the affinity or avidity 

 of the antibody for its homologous antigen. 



In our evaluation of these data, three methods of measurement 

 were used. In order to analyze the data, (1) the total number of pre- 

 cipitating antigen- antibody complexes was measured at each reading; 

 (2) in an attempt to evaluate the results quantitatively, each of the 

 precipitates was visually estimated and ranked with respect to the 

 total amount and homogeneity of precipitate; and (3) the time of 

 appearance of each of the precipitating bands was recorded. Pre- 

 cipitates were graded one through four based upon assigned criteria, 

 with the sharpest and most well-defined bands being assigned the 

 highest number. Quantitative evaluations were based on the number 

 of bands of precipitate times the intensity of each precipitate; for 

 example, a gel diffusion plate having four sharp, heavy bands of 

 precipitate and one faint, poorly-defined precipitate was assigned a 

 total value of 17 (4x4 + 1x1). All of the data, both qualitative and 

 quantitative, were subjected to statistical evaluation by the Mann- 

 Whitney U test. The results of one of these studies are given in 

 Tables I and II, 



From these tables it may be seen that a statistical evaluation of 

 both the qualitative data based on the number of precipitating bands 

 in the Ouchterlony gel diffusion plates and the quantitative data 

 based on the number of bands times the intensity of the precipitate 

 failed to reach a level of significance in the Mann- Whitney U test. 

 The U value at the 0.0 5 significance level is equal to 17. In study 

 groups in which the larger of two independent samples is smaller 

 than 9, tables for U values are not available and the P value is cal- 

 culated directly (Table III). It may be noted in Table III that differ- 

 ences which are significant at the 0.0 5 level are obtained in sample 2 



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