Search

Monoclonal antibodies are frequently shown to participate in unexpected cross reactions involving two apparently dissimilar antigens. This can be attributed either to partial epitope identity or to irrelevant interactions involving additional binding capacity of the antibody. The majority of such interactions appear to fall into the latter category. Such cross reactions are most commonly detected when one of the antigens has a high epitope density and the antibody is multivalent so that a spurious interaction of low intrinsic affinity is amplified by local concentration effects. In this review Souravi Ghosh and Ailsa Campbell discuss the selection of a monoclonal antibody for maximum affinity for the antigens it is designed to study and minimum cross-reactivity.

Lymphocytes from matched pairs of tumour-invaded and tumour-free lymph nodes from 22 stage II breast cancer patients have been analysed for expression of phenotypic and activation markers by flow cytometry. Although the relative proportions of T and B lymphocytes were similar in the two nodes, significant differences in the distribution of T cell subsets were observed between nodes that were invaded and those that were not. The CD4/CD8 ratio was markedly depressed in tumour invaded nodes (P0.001). This was due to an increase in the number of CD8+ T cells (P0.001) and a decrease in the CD4+ T cell population (P = 0.008) in invaded nodes in comparison with tumour-free nodes. The percentage of CD8+ T cells expressing HLA DR (P = 0.023) and IL-2 receptors (Tac) (P = 0.029) was significantly higher in invaded nodes and, while CD4+ T cells expressing HLA DR (P = 0.036) were also in a higher proportion of Tac expressing CD4+ T cells failed to reach significance. Although invaded nodes in a few patients were found to have a higher percentage of IgG-expressing B cells, no significant differences were observed between the two groups of nodes. These results suggest that the presence of metastatic tumour cells in a lymph node is associated with specific alterations in the T cell population.

Fresh tumour infiltrating lymphocytes (TILs) from 6 uveal melanomas and 4 breast cancers were analysed by flow cytometry with a panel of 6 monoclonal antibodies to V beta regions of the T cell receptor (V betas 5a, 5b, 5c, 6, 8a and 12a). With a single exception where one TIL sample lacked V beta 12a, lymphocytes from both tumour and blood contained cells reactive with all 6 probes, and no probe was highly dominant or missing. The proportions of reactive cells differed between tumour and blood within each patient. The data indicate that while tumour infiltrating lymphocytes have a capacity to locate selectively within the tumour they nonetheless comprise a population expressing a diversity of TCR V beta genes.

The quantitative distribution and phenotype of gamma/delta lymphocytes in the peripheral blood (PBL), tumour draining lymph node (LNL) and tumour infiltrating lymphocytes (TIL) from breast carcinoma patients were determined by one- and two-colour flow cytometry. The TCR-gamma/delta + cells generally expressed the T cell lineage antigen CD3. The proportions of such cells were variable but generally small from all the three sources. Phenotypic analysis revealed that the CD8 marker was consistently and predominantly observed on gamma/delta + CD3+ cells in the tumour infiltrate, whereas CD4 expression, while generally low, was noted on a significant percentage (median 10%) of LNL gamma/delta + lymphocytes. In both PBL and LNL the predominant gamma/delta cell population was CD4-8-.

This chapter deals with production and purification of antibodies. The main requirement for immunoassays is an antibody preparation that has a standard, reproducible, specific, and high-affinity interaction with the analyte under assay. Additional desirable qualities include stability in storage and the capacity for production in large amounts. The general procedure for monoclonal antibody production in rodents is illustrated. The usual animal is the Balb/c mouse, which is readily available from any laboratory supplier and routinely bred on all large university campuses. The usual tissue is the spleen, which is located on the right of the animal's body when it is viewed lying on its back. The mouse is immunized and then boosted 4 days before fusion, so that the B cells making the required antibodies have been stimulated into growth. This is vital because only growing cells fuse well. In case of humans, blood lymphocytes are used. Human monoclonal antibodies are projected to have considerable potential in therapy since they avoid the rejection problems experienced by animal antibodies and the dangers of HIV or hepatitis B transmission in the use of human polyclonal preparations. Affinity chromatography is described that is used only for polyclonal antibodies.

