Purified polyclonal IgG subclasses represent the whole or average properties of their subclasses in normal human serum, and thus are valuable for study of subclasses. We purified polyclonal IgG of four subclasses from pooled human gamma globulin without using any myeloma protein. Four semi-purified subclasses were first isolated from pooled human serum by a combination of chromatographies on DEAE-cellulose and protein A-Sepharose columns. The semi-purified subclasses were then used as immunogens and initial screening antigens to produce monoclonal antibodies with subclass-specific and subclass-restricted reactivities. These antibodies were bound to CNBr-activated Sepharose 4B to form immunoadsorbents. Purified polyclonal IgG of four subclasses sufficiently free from contamination by other subclasses or other proteins were prepared using a combination of immuno- and protein A-affinity chromatographies. These preparations showed characteristic patterns in isoelectric focusing.

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In order to evaluate the improved technique of capillary isotachophoresis, human sera from 10 normal subjects and 22 patients of monoclonal gammopathy (IgG type) were analyzed. The value of potential gradient was used to represent the mobility of each UV absorbing peak. All the sera from the patients were characterized by one or a series of sharp UV absorbing peak, which was at least 3-fold higher than the corresponding IgG peak of normal subject. Comparing the results with those obtained by polyacrylamide micro two-dimensional electrophoresis, it was as certained that the value of potential gradient of a monoclonal IgG molecule is related to its isoelectric point. Because of the highly simplified operation and the high resolution of proteins, the technique is promising for the routine analysis of serum proteins in clinical laboratories.

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Computer simulation of isotachophoresis of proteins using carrier ampholytes such as Ampholine or Sepaline as the spacers was performed on the basis of the author's theory of steady state electrophoresis. As carrier ampholytes behave as if they were weak acids in anionic isotachophoresis, the simulations were performed on systems composed of five weak acids with similar pK values in the absence or in the presence of up to two protein components. The results could explain those of agarose gel isotachophoresis experiments of proteins using carrier ampholytes as the spacers.

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In order to understand the mechanisms involved in the regulation of pepsinogen synthesis, we have investigated the changes of the expression and methylation of pepsinogen 1 (Pg1) genes during stomach development and also in carcinogen induced rat stomach cancers. Restriction analysis using methylation-sensitive restriction enzymes revealed progressive demethylation changes in Pg 1 genes region that almost coincided with the progressive increases in Pg 1 mRNA expression and Pg 1 protein synthesis during stomach development. Thus, there was an inverse correlation between the expression and methylation of Pg 1 genes and some control mechanism utilizing DNA methylation was suggested in the regulation of Pg 1 genes expression. In MNNG-induced stomach cancer, most of the composing cells were non-Pg 1 producing and there was no Pg 1 mRNA. The methylation patterns of Pg 1 genes in these cancers were different from those of normal tissues which do and do not synthesize Pg 1. This finding is in good agreement with the previous finding that DNA methylation is altered in cancer cells.

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