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C Milstein

Publications and source records attributed to C Milstein.

At least 109 records · Page 6Linked to original sources
PubMed1987

The regulation of membrane-bound and secreted immunoglobulins in the human lymphoid cell line LICR-LON and human hybridomas.

The synthesis of membrane-bound and secreted immunoglobulin was investigated in the human line LICR-LON-HMy2, a cell line often used for the derivation of human hybridomas. PAGE-SDS analysis of immunoprecipitates obtained from 35S-methionine labelled cell lysates shows that LICR-LON synthesize a hitherto undetected membrane form of IgG (with a heavy chain of mol. wt 62,000) in addition to the secretor form of IgG already described (55,000 heavy chain). Tunicamycin treatment, pulse-chase experiments and Western blot analysis showed that both chains are synthesized as independent proteins. Hybridomas obtained after fusion of LICR-LON and human peripheral blood lymphocytes retained the ability of the parental cell line to synthesize gamma m and gamma s. Some of these hybrids synthesize and secrete IgM which presumably originates from the parental B-lymphocytes. Precipitation and PAGE-SDS analysis of membrane proteins after iodination of intact cells revealed only one heavy chain band, corresponding in size to that of the gamma m. No indication of the synthesis of the membrane form of IgM was found in the hybrids. These data show that the parental (lymphoid) phenotype (m and s-IgG) is codominant with the more differentiated phenotype (s-IgM) of the fusion partner cell (plasma cell). These observations are compatible with a class-specific m-s regulation operating on a different chromatin structure at the expressed Ig loci of each parental chromosome.

Antibodies, Neoplasm↗
PubMed1987

Structure and expression of the human thymocyte antigens CD1a, CD1b, and CD1c.

The CD1 human antigens are a family of at least three components, CD1a, CD1b, and CD1c, that are characteristic of the cortical stage of thymocyte maturation. CD1a was originally named HTA1 or T6 and thought to be the human equivalent of mouse Tla. The genes coding for all three have now been identified by transfection into mouse cells. The transfectants express the surface antigens that can then be recognized by the corresponding cluster of monoclonal antibodies used to define the three members of CD1. The full sequence of the genomic DNA is described for all three. The intron-exon structure of CD1a is deduced by comparison with a near-full-length cDNA clone. Similar structures are proposed for the other two, largely based on sequence homology. An unusually long 5'-untranslated exon (280 bases long) is highly conserved between the three genes, suggesting an important but unknown function. CD1c has a duplicated form of this exon that is thought to be spliced out. The major homology between the three antigens is in the beta 2-microglobulin-binding domain. The general relatedness to major histocompatibility complex class I and class II molecules is significant but low, with no section of higher homology to mouse Tla.

Amino Acid Sequence↗
PubMed1986

Effect of purified human interferon-alpha on the expression of differentiation antigens and mitogen reactivity of cultured human thymic cells.

Human thymic cells were cultured in vitro either alone or with the addition of a highly purified preparation of human interferon-alpha. Immunofluorescence techniques using a series of monoclonal antibodies showed that 2-day cultured thymocytes express a more mature phenotype (low HTA 1, high T3 and HLA-A,B,C) than normal, uncultured thymocytes. Interferon addition to the cultures results in a strong increment in the number of HLA+ cells and in the total amount of HLA expressed by the cultured cells. Experiments with purified cell populations showed that the cortical, immature, thymocyte was the target cell for interferon action. Phytohemagglutinin responses--but not interleukin 2 responses--were diminished after pretreatment of thymic cells with interferon. We suggest that interferon may favor a pathway of intrathymic differentiation phenotypically characterized by a high content of Class I HLA antigens.

Antibodies, Monoclonal↗
PubMed1986

Advantages of bispecific hybridomas in one-step immunocytochemistry and immunoassays.

A chemical selection procedure has been used to prepare a hybrid hybridoma cell line (P4C1) following fusion of two previously established hybridomas secreting antiperoxidase and antisubstance P, respectively. P4C1 secretes bispecific monoclonal antibody alongside the two parental antibodies, with no visible inactive heterologous heavy-light chain pairs. The bispecific monoclonal antibody is thus easy to purify in excellent yields. The advantage of its monovalency for one antigen and simultaneous binding of a marker enzyme has been explored for its potential use in competitive immunoassays. Its use in immunocytochemistry led to major improvements in sensitivity, signal-to-noise ratio, simplification of staining procedures, and ultrastructural preservation of subcellular elements. Particularly remarkable was that, unlike conventional procedures, the immunoreaction with the bispecific monoclonal antibody was homogeneously distributed across the entire thickness of a 50-micron section.

Antibodies, Monoclonal↗
PubMed1986

Isolation of CD1 genes: a family of major histocompatibility complex-related differentiation antigens.

CD1 differentiation antigens are defined by a group of monoclonal antibodies that characterize immature human thymocytes. A cloned cDNA has been used to identify CD1 genes in a human genomic library. Five CD1 genes have been isolated, and Southern blot analysis suggests that these represent all the cross-hybridizing human CD1 genes. They share a highly conserved exon, which is homologous to the beta 2-microglobulin-binding domain (alpha 3) of major histocompatibility complex (MHC) class I antigens. In this domain, amino acid sequences are 71-88% homologous. However, the homology between CD1 and MHC class I alpha 3 domains is only 21%. This is the same degree of homology as between either of them and the class II beta 2 domain, which does not bind beta 2-microglobulin. The evolutionary implications of these results are discussed.