An antiserum was generated against a dodecapeptide whose sequence is found at the C-terminus of a cyclic AMP (cAMP)-specific, type-IVA phosphodiesterase encoded by the rat ‘dunc-like’ cyclic AMP phosphodiesterase (RD1) cDNA. This antiserum identified a single approximately 73 kDa protein species upon immunoblotting of cerebellum homogenates. This species co-migrated upon SDS/PAGE with a single immunoreactive species observed in COS cells transfected with the cDNA for RD1. Native RD1 in cerebellum was found to be predominantly (approximately 93%) membrane-associated and could be found in isolated synaptosome populations, in particular those enriched in post-synaptic densities. Fractionation of lysed synaptosomes on sucrose density gradients identified RD1 as co-migrating with the plasma membrane marker 5′-nucleotidase. Laser scanning confocal and digital deconvolution immunofluorescence studies done on intact COS cells transfected with RD1 cDNA showed RD1 to be predominantly localized to plasma membranes but also associated with the Golgi apparatus and intracellular vesicles. RD1-specific antisera immunoprecipitated phosphodiesterase activity from solubilized cerebellum membranes. This activity had the characteristics expected of the type-IV cAMP phosphodiesterase RD1 in that it was cAMP specific, exhibited a low Km cAMP of 2.3 microM, high sensitivity to inhibition by 4-[3-(cyclopentoxyl)-4-methoxyphenyl]-2-pyrrolidone (rolipram) (Ki approximately 0.7 microM) and was unaffected by Ca2+/calmodulin and low concentrations of cyclic GMP. The phosphodiesterase activities of RD1 solubilized from both cerebellum and transfected COS cell membranes showed identical first-order thermal denaturation kinetics at 50 degrees C. Native RD1 from cerebellum was shown to be an integral protein in that it was solubilized using the non-ionic detergent Triton X-100 but not by either re-homogenization or high NaCl concentrations. The observation that hydroxylamine was unable to cause the release of RD1 from either cerebellum or COS membranes and that [3H]palmitate was not incorporated into the RD1 protein immunoprecipitated from COS cells transfected with RD1 cDNA, indicated that RD1 was not anchored by N-terminal acylation. The engineered deletion of the 25 residues forming the unique N-terminal domain of RD1 caused both a profound increase in its activity (approximately 2-fold increase in Vmax) and a profound change in intracellular distribution. Thus, immunofluorescence studies identified the N-terminal truncated species as occurring exclusively ion the cytosol of transfected COS cells. The cDNA for RD1 thus appears to encode a native full-length type-IVA phosphodiesterase that is expressed in cerebellum.(ABSTRACT TRUNCATED AT 400 WORDS)

DNA aneuploidy and p53 or c-erbB-2 expression were simultaneously measured in 29 breast tumours by two-colour flow cytometry. (i) The majority of tumours had some cells expressing either p53 (5–68%) or c-erbB-2 (1–56%). (ii) Expression of p53 and c-erbB-2 was observed mainly in the aneuploid population of mixed aneuploid and diploid tumours but there was no significant correlation with a specific DNA index. Aneuploid tumours contained higher percentages of c-erbB-2 positive cells (average 25%) than purely diploid tumours (average 15%) but this just failed to reach significance (P = 0.074). No relevant trends were noted for p53 expression. (iii) Significantly increased c-erbB-2 expression was observed in stage 2 tumours (26%) compared to stage 1 tumours (12%) (P = 0.001) with no trend evident for p53 expression. (iv) The metastatic tumour in the axillary node contained similar or slightly higher percentages of positive cells than the matched primary tumour.

The phenotype and activation status of lymphocytes from the peripheral blood and axillary lymph nodes of 40 patients with breast cancer were analysed using flow cytometry and compared with lymphocytes from the blood and lymph nodes of 7 control subjects. There was little difference in the overall proportions of T and B lymphocytes but there was a much larger population of B cells bearing surface IgG and a greater number of CD4+ helper T cells, particularly in the regional nodes, in the breast cancer patients. Many more T cells in the cancer patients were found to be carrying the HLA DR and Tac antigens. The axillary lymph nodes were the major site of B cells and CD4+ T cells whilst the primary tumour was the source of the CD8+ suppressor/cytotoxic T cells. Any immune response appeared to be largely loco-regional and may therefore destroyed by conventional surgery or radiotherapy.

