Three immunogens with side chains of random amino acid sequence, poly (L Phe, l glu)-poly (DL Ala)--poly (L Lys) [(Phe, G)-A--L 223], poly (L Tyr, L Glu)-poly (DL Ala)--poly (L Lys) [(T, G)-A--L 509] and (T, G)-A-L 52, as well as two immunogens with side chains of defined amino acid sequences, GGT-A--L and TG-A--L, were sequenced using a Beckman automated sequenator. Despite the lack of a unique amino acid sequence for the amino terminus, reasonable results for the sequence studies were obtained using the Edman reaction. GGT-A--L and TG-A--L had 70% and 80% of their side chains respectively, with the desired sequence. The three compounds of random amino acid sequence were found to contain a large proportion of their A--L side chains unsubstituted. The side chains had a much greater probability of terminating in the aromatic amino acid than in the glutamic acid. The distribution of the length of side chains and their amino acid sequences was completely heterogeneous.
Mice were injected with a series of (T,G)-A--L[poly (L Tyr, L Glu)-poly DL Ala)--poly (L Lys)]-like compounds with side chains of homogeneous sequences: T-A--L, GT-A--L, GGT-A--L, and TG-A--L. T-A--L was not immunogenic. However, T-A--L was able to bind antibodies to (T, G)-A--L 509, and this binding could not be blocked by A--L. When complexed with bovine serum albumin, T-A--L, was immunogenic in both responder and nonresponder strains of mice. GT-A--L and GGT-A--L were both immunogenic and elicited the characteristic responder-nonresponder difference induced by (T,G)-A--L. TG-A--L was also immunogenic, but there was considerable overlap in the response of responder and nonresponder strains. On the average, responder mouse serum had a slightly higher antigen-binding capacity than nonresponder mouse serum. In contrast to antibodies against GGT-A--L, antibodies against TG-A--L bound heterologous antigens poorly. These data, along with the results of other investigators, are consistent with the hypothesis that there are multiple Ir- 1 genes which recognize different sequences. The specificity of the Ir- 1 genes is extraordinary. The polypeptides TG-A--L, TGTG-A--L and GTTG-A--L do not appear to be recognized by these genes.
We are interested in determining the range of variants present in a cell population that can actually be isolated. We have used subcloning and sublining to search for variants with increased antibody stability, increased cell line stability to freezing and defrosting, increased cell population viability, increased antibody production and the ability to grow in simpler media. This paper presents the case histories of several different hybridoma cell lines which required some property changed before they became production ready clones. We found that switching the class of an antibody from IgG3 to IgG1 did increase its stability, decrease its tendency to aggregate and allowed it to be used in a commercial diagnostic kit. We could isolate subclones that produced twice the level of antibody with a frequency of 1-3%. It was straight forward to isolate clones that were stable to freezing and defrosting or grew in a simpler media. We were not successful in increasing the maximum viability of a cell line. In conclusion, we have found that any population of hybridoma cells has natural variants with significantly enhanced properties that can be isolated.
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