Roger H. Kennett

Hybridomas: A new dimension in biological analyses

SummarySince the first report of hybridomas producing monoclonal antibodies by Kohler and Milstein in 1975, this technique has spread to nearly all areas of biological, biochemical, and biomedical research. Watching the use of these methods spread from immunologists to cell biologists, developmental biologists, biochemists and to other biological disciplines and observing the nearly logarithmic increase in publications using these reagents has been in itself fascinating and informative. An overview of the development of this technology and its applications is presented including the use of monoclonal antibodies to study cell surface molecules, differentiation antigens, receptors, and histocompatibility antigens. The use of these antibodies to analyze microorganisms and parasitic antigens as well as their use in the genetic analysis of human cell surface antigens and the detection of polymorphic variation in enzymes and other proteins is discussed. Examples of the application of monoclonal reagents to the study of tumor cell biology including the labeling of metastatic tumor cells and the detection of cell surface molecules implicated in the regulation of growth control and cell division are provided.

The independent expression of HLA and? 2-microglobulin on human-mouse hybrids

Direct cytotoxicity and absorption analysis were used to detect HLA antigens on man/mouse somatic cell hybrids. Confirmation of the assignment of the Major Histocompatibility Complex to chromosome 6 was obtained. The same series of hybrids were used to study the interaction between HLA and β2m on the cell surface. Results obtained from a series of clones indicated that there was complete independence of expression of HLA and β2m. Human β2m was found to be unnecessary for the normal serological expression of HLA, suggesting that mouse β2m could possibly replace human β2m as a subunit of the HLA molecule. Evidence is presented demonstrating that this is in fact the case. Heteroantisera to human β2m and alloantiserum to HLA-A2 were used, together with complement, to selectively remove the chromosome coding for β2m and HLA from a hybrid clone. Manipulation of the karyotype in this way further confirmed the independence of HLA and β2m on somatic cell hybrids.

Glutaraldehyde fixation of the primary antibody-antigen complex on nitrocellulose paper increases the overall sensitivity of immunoblot assay

A simple method to increase the overall sensitivity of immunoblot assays is described. The method is based on fixation of the primary monoclonal antibody-antigen complex on nitrocellulose paper by a divalent crosslinker, glutaraldehyde. The fixation of the antibody-antigen complex significantly increases the sensitivity of the assay. Unexpectedly, the glutaraldehyde treatment also improves the signal/noise ratio by decreasing non-specific binding of secondary antibodies and/or tertiary reagents. Thus, a significant overall improvement of immunoblot assays is achieved by this method.

Monoclonal antibodies specific for cell culture mycoplasmas

SummaryMycoplasma infection of cell cultures is still a major problem in some laboratories. Although several methods can be used for their detection, identification is normally by serological procedures. As no commercial source for the necessary antibodies is available we have prepared monoclonal antibodies to the five mycoplasma species that account for the majority of cell culture infections. These antibodies have been characterized by the growth inhibition test (GIT), immunofluorescence, and enzyme linked immunosorbent assay (ELISA) and have shown perfect correlation in all tests when compared to conventional antisera raised in rabbits or donkeys. In addition, a monoclonal antibody toMycoplasma pneumoniae was produced.M. pneumoniae is an infrequent cell culture contaminant but is a human pathogen, and the monoclonal antibody described here could be useful in the clinical diagnosis ofM. pneumoniae infection in man.

The effects of gamma interferon on the natural killer and tumor cells of children with neuroblastoma. A preliminary report

Human neuroblastoma cells lack HLA‐A,‐B,‐C molecules which can be induced in vitro by gamma interferon (γIFN). To test the hypothesis that the same induction would occur in vivo leading to tumor regression, a Phase I study was initiated. Seven patients with neuroblastoma were entered on a Phase I study of recombinant γIFN in children. Three received 0.05 mg/m2 intravenously (IV) three times a week, three received 0.1 mg/m2 for 4 weeks, and one patient withdrew from study before receiving adequate treatment for evaluation. No significant clinical response was seen. The side effects were fever and chills, and no serious toxicity occurred. Natural killer (NK) and lymphocyte activated killer (LAK) precursor activity of peripheral blood mononuclear cells was determined before and during treatment, and expression of HLA‐A,B,C molecules was looked for on the tumor cells in the bone marrow of five patients. The NK activity initially low, reached control levels in six patients, but the increase was transient. The LAK precursor activity remained normal. Expression of HLA‐A,B,C, initially absent, was induced on the neuroblastoma cells in four of six patients.

