Herbert Lazarus

A Radioimmunoassay Using a Monoclonal Antibody to Monitor the Course of Epithelial Ovarian Cancer

The murine monoclonal antibody OC 125 reacts with an antigen (CA 125) common to most nonmucinous epithelial ovarian carcinomas. An assay has been developed to detect CA 125 in serum. By this assay, only 1 per cent of 888 apparently healthy persons and 6 per cent of 143 patients with nonmalignant disease had serum CA 125 levels above 35 U per milliliter. In contrast, 83 of 101 patients (82 per cent) with surgically demonstrated ovarian carcinoma had elevated levels of antigen. In 38 patients with epithelial ovarian carcinoma monitored on 2 to 18 occasions during 2 to 60 months, antigen levels ranged from less than 1 to more than 8000 U per milliliter. Rising or falling levels of CA 125 correlated with progression or regression of disease in 42 of 45 instances (93 per cent). Determination of CA 125 levels may aid in monitoring the response to treatment in patients with epithelial ovarian cancer.

Reactivity of a monoclonal antibody with human ovarian carcinoma.

A murine monoclonal antibody (OC125) has been developed that reacts with each of six epithelial ovarian carcinoma cell lines and with cryopreserved tumor tissue from 12 of 20 ovarian cancer patients. By contrast, the antibody does not bind to a variety of nonmalignant tissues, including adult and fetal ovary. OC125 reacts with only 1 of 14 cell lines derived from nonovarian neoplasms and has failed to react with cryostat sections from 12 nonovarian carcinomas.

Application of endonuclease mapping to the analysis and prenatal diagnosis of thalassemias caused by globin-gene deletion.

We applied a recently developed and more direct technic to diagnose thalassemia syndromes associated with deletion of particular globin structural genes and to assess a fetus at risk for one of those conditions, deltabeta-thalassemia. The method allows assessment of the globin genes present in total cellular DNA and is applicable to amniotic-fluid cell DNA. Cellular DNA fragments produced by cleavage using two specific restriction endonucleases are separated on the basis of size by agarose-gel electrophoresis, and the distribution of specific sequences among the DNA fragments determined by molecular hybridization. We observed the total deletion of alpha-globin genes in homozygous alpha-thalassemia (hydrops fetalis with hemoglobin Barts) and the deletion of particular beta and beta-like sequences in cases homozygous for hereditary persistence of fetal hemoglobin and deltabeta-thalassemia. Analysis of amniotic-fluid cell DNA from a fetus at risk for deltabeta-thalassemia demonstrated the feasibility of these improved methods for antenatal diagnosis. The molecular studies confirmed the diagnosis predicted by analysis of fetal blood and established at birth.

The molecular basis of α-thalassemias: Frequent occurrence of dysfunctional α loci among non-Asians with Hb H disease

Abstract Study of Asians has previously indicated that deletion of α-globin structural genes is the predominant lesion in α-thalassemias and that Hb H disease occurs when three of four normal α loci per cell are deleted. To test the generality of this model, Hb H disease DNAs of both Asian and non-Asian origin were analyzed by restriction endonuclease mapping using the technique of Southern (1975). Whereas in normal DNA, α sequences are present in a single Eco RI fragment of cellular DNA approximately 22.5 kb long, fragments of 22.5, 20 and 2.6 kb were found in various Hb H disease DNAs. The 20 kb Eco RI fragment alone, in which a single α-globin structural locus resides, was found in Asian Hb H disease DNA. This finding is consistent with the deletion model of α-thalassemia. In contrast, seven of eight non-Asian Hb H disease DNAs displayed a more complex molecular composition. The fragment patterns observed were 22.5 kb alone, 22.5 plus 2.6 kb, 20 plus 2.6 kb and 20 kb alone. Non-Asian Hb H disease DNAs contained one, two or three α loci per cell in contrast to the one locus predicted by the simple deletion model of α-thalassemia. The data are best explained by the existence of defective α loci in certain individuals with α-thalassemia, particularly outside the Asian population. Restriction mapping of the 20 kb Eco RI fragment found in Asian and some non-Asian Hb H disease DNAs demonstrated a striking similarity in the placement of restriction sites about the single α gene compared with sites about the two genes in the 22.5 kb Eco RI fragment seen in normal DNA. These data are consistent with origin of the 20 kb fragment from the 22.5 kb normal Eco RI fragment by either unequal crossing-over or a deletion event. The molecular heterogeneity and frequent occurrence of defective α loci in non-Asian Hb H disease DNAs described here may explain, in part, the clinical heterogeneity of α-thalassemias and the absence of the homozygous deletion state (hydrops fetalis) in non-Asians. Further study of cellular DNA fragments containing the defective α loci identified in this work may indicate the types of specific mutations responsible for abnormal globin gene expression and complement similar studies on abnormal β genes in β-thalassemias.