Fresh, paired primary tumours and lymph node metastases from breast cancer patients were compared by DNA flow cytometry. Although 65% of primary tumours were aneuploid, the detection of aneuploid peaks in corresponding nodal metastases was rare (only 6 cases out of 25) in single-parameter DNA analysis. Detection of aneuploid subpopulations in lymph nodes was greatly improved in dual-parameter DNA analysis using an anticytokeratin (CK) antibody which allowed ploidy determination on CK+ epithelial cells alone. Examination of 12 lymph nodes for CK+ cells revealed the presence of both diploid and aneuploid tumour cells in tumour invaded nodes. In patients with multiploid primary tumours, a subpopulation of the primary aneuploid cells was dominant in the nodal metastases. This suggests that aneuploidy is an integral property of metastatic cells and that within a primary tumour a subpopulation may have a higher metastatic potential.

An enzyme-based double monoclonal field diagnostic system detecting both serotypes of Vibrio cholerae has been developed. The system uses nitrocellulose as a solid support, 1.25% skimmed dried milk as blocking reagent, water as washing reagent, and alkaline phosphatase cross-linked to antibody by means of glutaraldehyde as detecting reagent. The sensitivity of the system was 10(5) vibrios/ml. The biotin-avidin system gave sensitivity an order of magnitude weaker. There were no cross-reactions with the range of other bacteria tested.

Objective To quantify vascular smooth muscle polyploidy and growth kinetics in aortic cells from stroke-prone spontaneously hypertensive rats (SHRSP) and from normotensive Wistar-Kyoto (WKY) rats, and to examine the effects of treatment with the angiotensin converting enzyme (ACE) inhibitor perindopril on these parameters. Design The following experimental groups were used: young (age 20 weeks) untreated WKY rats and untreated SHRSP; SHRSP treated with perindopril, and age- and sex-matched control SHRSP; and SHRSP treated with hydralazine and hydrochlorothiazide and age- and sex-matched control SHRSP. The effects of treatment of the SHRSP with perindopril for 30 days on vascular smooth muscle polyploidy and growth kinetics were measured and compared with the effects of equivalent antihypertensive doses of hydralazine and hydrochlorothiazide. Methods Vascular smooth muscle polyploidy was measured using flow-cytometry DNA analysis of freshly harvested cells. Growth curves were performed on cultured aortic cells. Plasma renin activity was measured by an antibody-trapping method, plasma angiotensin II (Ang II) by radioimmunoassay and plasma ACE activity by a colorimetric method. Cardiac hypertrophy was evaluated by measuring the heart weight:body weight and left ventricle + septum weight:body weight ratios. Results The SHRSP had markedly and significantly elevated G2 + M phase of the cell cycle. Treatment with perindopril resulted in a significant reduction in polyploidy in the SHRSP, whereas treatment with hydralazine and hydrochlorothiazide had no effect on the percentage of cells in the G2 + M phase of the cell cycle. The regression of polyploidy after treatment with perindopril was associated with a significant reduction in the concentration of Ang II and ACE activity, and with a significant regression of cardiac hypertrophy. Increased mitogenesis of cultured vascular smooth muscle cells from the SHRSP was not altered by treatment with perindopril. Conclusions ACE inhibition reduces vascular smooth muscle polyploidy in large conduit arteries. This type of vascular protection is mediated by the reduced Ang II and possibly by increased kinins level, rather than by the hypotensive effect alone.

Recent evidence indicates that substantial areas of the eukaryote chromosome exist in a superhelical configuration. Supercoils are therefore widely distributed in various biochemical systems which differ substantially in their methods of DNA packaging. This suggests that they may play a more general role in gene expression. Evidence obtained from the use of small circular supercoils suggests that the presence of superhelical torsion on DNA can lead to the opening up of specific and highly reactive sites under certain conditions. Supercoiling may therefore be a method of controlling gene activity through changes in DNA structure.