Antigens, Differentiation, T-Lymphocyte↗
PubMed1986

Luminescent detection method for immunodot, Western, and Southern blots.

An anti-peroxidase-anti-biotin hybrid hybridoma rat cell line, capable of producing a bispecific monoclonal antibody, has been derived to explore its use in conjunction with a luminol immunodetection system. Luminescence was detected using x-ray film. The method was sufficiently sensitive and effective, but was less sensitive than autoradiographic methods using high-specific-activity 32P-labeled probes. Exposure times, on the other hand, were of the order of seconds rather than days. The direct binding of both peroxidase and biotin by the bispecific monoclonal antibody is simpler but less sensitive than the more conventional indirect method using a commercial peroxidase coupled with anti-rat antibody as a developing antibody.

Animals↗
PubMed1985

Light chain germ-line genes and the immune response to 2-phenyloxazolone.

Direct sequencing of mRNA has shown that the early primary response of the BALB/c mouse to the hapten 2-phenyloxazolone is dominated by antibodies with a particular light chain, V kappa-Ox1. Although the V kappa-Ox1 sequence is still commonly expressed later in the response it now includes a number of nucleotide changes. From two independent BALB/c germ-line DNA libraries 13 different genes hybridizing to a V kappa-Ox1 probe were isolated and characterized. Two are identical to mRNA sequences found in the early primary response, one of which is the V kappa-Ox1 sequence. None of the germ-line clones show the characteristic nucleotide changes contained in the late anti-phenyloxazolone light chain mRNAs. These results demonstrate that the V kappa-Ox1 sequence used in the early primary response is entirely encoded by the germ-line and further substantiate the importance of somatic mutations in the maturation of the anti-phenyloxazolone response. The statistical analysis of the data shows that the V kappa-Ox1 related germ-line gene family contains greater than 20 and probably less than 50 genes.

Animals↗
PubMed1985

A direct antigen-binding assay to screen hybridoma supernatants.

A simple and convenient method of directly assaying hybridoma supernatants for the desired monoclonal antibodies is described which obviates the need for labeled second or third antibody conjugates. Culture supernatants (1-5 microliters) were directly spotted onto a nitrocellulose sheet, and additional protein binding sites blocked with bovine serum albumin and incubated with enzyme-labeled, radioactive, or fluorescent antigen. Positive hybridoma supernatants were identified after washing and detection of bound antigen by appropriate means.

Antibodies, Monoclonal↗
PubMed1985

Preliminary crystallographic study of the Fab fragments of two monoclonal anti-2-phenyloxazolone antibodies.

We report on the preparation, crystallization, and preliminary x-ray crystallographic study of the Fab fragments of two monoclonal anti-2-phenyloxazolone antibodies obtained from the secondary response to this hapten. The Fab fragment from one of these (NQ10/12.5) has been crystallized from polyethylene glycol 8000 solutions in a form suitable for high-resolution x-ray crystallographic studies. These crystals are monoclinic, space group C2, with a = 129.2 A, b = 79.4 A, c = 57.7 A, beta = 96.2 degrees, and one Fab/asymmetric unit. Determination of the three-dimensional structure of Fab NQ10/12.5 should help clarify the role of somatic mutation in the maturation of an immune response. This antibody and an anti-lysozyme antibody also under study apparently use the same germ-line encoded VK and a similar VH gene, respectively, as the idiotypic anti-oxazolone antibodies characteristic of the primary response. A comparative study of the two structures should shed light on the role of the pairing of heavy and light chains in the antigen-binding function of antibodies.

Animals↗
PubMed1985

Stimulation of HLA-A,B,C by IFN-alpha. The derivation of Molt 4 variants and the differential expression of HLA-A,B,C subsets.

Spontaneous mutants with altered HLA-A,B,C response to interferon-alpha (IFN-alpha) were isolated from the human thymus leukemia cell line Molt 4. Using fluorescein isothiocyanate (FITC)-conjugated W6/32 (a monoclonal antibody to HLA-A,B,C) and the fluorescence-activated cell sorter, the cells with highest and lowest fluorescence after 24-48 h of IFN-alpha treatment were selected and expanded. After several cycles of selection, mutant clones with low (greater than 10% of wild-type) and high (three times better) response were obtained. A similar protocol was employed to derive high responder mutants with the monoclonal antibody YT76, which recognises a subset of HLA strongly induced by IFN-alpha. Stable clones were derived for which YT-HLA induction was 7-fold that of Molt 4 cells and for which HLA induction occurred at 100-fold lower concentrations of IFN-alpha. The high response phenotype of the mutants was not accompanied by a significant increase in the constitutive level of expression of HLA-A,B,C (in the absence of IFN). The increase in the level of HLA-A,B,C expression after IFN-alpha treatment is mostly accounted for by the increase in the expression of a subset of HLA molecules, detected by the monoclonal antibody YT76 including HLA-B molecules.

Cell Line↗