Monoclonal antibodies recognizing CD5, CD10 and CD23 in formalin-fixed, paraffin-embedded tissue: production and assessment of their value in the diagnosis of small B-cell lymphoma.

AIMS: Assessment of the expression of antigens CD5, CD10 and CD23 can be of value in the differential diagnosis of small B-cell lymphoma. Correct subclassification is important since optimal treatment regimes differ between the subtypes. The aim of this study was to generate monoclonal antibodies recognizing these antigens in paraffin-embedded tissue and to assess their efficacy using a panel of cases of small B-cell lymphoma of various subtypes. METHODS AND RESULTS: For each antibody synthetic recombinant protein and conventional murine hybridoma technology was employed. Monoclonal antibodies effective in formalin-fixed, paraffin-embedded tissue were successfully generated, designated NCL-CD5-4C7, NCL-CD10-270 and NCL-CD23-1B12, respectively. A series of 58 cases of small B-cell lymphoma including examples of each subtype (lymphocytic, follicle centre cell, mantle cell, marginal zone and lymphoplasmacytoid) was assembled and immunostaining for the respective antigens carried out using the monoclonal antibodies produced. Our results indicate that the antibodies are specific for their respective antigens and give the predicted phenotypic profile in the small B-cell lymphoma subtypes. CONCLUSIONS: These novel monoclonal antibodies may be of value in routine diagnostic practice.

New monoclonal antibodies to the T cell antigens CD4 and CD8. Production and characterization in formalin-fixed paraffin-embedded tissue.

We have generated a recombinant protein representing part of the CD4 molecule and a peptide representing an epitope of predicted high antigenicity on the CD8 molecule and employed these to generate mouse monoclonal antibodies using standard hybridoma protocols. The extracellular domain of the CD4 molecule was obtained by reverse transcription of mRNA from peripheral blood lymphocytes followed by polymerase chain reaction. The amplified gene fragment was cloned into an expression vector to allow a histidine-tagged fusion protein to be produced in Escherichia coli. Purified fusion protein was used to immunize mice. The CD8 monoclonal antibody was raised against a peptide consisting of 13 amino acids within the carboxyl-terminal region of the CD8 cytoplasmic domain. The antibodies showed appropriate reactivity on Western blotting. By heat pretreatment, these antibodies have been shown to be highly effective on paraffin-embedded tissue. In normal lymphoid tissue, the expected distribution of CD4 and CD8 lymphocytes was observed. In a series of 16 T cell lymphomas and B cell lymphomas, immunostaining results were compared with those obtained using reagents effective only in frozen tissue. A high degree of correlation was observed. These results suggest that NCL-CD4 and NCL-CD8 may be of value in the characterization of T cell disorders.

Production and characterization of a new monoclonal antibody effective in recognizing the CD3 T-cell associated antigen in formalin-fixed embedded tissue.

Phenotypic analysis of lymphoproliferative disorders is now considered mandatory for accurate classification which is the basis for optimum patient management. This is presently carried out in most cases using a range of antibodies recognizing B and T-cell antigens effective in paraffin sections, and an antibody to CD 3 is currently a key member of such panels, indicating T-cell phenotype. Current antibodies to CD3 are polyclonal with the inherent disadvantages of this type of reagent compared to monoclonal antibodies. In this study, we have used a recombinant fusion protein representing part of the epsilon subunit of the CD3 molecule to generate a novel monoclonal antibody (NCL-CD3-PS1) effective in paraffin sections. The antibody has been characterized biochemically and by immunohistochemistry using a wide range of normal and pathological tissues. Lineage and phenotype specificity have been supported in our study and results from other laboratories are awaited with interest.

Expression of the glial glutamate transporter EAAT2 in the human CNS: an immunohistochemical study.