The self-association of oligonucleosomal chromatin particles in solution has been studied by light scattering and sedimentation. In the absence of magnesium ions no association is observed. In the presence of 70mM sodium or 2mM magnesium ions mono, di, tri and tetranucleosomes self-associate only if they contain bound histone 1. This association leads to the formation of compact aggregates and is continuous and non-cooperative. The relevance to higher order arrangements of nucleosomes is discussed.

Laser light scattering measurements have been made on a series of polynucleosomes containing from 50 to 150 nucleosomes. Radii of gyration have been determined as a function of polynucleosome length for different ionic strength solutions. The results suggest that at low ionic strength the chromatin adopts a loosely helical structure rather than a random coil. The helix becomes more regular on increasing the ionic strength, the dimension resembling those proposed by Finch and Klug for their solenoid model.

This report describes the ultrastructural localization of S-antigen in pig and human retinas by means of a highly specific rat IgG monoclonal antibody, S2.4.C5, followed by a secondary antibody adsorbed to colloidal gold. This monoclonal antibody gave definitive staining with negligible background. The protein was detected in both the rod outer and inner segments. Connecting cilia and the rod outer segment disc membranes were labelled. The outer segment plasma membrane was not obviously labelled. Cones were labelled at background level. S-Antigen was not detected in any other cells of the neural retina. The fate of S-antigen was also followed to the pigment epithelial phagosomes where intracytoplasmic ROS debris was stained with the antibody. No label, however, was detected in the choroid, suggesting that trans-cellular transport of the S-antigen did not occur.

Laser light-scattering studies of bacteriophage PM2 DNA showed the molecule to have mol.wt. 5.9 } 10(6) and root-mean -square radius 125 nm at an ionic strength of 0.2 mol/litre. Computer-generated curves compatible with these data were compared with the experimental interference curve for several structural models of the molecules. The data fit best to an asymmetric four-armed planar molecule in which all four arms emerge from or close to the one area of the molecule. This contrasts with the smaller DNA molecules investigated, which have shown a three-armed molecule, whose symmetry varies with primary structure.

In a previous study, we have described unusual cross-reactions among monoclonal antibodies (Mabs) to bacteria and in particular to the Inaba and Ogawa serotypes of Vibrio cholerae. In this study, the extent to which the binding sites of both antibodies and antigens overlap has been investigated by competitive binding and idiotypic analysis. The competitive binding data indicate that the cross-reactive binding of the Inaba Mabs to the Ogawa vibrios can be abolished by incubation with higher affinity Ogawa Mabs. However, rabbit antiserum raised against the Inaba series does not react with the Ogawa series, indicating that anti-Inaba Mabs do not share idiotypic determinants with anti-Ogawa Mabs. The results therefore suggest that the two sets of antibodies recognise different determinants which are closely related in spatial terms, and which consequently do not permit simultaneous binding of the two types of monoclonal antibody.

The naturally occurring humoral immune response of Campbell and Hunter strains of Royal College of Surgeons (RCS) rat to bovine retinal S-antigen and bovine rod outer segments was tested by a sensitive ELISA (enzyme linked immunosorbent assay) in animals up to the age of 9 weeks. Controls of Piebald Virol Glaxo, Wistar and DA rats were also employed. The ELISA results indicate that rats of all strains had a response to both antigens. The magnitude of the response in any particular strain varied with the age of the animal and the antigen and serum dilution employed in the assay. Considerable variation in response was observed among animals of the same strain at any one particular age. Immunoblot analysis of the strongest ELISA positive sera detected S-antigen and other autoantigenic proteins in both RCS and Wistar rats. The data suggest that the antibody response observed may have little significance to the retinal degeneration observed with RCS rats.

A murine monoclonal antibody which reacts strongly with the nucieoli of human epithelial cells has been isolated. The antibody is of the IgM class and the antigen has a molecular weight of 45 000. The antibody appears to react with interphase chromatin only and to have specificity for epithelial cells.