Absence of messenger RNA and gene DNA for β-globin chains in hereditary persistence of fetal hemoglobin

The relative amounts of alpha-amd beta-globin mRNA and globin gene DNA were measured in reticulocyte RNA and lymphocyte DNA of an individual with homozygous hereditary persistence of fetal hemoglobin whose red blood cells contain 100% fetal hemoglobin (hb F: alpha2gamma2.) Molecular hybridization assays used as probes full-length DNA copies of human alpha- and beta-globin messenger RNA. The results of these hybridization assays demonstrated the expected amounts of alpha-globin mRNA and gene DNA, but absence of beta-globin mRNA and absence of beta-globin gene DNA. In the individual studied, hereditary persistence of fetal hemoglobin is associated with total deletion of the beta-globin structural gene.

An enzyme-linked immunosorbent assay (ELISA) for the detection of monoclonal antibodies to cell surface antigens on viable cells.

A rapid and inexpensive ELISA method is described which is suitable for the large scale screening of monoclonal antibodies to cell surface antigens. The use of acrylic plates and viable cells eliminates background and false positive reactions, and avoids modification of surface antigens caused by fixation. This facilitates easy and rapid detection of positives by visual inspection of the plates. The specificity and sensitivity of the methods is comparable to indirect immunofluorescence or radioimmunoassay. The advantages of this ELISA system when compared to these methods and to previously described ELISA systems utilizing fixed cells are discussed.

Comparison of a rabbit heteroantiserum and a murine monoclonal antibody raised against a human epithelial ovarian carcinoma cell line

An ovarian carcinoma cell line (OVCA 432) and a B-lymphocyte line (LAZ 446) were established from the same donor. A heteroantiserum (D-100) was prepared in rabbits against OVCA 432 and absorbed with LAZ 446, human AB erythrocytes, and the CX-1 colorectal cell line. After absorption, D-100 reacted by indirect immunofluorescence with six of six epithelial ovarian carcinoma cell lines and cryostat sections of 18 of 18 epithelial ovarian tumors but bound to zero of 12 nonovarian tumor cell lines. Despite a lack of reactivity with nonovarian tumor cell lines, D-100 reacted with epithelial components of normal ovary, fallopian tube, endometrium, endocervix, breast, colon, and epididymis. A murine monoclonal antibody (OC 133) was also raised against OVCA 432 and selected for lack of reactivity with the autologous B-lymphocyte line LAZ 446. OC 133 reacted with six of six ovarian carcinoma cell lines and cryostat sections of seven of 19 ovarian tumors, but it also reacted with five of five nonovarian tumor cell lines. Of 12 normal tissues examined, OC 133 reacted with endometrium and endocervix only. Whereas D-100 bound to serous, mucinous, endometrioid, and clear cell ovarian neoplasms, OC 133 bound only to serous tumors. In developing monoclonal reagents, cell lines may provide a useful source of antigen, but promising antibodies should be screened for reactivity with sections of normal and malignant human tissues.

IgM-restricted production of immunoglobulin by lymphoid cell lines from patients with immunodeficiency with hyper IgM (dysgammaglobulinemia)☆

Abstract Patients with immunodeficiency with hyper IgM have elevated levels of serum IgM and sometimes IgD, with absent IgG and IgA. To examine the cellular basis of this deficiency, B lymphocytes from five patients with this disease were transformed by Epstein-Barr virus (EBV). The resulting lymphoid cell lines bore surface IgM and IgD, but no IgG or IgA. IgM was secreted by cell lines for all five individuals. No secretion of IgG, IgA, or IgD was detected. The restricted expression of IgM and IgD as surface molecules and secretion of IgM only contrasts with the expression of multiple classes of immunoglobulin by recently established cell lines from normal individuals. These results demonstrate there is a primary B lymphocyte defect responsible for immunodeficiency.

Patterns of late replication in X chromosomes of human lymphoid cells

Two predominant patterns of late X replication were observed in both short-term and established human lymphoid cultures. One pattern was found in a minority of short-term cultured T-cell metaphases, in most lectin-stimulated B cells, and, with minor variations, in established B-cell lines. In these cells, DNA replication terminated in the distal part of the long arm of the late X. A different pattern was found in the majority of lectin-simulated T cells and in the T-cell line CCRF-CEM. These cells exhibited terminal replication in a region of the long arm of the late X that was nearer to the centromere. It is speculated that the variations in replication patterns correlate with phenotypic and functional characteristics of human lymphoid subsets.

Human cell lines express multiple populations of Ia-like molecules

Three monoclonal antibodies have been used to isolate Ia-like antigens from three human cell lines; two of which are thought to be homozygous at the HLA-D/DR locus. Complete extraction of the Ia antigens identified by one antibody leaves those recognized by the two remaining antibodies in three parallel sets of experiments, indicating that the antigenic determinants recognized by these antibodies are present on three different populations of Ia molecules from cells of single individuals. These three populations of Ia-like molecules may reflect serologic variants of the product of a single genetic locus or may represent the products of as many as three nonallelic genetic regions. Demonstration of the existence of these multiple populations of Ia-like molecules on presumed homozygous typing cells indicates that this antigenic system is much more complex than has been generally realized. Further study may clarify the relationship between HLA-D/DR type and susceptibility to a variety of diseases and ultimately lead to better matches and improved survival for allogenic transplants. Since the HLA-D region is intimately involved in regulation of the immune response and susceptibility to a variety of diseases, use of monoclonal antibodies specific for discrete Ia antigens, the only identified products of the HLA-D region, may facilitate dissection of its many biological functions.