THE manufacture of predefined specific antibodies by means of permanent tissue culture cell lines is of general interest. There are at present a considerable number of permanent cultures of myeloma cells1,2 and screening procedures have been used to reveal antibody activity in some of them. This, however, is not a satisfactory source of monoclonal antibodies of predefined specificity. We describe here the derivation of a number of tissue culture cell lines which secrete anti-sheep red blood cell (SRBC) antibodies. The cell lines are made by fusion of a mouse myeloma and mouse spleen cells from an immunised donor. To understand the expression and interactions of the Ig chains from the parental lines, fusion experiments between two known mouse myeloma lines were carried out.

Continuous cultures of fused cells secreting antibody of predefined specificity

High resolution two-dimensional electrophoresis of proteins.

In the genome of a germ-line cell, the genetic information for an immunoglobulin polypeptide chain is contained in multiple gene segments scattered along a chromosome. During the development of bone marrow-derived lymphocytes, these gene segments are assembled by recombination which leads to the formation of a complete gene. In addition, mutations are somatically introduced at a high rate into the amino-terminal region. Both somatic recombination and mutation contribute greatly to an increase in the diversity of antibody synthesized by a single organism.

Somatic generation of antibody diversity

Cell fusion techniques have been used to produce hybrids between myeloma cells and antibody-producing cells. The hybrid lines derived are permanently adapted to grow in tissue culture and are capable of inducing antibody-producing tumors in mice. Spleens from mice immunized against sheep red blood cells (SRBC) were fused to an 8-azaguanine-resistant clone (X63-Ag8) of MOPC 21 myeloma. Over 50% of the derived hybrid lines produce and secrete immunoglobulins different from the MOPC 21 myeloma. About 10% of the hybrid lines exhibit anti-SRBC activity. The high proportion of antibody-producing hybrids suggests that the fusion involves a restricted fraction of the spleen cell population, probably cells committed to antibody production. In order to avoid the presence of the MOPC 21 heavy chain in the specific hybrids, another myeloma cell line (NSI/1-Ag4-1) has been used. This is a nonsecreting variant of the MOPC 21 myeloma which does not express heavy chains. Three anti-SRBC (probably of the mu, gamma2b and gamma1 classes, respectively) and two anti-2,4,6-trinitrophenyl (of the mu class) antibody-producing hybrids have been repeatedly cloned. By random selection and by selection of specific clones according to their lytic activity (clone plaque selection), a number of different lines have been constructed. Such lines express different combinations of the four possible chains of each hybrid line: the myeloma gamma and K chains and the specific antibody heavy and light chains. In three cases (Sp1, Sp2 and Sp7) it is shown that only the specific H and L combination has activity and that the myeloma chains are unable to substitute for them. In most cases lines have been derived which no longer express the MOPC 21 chains but only the specific antibody chains.

Derivation of specific antibody-producing tissue culture and tumor lines by cell fusion

Publisher Summary
This chapter reviews the somatic generation of antibody diversity. It was shown that a typical antibody molecule is composed of two identical light chains and two identical heavy chains. It had also been found that each of these two types of chain exhibits great sequence variability in the amino terminal region between one antibody molecule and the next and no sequence variability in the carboxyl terminal regions. Use of restriction enzymes and recombinant DNA methods allowed resolution of a long-standing and central issue in immunology, the genetic origins of antibody diversity. It turned out that an organism does not inherit even a single complete gene for antibody polypeptide chains. Rather, the genetic information is transmitted in germline as no more than several hundred gene segments. Through a series of specialized somatic recombination occurring specifically during the differentiation of B lymphocytes, these gene segments are assembled into tens of thousands of complete genes. In the immune system, organisms have exploited two major processes for modification of DNA, recombination and mutation, as a means to diversify somatically the limited amount of inherited genetic information to cope with the vastly diverse antigen universe. Somatic diversification allows the individual organism to generate a virtually limitless number of lymphocyte variants.

Somatic Generation of Antibody Diversity1

THE principle of isoelectric fractionation was applied to the separation of amino-acids as early as 1912 by Ikeda and Suzuki (Japanese patent quoted in ref. 8), but the technique did not become practical for the fractionation of macromolecular ampholytes such as proteins until the introduction of natural pH gradients1 and suitable carrier ampholytes2–4. Svensson described an apparatus for isoelectric focusing in shallow pH gradients formed by low molecular weight ampholytes and stabilized by sucrose density gradients3. In this form the technique has been successfully applied to studies on the heterogeneity of myoglobins4,5, cytochrome c6, lactoperoxidase7 and a number of other proteins (see review by Haglund8).