Glutamate transporters play an essential role in terminating the excitatory glutamatergic signal at post-synaptic receptors and in protecting neurones from excitotoxic effects, as well as replenishing the neurotransmitter supply at glutamatergic synapses. The distribution and density of glutamate transporters may be important determinants of vulnerability to glutamate-mediated injury. There is emerging evidence that glutamate transporter dysfunction may be present in motor neurone disease (MND). In this study, a monoclonal antibody, suitable for immunohistochemistry (IHC) in human post-mortem tissue, was produced to the human astrocytic glutamate transporter EAAT2 (excitatory amino acid transporter 2). Western blotting of homogenates of human cortical tissue with the EAAT2 antibody produced a discrete band at 66 kDa. Detailed IHC analysis of the expression of the EAAT2 protein in the human CNS was undertaken. EAAT2 was exclusively localised to astrocytes, with preferential expression in the caudate nucleus, nucleus basalis of Meynert, spinal ventral horn, cerebral cortex and hippocampus, but with lower levels of expression throughout many other CNS regions. Motor neurone groups vulnerable to neurodegeneration in MND appeared distinctive in being surrounded by extensive, coarse, strongly immunoreactive perisomatic glial profiles. Motor neurone groups which tend to be spared in MND, such as those present in the oculomotor nucleus, showed a lower expression of EAAT2, with fewer perisomatic profiles. The EAAT2 antibody will provide a useful tool for increasing our understanding of the role of EAAT2 in excitatory neurotransmission in health and disease states.

Enzyme linked immunosorbent assay for detecting benzodiazepines in urine.

A relatively simple ELISA technique was developed for the detection of a range of benzodiazepines (BZs) in urine. The assay employs a mouse anti-oxazepam antibody that is highly specific for the BZs. The limit of detection using 10 microliters samples of urine was 0.3 microgram ml-1 oxazepam. N-Desmethyldiazepam showed equal cross-reactivity to oxazepam, 11 BZs cross-reacted weakly and flurazepam and chlordiazepoxide did not cross-react at levels reported to be found in urine. No cross-reactivity was observed with drugs of abuse and a range of therapeutic drugs commonly found in urine. The assay was used as a screen to detect the presence of BZs in urine from 88 addicts that had been screened by the EMIT technique and a radioreceptor assay (RRA) for BZs. The ELISA produced two false negatives that were EMIT and RRA positive whereas the EMIT produced four different false negatives that were positive by both ELISA and RRA. Thirty-three positives were common to all three assays. The ELISA was also used to monitor nitrazepam-like activity in the urine of a greyhound receiving 5 mg oral medication and the results were compared with those obtained by RRA. Both assays were able to detect nitrazepam-like activity for up to 10 h post-administration.

Characterization of a subtype of primary osteoclastoma: extracellular calcium but not calcitonin inhibits aggressive HLA-DR-positive osteoclastoma possessing 'functional' calcitonin receptors.

We report here a case of primary osteoclastoma that despite possessing HLA-DR-positive status and 'functional' calcitonin receptors, exhibited aggressive in vitro and in vivo bone resorptive activity. In the osteoclast bone slice assay employing scanning electron microscopy, the giant cell-mediated bone resorption was uninhibited by salmon calcitonin (10 nM) and significantly inhibited by raised extracellular calcium (20 mM). In Fura-2AM based microspectrofluorimetric assays, the presence of the 'functional' calcitonin receptors was ascertained by a rise in intracellular calcium induced by calcitonin and high extracellular calcium. These findings provide evidence for a hitherto unrecognized subtype of giant cells that have HLA-DR-positive status, exhibit avid bone resorptive activity, but remain insensitive to calcitonin despite possessing calcitonin receptors.

Mitogenic activity of procathepsin D purified from conditioned medium of breast-cancer cells by affinity chromatography on pepstatinyl agarose.

A simple procedure for the affinity purification of procathepsin D from tissue culture medium conditioned by breast-cancer cells is described. This procedure yielded 2 micrograms of procathepsin D/100 ml medium. The procathepsin D was approximately 95% pure as judged by silver staining of polyacrylamide gels, the major contaminant being mature cathepsin D. The ability of procathepsin D to stimulate the proliferation of oestrogen-responsive MCF-7 breast cancer cells was determined. The purified procathepsin D had no mitogenic effect alone or in combination with oestradiol or other growth factors. These data suggest that procathepsin D does not act as an oestrogen-regulated autocrine growth factor for malignant breast epithelial calls.

Studies of c-jun oncogene expression in human breast using a new monoclonal antibody, NCL-DK4.