Lipopolysaccharide (LPS) from Vibrio cholerae has been analysed by sodium dodecyl sulfate-potyacrylamide gel electrophoresis. Under normal conditions of electrophoresis which resolve Escherichia coli LPS, V. cholerae LPS shows two diffuse and unresolved bands. However, on long gels at low concentration it can be resolved into two major band types. There are at least 10 slow moving, discrete bands of regular periodicity and three fast moving bands. Comparison with LPS from E. coli indicates that the heterogeneity occurs over a much smaller range of molecular weight in V. cholerae LPS, with the entire spectrum of discrete bands being contained within the space of four E. coli repeating units.

A murine monoclonal antibody produced against heat inactivated spores of Bacillus anthracis Ames, reacted with live or inactivated spores of several anthrax strains in indirect immunofluorescence (IF) tests. The reactive anthrax strain gave only a moderate degree of reaction. No staining of anthrax vegetative cells was observed. The monoclonal did not react with spores of non-anthrax Bacillus strains that gave cross reactions with mouse hyperimmune antiserum raised against Ames spores. The staining of individual spores in B. anthracis preparations was more heterogeneous with the monoclonal antibody than with the hyperimmune serum. Evidence is produced that the epitope for this monoclonal is not stable during long-term storage of inactivated spore preparations, and is not fully available for reaction with antibody until late in spore maturation. The monoclonal did not react by immunoblotting (Western blotting) of spore extracts. A monoclonal antibody produced against Ames spore extracts reacted with about 1% of Ames spores in IF tests, but not reproducible reactions with other anthrax strains were recorded. This monoclonal interacted with three bands in Western blots of anthrax spore extracts.

SynopsisStudies have been made on the incorporation of [8H]-deoxythymidine into the DNA of the livers ofRana catesbeianatadpoles. When metamorphosis was induced with tri-iodothyronine, the specific activity of the nuclear DNA rose 5 days after administration of the hormone. In contrast, the specific activity of the DNA from the mitochondrial fraction rose grave–2 days after hormone administration.In order to determine whether thein vivochange was due to alterations in the pool sizes of the DNA precursors,in vitrostudies on DNA polymerase were carried out. It was found that under conditions where the enzyme activity was not limited by availability of template or substrates, there was a rise in the DNA polymerase activity in crude cell extracts from the tadpole liver. Fractionation of the cell components showed that little of this increment in activity appeared to be located in the nucleus, but that a large percentage alteration in activity occurred in the mitochondrial and cell sap fractions.A possible interpretation of these results is that an increase in the mitochondrial DNA polymerase is one of the early effects of thyroid hormone. This possibility is discussed in relation to the other known effects of thyroid hormones in tadpoles, with particular reference to nucleic acid metabolism and also to mitochondrial hyperplasia.

Previous investigations on the persistence length of DNA in solution have revealed large discrepancies between hydrodynamic results and those from light-scattering techniques which have potentially a greater resolving power. The information obtained from experiments on a small circular DNA molecule has resolved these discrepancies. The non-superhelical circular double-stranded DNA molecule from bacteriophage [unk]X174-infected cells is small enough to permit accurate light-scattering extrapolations, and its solutions have negligible anisotropy. The persistence length obtained from experimental investigations on this molecule is comparable with that obtained by hydrodynamic techniques, even with variation of the excluded-volume factor.

The DNA-binding site of a previously characterized non-histone chromosomal protein antigen(s) from HeLa cells was investigated for its species specificity. Treatment with large amounts of micrococcal nuclease abolishes immunoactivity, which can then be recovered by the subsequent addition of human or HeLa DNA to reconstitute the immune complex. Neither rat nor calif DNA exhibits this property, but DNA from monkey cells gives considerable activity. The antigen is not, however, detectable in monkey chromatin.

In this study the immunoblotting technique has been employed to analyse the specificities of autoimmune sera from patients with systemic rheumatic disease for nuclear and cytoplasmic antigens from both human liver and cultured HeLa cells. The results show the heterogeneity of the autoimmune response but also indicate restricted antigenic specificities among some sera. Some evidence of tissue-specificity is presented and the advantages of the immunoblot technique are discussed.