Isoelectric Focusing in Polyacrylamide Gel and its Application to Immunoglobulins

Plasma-cell tumour 5563 forms a single molecular species of immunoglobulin IgG(2)a, i.e. one variant of heavy chain and one variant of light chain. The molecules formed are labile and undergo alterations in charge properties, which rapidly lead to heterogeneity of the myeloma protein after synthesis. The single immunoglobulin species originally formed is found only after the shortest time-intervals tested, i.e. 10min incubation. Two types of changes in charge properties take place: (1) The originally formed protein (component o) is converted via an intermediate o' into the most basic form of 5563 myeloma protein found in serum (component a). Charge differences between these components are suppressed at pH8.9, but can be studied by chromatography at pH6.5 or by analysis of isoelectric points by isoelectric focusing in polyacrylamide gel. The conversion of components o and o' into component a apparently commences soon after assembly of the molecules and proceeds to completion extracellularly. (2) The second type of charge difference that distinguishes components a, b, c and d is exhibited over the pH range 6.0-8.9, but not at acid pH, and has been studied by electrophoresis at pH8.9, by chromatography and by isoelectric focusing. Conversion of component a into components b, c, d and e is only partial so that all five components can be found at decreasing concentrations in serum. Both types of charge alteration can be effected in vitro in the presence of serum, with optimum pH8.0. None of the charge differences could be attributed to the secretion process, since a component with the same isoelectric point as component o was found in secreted myeloma protein (1h). We have found no evidence to support the idea that the first type of change from component o to component a is due to ring formation of N-terminal [(14)C]glutamine into pyrrolid-2-one-5-carboxylic acid; however, our findings do not exclude this process happening very rapidly to a precursor of component o, possibly the polypeptide chain during or immediately after synthesis. In studying this point we noted that not only the heavy chains but also the kappa-type light chain of mouse 5563 myeloma protein have a blocked N-terminus.

One cell–one immunoglobin. Origin of limited heterogeneity of myeloma proteins

Proteins purified by a variety of procedures have almost invariably shown heterogeneity when analyzed by isoelectric focusing. Multicomponent isoelectric spectra for homogeneous proteins have been a cause for concern and inevitably the analysis technique was questioned. With one or two exceptions, isoelectric focusing has not been proven to introduce heterogeneity into proteins. On the contrary, the use of this high-resolution equilibrium separation method has caused us to reexamine our criteria for protein homogeneity. A protein is usually defined by a particular function or property, though clearly this particular feature may be shared by several similar or even disparate proteins. One name may mean one protein but it may cover a group of proteins distinguishable by criteria not included in the original definition. I n the latter case, it would later be discovered that several molecular species were present and the “pure” protein would be said to be “heterogeneous.” There are a wide variety of reasons why a protein might be heterogeneous. I will list some of them and then discuss the interpretation of isoelectric spectra of proteins in terms of these various categories of heterogeneity. Basically, the causes of protein heterogeneity are either synthetic or postsynthetic.

Microheterogeneity and Allomorphism of Proteins

1. An analytical technique of isoelectric focusing in thin layers of polyacrylamide gel has been used to determine the isoelectric point, pI, of several proteins in the presence and in the absence of concentrated urea. 2. The presence of urea did not greatly affect pI except for bovine plasma albumin, where an increase of approx. 1pH unit was found. 3. Evidence is presented that this change in the pI of bovine plasma albumin is due to the normalization of certain ionizable groups on unfolding of the protein in urea. 4. Evidence is also presented that prolonged exposure of bovine plasma albumin to urea results in intramolecular disulphide interchange and that, on removal of urea, the new patterns of disulphide bonding stabilize abnormal conformations with pI values intermediate between those of the native and denatured states. 5. The studies demonstrate heterogeneity in bovine plasma albumin based on primary-sequence differences. 6. Isoelectric focusing of proteins in urea appears to be useful in the study of various aspects of protein structure.

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Isoelectric focusing of proteins in the native and denatured states. Anomalous behaviour of plasma albumin

The number of different anti-NIP (4-hydroxy-5-iodo-3-nitro-phenacetyl) molecules which inbred CBA/H mice can make was estimated. Spleen cell transfer has been used and the isoelectric spectra of antibodies present in the sera of recipient mice determined. Comparison between the sets of monoclonal antibody spectra deriving from different donor mice gave a repeat frequency for individual spectra. This frequency permitted a statistical estimate (see Addendum) of the minimum number of different anti-NIP molecules potentially capable of being elicited in CBA/H mice. The most likely value of this pool is 8000 with 98 % probability that pool exceeds 3000 and 95 % probability that pool is less than 30 000.

A. R. Williamson

Papers

Isoelectric Focusing in Polyacrylamide Gel and its Application to Immunoglobulins

One cell–one immunoglobin. Origin of limited heterogeneity of myeloma proteins

Microheterogeneity and Allomorphism of Proteins

Isoelectric focusing of proteins in the native and denatured states. Anomalous behaviour of plasma albumin

The extent of diversity of anti-hapten antibodies in inbred mice, anti-nip (4-hydroxy-5-iodo-3-nitro-phenacetyl) antibodies in cba/h mice.