A comparison of ELISA assays as routine diagnostic test for detection of autoantibodies against extractable nuclear antigens.

OBJECTIVE: In an analytical evaluation, commercially available ELISA test kits for detection of antibodies directed against extractable nuclear antigens (ENA) were compared with the currently used combination of counterimmunoelectrophoresis and immunoblotting. DESIGN: Three screening ELISAs and two typing ELISAs were tested. These methods were fairly simple, easy to perform and "user friendly," because most of the reagents were ready to use. RESULTS: The agreement with the current methods was good, but the screening as well as typing ELISAs proved to be more sensitive, especially with regard to detection of SS-A auto-antibodies. The cut-off range of one screening assay was not well established and one typing assay suffered from problems with inaccuracy of package insert, purity of antigen and standardisation of reactivity (possibly caused by differences in amount of coated antigen). The other three ELISAs were reliable and sensitive for detection of ENA auto-antibodies. CONCLUSIONS: The ELISA ENA screen assays ENA-LISA polyvalent and Milenia ENA screen and typing assays ENA-LISA are reliable and sensitive for detection of autoantibodies in clinical specimens without substantial false negatives.

Large discrepancy between prostate-specific antigen results from different assays during longitudinal follow-up of a prostate cancer patient.

A case is presented of a patient with stage D prostatic carcinoma, from whom a serum sample proved to be an outlier in a correlation study performed with a 2nd-generation prostate-specific antigen (PSA) assay on the Immulite system (6.4 micrograms/L) and IMx (101 micrograms/L). Clearly, the PSA result reported by Immulite was falsely low. For nine longitudinal samples, Immulite results were approximately 20-fold lower than the IMx value (range of IMx results 5-275 micrograms/L). A selection of the samples was analyzed with the ACS:180, ES-600, and IMx (all > 180 micrograms/L); Immulite, DPC Coat-A-Count IRMA, Immuno 1, AIA-pack, and Tandem-R (all <70 micrograms/L); and Immulite free PSA assay (41 micrograms/L). Gel filtration demonstrated that apart from the alpha1-antichymotrypsin (ACT) complex, no other complexes were found. However, the sample consisted of 53% free PSA (IMx). Possibly, a change of conformation of the PSA molecule resulted in a decreased binding to ACT and a reduced affinity of the antibodies used in the affected assays.

Marked interference of hyperglycemia in measurements of mean (red) cell volume by Technicon H analyzers.

Severe hyperglycemia can result in falsely high results for mean cell (erythrocyte) volume (MCV), which will also cause false results for erythrocyte indices calculated on the basis of MCV. Falsely high MCV results were obtained with the Technicon H1 and H2 analyzers and (to a lesser extent) with the Coulter T660. The H analyzers were more susceptible to this interference than was the Coulter T660. This difference in sensitivity of MCV to hyperglycemia can be explained by the use of sodium dodecyl sulfate in the Technicon erythrocyte diluent and by differences in incubation times. In severe hyperglycemia, results for MCV, mean cell hemoglobin concentration, and hematocrit obtained from electronic cell counters, especially Technicon H systems, are unreliable.

Differential expression of the neuroendocrine polypeptide 7B2 in hypothalami of Prader-(Labhart)-Willi syndrome patients.

Prader-(Labhart-)Willi syndrome (PWS) is characterized by infantile hypotonia, early childhood obesity, mental deficiency, short stature, small hands and feet and hypogonadism. In 70% of the cases this syndrome is associated with a defect of chromosome 15 at 15q11-q13, close to the location of the 7B2 gene (15q13-q14). The majority of the remaining PWS patients display maternal uniparental disomy on chromosome 15. Since the 7B2 gene products are expressed in neuroendocrine cells that are probably affected in PWS, e.g. by a pleiotrophic influence of the neighboring deletion, the presence of 7B2 was studied in the supraoptic and paraventricular nucleus of the hypothalamus of five subjects clinically diagnosed as PWS patients using five antibodies against various parts of the 7B2 precursor polypeptide. Three of the five PWS patients studied showed no reaction to the 7B2 antibody MON-102, whereas all 30 control patients did. However, one of the three MON-102 non-reacting PWS patients reacted to other 7B2 antibodies. In conclusion, the vanishing of 7B2 gene products is not obligatory for PWS, possibly due to the variable genetic background of PWS patients. However, in most patients there is a clear modification of 7B2 expression, pointing to altered neuroendocrine functions.

Structure and function of eukaryotic proprotein processing enzymes of the subtilisin family of serine proteases.