Assignment of the structural genes for the α subunit of hexosaminidase A, mannosephosphate isomerase, and pyruvate kinase to the region q22-qter of human chromosome 15

Concordant segregation of the expression of the α subunit of human hexosaminidase A, human mannosephosphate isomerase, and pyruvate kinase was observed in somatic cell hybrids between either thymidine kinase-deficient mouse cells or thymidine kinase-deficient Chinese hamster cells and human white blood cells carrying a translocation of the distal half (q22-qter) of the long arm of chromosome 15 to chromosome 17. A positive correlation was established between the expression of these human phenotypes and the presence of the distal half of the long arm of human chromosome 15.

Use of monoclonal antibodies in an enzyme-linked inhibition assay for rapid detection of streptococcal antigen

We have developed an enzyme-linked monoclonal antibody inhibition assay to detect bacterial antigens in cerebrospinal fluid. The monoclonal antibody used in this immunodiagnostic test was produced by continuous cultures of hybrid myeloma cell lines. Using this assay, type III GBS antigen was detected in CSF specimens from 11 culture-proven cases of GBS meningitis and in the knee aspirate from an infant with GBS septic arthritis. Five spinal fluid specimens from meningitis due to other bacterial pathogens and ten other control samples were negative. The ELMIA detected streptococcal antigen at a concentration of 10 ng/ml, and is more sensitive and specific than currently used immunodiagnostic tests.

Monoclonal Antibodies against Human Tumor-Associated Antigens

It is now well established that certain tumor cells express surface antigens that are not present on cells in the tissue from which the tumor was initially derived (Ruddon, 1978; Greaves and Janossy, 1978). Such antigens have several possible origins: (1) new genetic information resulting from either mutation or oncogenic viral infection, (2) expression of normal genetic information out of temporal or cellular context, such as the expression of fetal antigens on cells in an adult, (3) rearrangement or modification of normal cell surface structures by mechanisms such as glycosylation, protease activity, or intermolecular interactions due to changes in membrane structure or fluidity. The production of specific antibody reagents against these antigens would facilitate analysis of their structure and genetic origin and also provide a means of detection and perhaps even selective destruction of tumor cells (Table I).

3,4-Dihydroxyphenylalanine (DOPA) metabolism and retinoic acid induced differentiation in human neuroblastoma

In mature cells of the sympathetic nervous system and the adrenal gland, the activity of dihydroxyphenylalanine decarboxylase (DDC) is higher than that of tyrosine hydroxylase and 3,4-dihydroxyphenylalanine (DOPA) does not accumulate in the cells. On the other hand, it is known that in some neuroblastoma cells there is a relative deficiency of DDC, resulting in accumulation and secretion of DOPA. Such a relative deficiency of DDC is a characteristic of neural cells at an early stage of neural crest development, suggesting the neuroblastoma are cells arrested in early neural crest development. If this were the case, it is possible that agents such as retinoic acid (RA) could induce neuroblastoma to differentiate into mature cells with respect to their metabolism of catecholamines. We have measured the effect of RA on the metabolism of DOPA and expression of tyrosine hydroxylase and DDC in human neuroblastoma cell lines, CHP-126, CHP-134, IMR-32, NB-59, and LA-N-5. When the cell cultures were treated with RA, they showed wide variations in response as measured by morphological change, growth inhibition, enzyme activities and DDC, but does not increase DDC relative to tyrosine hydroxylase. It is concluded that RA does not induce biochemical differentiation of the neuroblastoma into mature cells even when there are extensive morphological changes and suppression of growth rate.

Germ-Cell-Related and Nervous-System-Related Differentiation and Tumor Antigens

In recent years increasing attention has been given to the cell surface as an important functional part of the cell. Its component molecular structure and the mechanisms for its involvement in the interactions of cells with their environment are currently the subject of intensive study. Considerable evidence suggests that important aspects of many complex cell-environment interactions involve specific components of the cell surface. A graphic example of this is the species-specific aggregation factors of sponge (Frazier and Glaser, 1979). The factor (molecule), eluted from the sponge cells in Ca2+, Mg2+-free seawater, will cause species-specific aggregation of dissociated sponge cells. While the role of this molecule in the intact sponge is unknown, it may function as one part of the complex system that maintains the cellular associations of the live sponge. Examples of specific surface function in higher organisms are tissue-specific aggregation of cells (Culp, 1978), the sorting out of cells from different tissues (e.g., mesonephric and chondrogenic cells) in cultures (Steinberg, 1978), and the requirement in kidney tubule induction for cell-surface contact between meta-nephrogenic mesenchyme and ureteric bud cells (Wartiovaara et al, 1974; Leh-tonen et al., 1975). Several mechanisms for cell-surface interactions have been proposed, but few of the interactions are well understood.