Publisher Summary This chapter discusses analytical ultracentrifugation. Analytical ultracentrifugation has had a considerable effect on the study of biological macromolecules, in particular proteins and nucleic acids. Theoretical and practical advances have been made in recent years both in the equilibrium and velocity techniques, and data from analytical ultracentrifuge experiments are highly accurate because of the introduction of automatic data-processing equipment and the photoelectric scanner. Sedimentation equilibrium analysis continues to supplement other absolute methods, such as osmotic pressure and light-scattering methods, which have their basis in equilibrium thermodynamics. The development of three-component theory that applies to such conditions has allowed a clear understanding of the meaning of molecular-weight data obtained from centrifuge experiments. A most important area of progress has been the study of associating systems at chemical and sedimentation equilibrium, and alternative theoretical approaches are at present available to confirm the assignment of particular types of model. The analysis and simulation of boundary and band shapes in interacting systems has undergone important development, and the dangers of misinterpretation have been emphasized in recent years. The introduction of boundary analysis in active enzyme–substrate complexes represents a most interesting extension of the velocity technique. These developments have greatly expanded the potential for analyzing biological macromolecules in solution, and it is clearly desirable to use as many of these as possible to define completely the properties of the system under examination.

A solid-phase radioimmunoassay has been developed for the detection of cell specific chromosomal proteins which require the presence of DNA for their immunoreactivity. The assay involves the binding of chromatin particles sonicated under highly controlled conditions to PVC plates followed by first and second antibody with extensive detergent washing. The sensitivity and specificity of the assay system compares favourably with the complement fixation technique.

Two murine monoclonal antibodies of the IgG3 class have been isolated after immunization with Brucella abortus. An indirect immunofluorescence test was used to screen hybridoma supernatants and subsequently to determine the cross-reactivity of the monoclonal antibodies with other bacteria. One monoclonal antibody reacted with all the smooth Brucella biotypes tried and with Yersinia enterocolitica serogroup O:9, though not with rough Br. ovis or with strains of Escherichia, Proteus, Salmonella, Pseudomonas, Francisella and Bordetella. The other monoclonal antibody displayed a high degree of specificity for brucellae carrying the A lipopolysaccharide-protein surface antigen. The implications for the diagnosis of brucellosis are discussed.

1. The size and shape of superhelical double-stranded circular DNA from bacteriophage ØX174 were investigated by light-scattering. The molecular weight of the DNA is 3.17×106 and the root-mean-square radius is 103.5nm. 2. The light-scattering envelopes of various theoretical three-dimensional models for such DNA molecules were calculated by repetitive computational techniques, and the results were compared with the experimental findings. 3. It is concluded that the structure of supercoiled DNA containing -12 superhelical turns in buffer of I0.2 corresponds best to one of the more compact models for superhelix structure such as the branched model, and the commonly employed straight interwound superhelix model is incompatible with the experimental results, at the superhelix density found.

It has been shown previously that supercoiled [unk]X174 bacteriophage intracellular DNA (mol.wt. 3.2x10(6)) with superhelix density, sigma=-0.025 (-12 superhelical turns) at 25 degrees C is best represented as a Y shape. In this work two techniques have been used to unwind the supercoil and study the changes in tertiary structure which result from changes in the secondary structure. The molecular weights from all experiments were in the range 3.2x10(6)+/-0.12x10(6). In experiments involving temperature change little change in the Y shape was observed between sigma=-0.027 (-13 superhelical turns, 14.9 degrees C) and sigma=-0.021 (-10 superhelical turns, 53.4 degrees C) as evidenced by the root-mean-square radius and the particle-scattering factor P(theta). However, at sigma=-0.0176 (-8 superhelical turns, 74.5 degrees C) the root-mean-square radius fell to between 60 and 70nm from 90nm indicating a large structural change, as did alterations in the P(theta) function. In experiments with the intercalating dye proflavine from values of bound proflavine of 0-0.06mol of dye/mol equiv. of nucleotide which correspond to values of sigma from -0.025 to -0.0004 (-12 to 0 superhelical turns) a similar transition was found when the superhelix density was changed by the same amount, and the molecule was shown to go through a further structural change as the unwinding of the duplex proceeded. At sigma=-0.018 (-9 superhelical turns) the structure was compatible with a toroid, and at sigma=-0.0004 it was compatible with a circle but at no point in the sequence of structure transitions was the structure compatible with the conventional straight interwound model normally visualized as the shape of supercoiled DNA.