Georges Köhler

Deletions in immunoglobulin mu chains.

Eight mutant hybridoma lines are described, which synthesize short immunoglobulin mu chains. Four internal deletions were mapped by Southern blot analysis. They are shown to remove DNA from either part or all of the first, and first and second, constant mu exons. The sizes of the deletions range between 0.6 and 5 kb, leaving an equal or unequal number of splice signals. Shorter mu RNA of one size was found irrespective of whether an exon was completely or only partially deleted. These results preclude exclusive 3′ (constant region) to 5′ (variable region) directional splicing of the mu RNA. No important signals seem to reside in the deleted DNA stretches affecting the transcription or the correct RNA splicing of the remaining exons. The internal mu protein deletions revealed unusual covalent light chain attachment demonstrating functional homology between the first (normally used) and fourth mu constant domain. The other mu protein deletions (10, 11, and 12 kd) involved neither gross DNA nor RNA lesions and are considered to be due to premature chain termination. Since secretion is found in most of the mutant IgM‐producing lines, no single one of the four mu constant domains (including the C‐terminal one which contains the so‐called secretory piece) is necessary for secretion.

Resolving power of isotachophoresis and isoelectric focusing for immunoglobulins.

Among the various techniques which are able to discriminate between individual members of a protein family like the immunoglobulins, isoelectric focusing (IEF) (for a recent review, see [ 1 ] ) has been very frequently used since the introduction of polyacrylamide slab-gels. The advantages of IEF include high resolving power and flexibility with regard to sample detection. One of the drawbacks of IEF in polyacrylamide gels is the restriction exerted on very large molecules, but this problem has recently been solved by the use of polymerizable thin layers containing Sephadex as anti-convective medium [2]. Another disadvantage is of a more serious nature, namely the low ionic strenght in the medium in which focusing is carried out. The sensitivity of many proteins to low ionic strenght and their tendency to precipitate at a pH near their isoelectric point therefore preclude the analysis of an appreciable number of proteins by IEF. We have tried to overcome these problems by employing isotachophoresis (ITP), an electrophoretic method, which has so far found only very limited application in biochemistry, especially in the analysis of protein molecules. The two most attractive features of ITP derive from its theoretical basis [3-71: the ionic strenght in the system can be regulated over a wide range and the sample ions carry a net charge. Thus, precipitation of proteins at their isoelectric point is avoided. Usually, ITP is done either in columns or in capillary tubes, both of which make a comparison of similar samples difficult and time-consuming. We therefore introduce here a simple and inexpensive

Derivation and Diversification of Monoclonal Antibodies

A mouse can make ten million different antibodies, each synthesized by its own B lymphocyte. About 1000 different antibodies are able to recognize one single antigcnic determinant. When a conventional mouse response to such a determinant is analysed, only 5-10 different antibody species are seen, representing probably a random sample of the total repertoire. So even when appropriate absorptions or allogeneic immunizations have confined the antibody heterogeneity to one single determinant the sera obtained have four disadvantages: (I) the titres are low; (2) the antibodies, while specific for a single determinant, are nevertheless heterogeneous; (3) the supply is limited: and (4) the same combination of specific antibodies is impossible to reproduce in a new animal.