A new monoclonal antibody to human c-jun oncoprotein, designated NCL-DK4, has been produced. NCL-DK4 has been proved to be highly effective for use on formalin-fixed, paraffin-embedded tissues, enabling the study of c-jun expression at a cellular level in both normal and neoplastic human tissues. The expression of c-jun oncogene has been examined in normal, benign, and malignant breast tissues, and c-jun-specific immunoreactivity in carcinomas has been related to histological type, tumour grade, c-erbB-2, oestrogen receptor, progesterone receptor, and epidermal growth factor receptor expression. Normal and benign breast tissues showed c-jun-specific immunostaining, which was weaker and in fewer cells compared with the c-jun immunoreactivity observed in breast carcinomas. No relationship was found between the degree of immunostaining and the extent of proliferative changes in benign breast tissues. Ninety per cent of all breast carcinomas studied showed c-jun-specific nuclear staining. There were no statistically significant differences in the intensity of c-jun immunoreactivity among grade I, II, and III infiltrating ductal carcinomas. There was no significant relationship between c-jun oncoprotein expression and c-erbB-2, oestrogen, progesterone, and epidermal growth factor receptor immunoreactivity.

Expression of the pNR-2/pS2 protein in diverse human epithelial tumours.

The pNR-2/pS2 protein is regulated by oestrogens in breast cancer cell lines. This report describes a systematic survey of pNR-2/pS2 expression in a number of common epithelial tumours. Expression was evaluated immunohistochemically in an archival series using antisera raised against the C-terminus of the pNR-2/pS2 protein. Expression of pNR-2/pS2 by malignant epithelial tumours was widespread. Intense immunohistochemical staining was found in tumour cells in a proportion of pancreatic (6/8), large intestinal (7/12), gastric (9/16) and endometrial (4/12) carcinomas. Positive staining for the pNR-2/pS2 protein was also found in both benign and malignant ovarian epithelial tumours and was very significantly associated with mucinous differentiation (P less than 0.00001). Small numbers of carcinomas of bladder (2/10) and prostate (2/7) showed less intense staining and single examples of cervical carcinoma (1/7) and lung carcinoma (1/19) stained positively. None of the renal carcinomas (0/16) examined stained positively. Positive staining showed no correlation with gender. Although there are reports of oestrogen receptor expression in most of the tumour types considered, the possibility of other regulatory influences must also be considered. The pNR-2/pS2 protein may well have a more general role in human epithelial neoplasia than hitherto realised.

pNR-2/pS2 immunohistochemical staining in breast cancer: correlation with prognostic factors and endocrine response.

Expression of the oestrogen-regulated pNR-2/pS2 protein has been studied in paraffin sections of a series of 172 primary breast cancers using an immunohistochemical technique. Positive staining of tumour cells was found in 117 tumours (68%): most of these tumours contained only a small proportion of positive cells. pNR-2 immunohistochemical staining correlated positively and significantly with the presence of oestrogen receptor. Mean percentages of pNR-2 positive cells were lower in tumours from postmenopausal women. Smaller, better differentiated tumours were significantly more likely to stain positively for pNR-2. The percentages of pNR-2 positive tumour cells in primary tumours and synchronously excised lymph node metastases were very similar. pNR-2 expression showed an unexpected positive association with lymph node metastasis. We were unable to find any significant association between pNR-2 immunohistochemical staining and either time to relapse or overall survival. There was a significant association between pNR-2 expression in primary tumours and response to endocrine therapy on relapse: positive pNR-2 immunohistochemical staining in primary tumours is predictive of response to hormonal therapy on relapse.

Antipeptide antibodies against the pNR-2 oestrogen-regulated protein of human breast cancer cells and detection of pNR-2 expression in normal tissues by immunohistochemistry.

Five peptides, corresponding to regions of the predicted protein sequence of the oestrogen-regulated pNR-2 protein which is expressed in oestrogen-responsive human breast cancer cells, were synthesized. Two peptides were immunogenic in rabbits and antisera against one peptide reacted with the pNR-2 protein in sections of formalin-fixed, paraffin-embedded breast tumour. There was a significant correlation between the extent of pNR-2 protein expression detected by immunohistochemistry and pNR-2 mRNA levels determined by hybridization with a cDNA probe in a series of primary breast tumours. pNR-2 expression was assessed immunohistochemically in a panel of normal tissues. Expression was detected in normal breast, small intestine, and stomach (body and antrum).