Production of a broad spectrum of regulatory proteins in eukaryotes occurs via an intricate cascade of biosynthetic and secretory processes. Often these proteins initially are synthesized as parts of higher molecular weight, but inactive, precursor proteins. Specific endoproteolytic processing of these proproteins is required to generate the regulatory proteins in a mature and biologically active form. Such endoproteolysis generally occurs at cleavage sites consisting of particular sequence motifs of basic amino acids, often paired basic residues. This phenomenon, first observed almost 25 years ago, has intrigued scientists ever since then. Nevertheless, the responsible enzymes remained elusive for long. The first known eukaryotic enzyme with the exquisite cleavage specificity for paired basic amino acid residues was the prohormone processing enzyme kexin (EC 3.4.21.61), a subtilisin-like serine protease that is encoded by the KEX2 gene of yeast Saccharomyces cerevisiae. Recently, a number of kexin-like mammalian proprotein-processing enzymes were discovered. The enzyme furin, which is encoded by the fur gene, was the first and can be considered the prototype of a mammalian subclass of subtilisin-like serine proteases. It is predicted to contain a "prepro" domain, a subtilisin-like catalytic domain, a middle domain, a cysteine-rich region, a transmembrane anchor, and a cytoplasmic domain. Furin is expressed in a wide variety of tissues, perhaps even in all tissues. In all likelihood, it is the enzyme responsible for the proteolytic bioactivation of a wide variety of precursor proteins. Two other novel mammalian proprotein-processing enzymes are PC1 (also known as PC3) and PC2. Some of the protein domains of these enzymes resemble those in kexin and furin, however, there are also differences. The PC1/PC3 and PC2 enzymes exhibit a more restricted expression pattern than furin. It has been suggested that PC1/PC3 and PC2 are involved primarily in the processing of prohormones within the regulated secretory pathway of cells of endocrine and neural tissue. Recently, the coding sequences for two other candidate mammalian proprotein-processing enzymes were identified. They were called PACE4 and PC4. Like that of furin, the tissue distribution of PACE4 is widespread. PC4, however, may represent a candidate for a precursor-processing endoprotease that is specifically expressed in the testicular germ cells. Finally, DNA sequences encoding kexin- and furin-like candidate pro-protein-processing enzymes have been identified in Drosophila melanogaster, Dfur1 and Dfur2 genes; in Xenopus laevis, Xen-14 gene; and in Caenorhabditis elegans, bli-4 gene.(ABSTRACT TRUNCATED AT 400 WORDS)

Monoclonal antibody MON-114: detection of a marker for neuroendocrine differentiation in human lung cancer.

Mouse monoclonal antibody MON-114 was generated upon immunization with a human small cell lung carcinoma cell line GLC-19. Immunohistochemical analysis of normal tissues with MON-114 showed staining of the adrenal gland, brain and peripheral nerves. With respect to human lung carcinomas, 7 out of 8 small cell lung carcinomas were positively stained as well as 5 out of 5 carcinoid tumors, whereas only 4 out of 31 squamous cell carcinomas and 3 out of 19 adenocarcinomas were weakly stained. Furthermore, 1 large cell carcinoma was negative for MON-114 staining. Apparently, MON-114 stains cells of neuroendocrine differentiation.

Development and characterization of a panel of monoclonal antibodies against the catalytic domain of the human fes proto-oncogene product.

In developing monoclonal antibodies (Moabs) against the human fes proto-oncogene product, recombinant DNA technology was used to target reactivity of the Moabs towards the catalytic domain of it. Therefore, sequences of human fes exons 15-19 encoding amino acid residues 612 to 822 which harbor the catalytic domain except the presumed ATP-binding region, were fused in phase to the bacterial trp E gene which encodes anthranilate synthase. After partial purification of it, the bacterially produced hybrid product of this trp E-delta fes fusion gene was used as immunogen. A series of twelve mouse Moabs was obtained which recognized the human p92fes protein and the viral oncogene product p85gag-fes encoded by the Snyder-Theilen strain of feline sarcoma virus. Reactivity appeared to be directed towards the catalytic domain of the human fes proto-oncogene product. This was demonstrated by in vitro transcription and translation experiments using human fes coding sequences from exons 16-19. Upon testing their functional activity in divers immunological techniques, the whole panel of Moabs appeared to be useful in immunoprecipitation, Western blot and immunohistochemical analysis. Immunocytochemical analysis indicated that p85gag-fes is predominantly a cytoplasmic protein.

Development and characterization of a panel of monoclonal antibodies against the novel subtilisin-like proprotein processing enzyme furin.

Monoclonal antibodies were raised against the recently discovered subtilisin-like proprotein processing enzyme furin. As immunogen, a bacterially expressed hybrid protein was used which consisted of glutathione S-transferase fused to almost the entire human furin protein. Ten monoclonal antibodies were obtained and these could be divided into four categories on the basis of their reactivity towards a number of bacterially expressed hybrid proteins, each of which contained a different portion of human furin. Four of the monoclonal antibodies did not recognize mouse furin. All monoclonal antibodies were tested for their applicability in Western blot and immunofluorescence analysis. Western blot analysis was performed with COS-1 cells in which biologically active forms of human and mouse furin were expressed transiently under control of the SV40 late promoter. This approach was necessary, since physiological levels of fur gene encoded proteins appeared to be very low. In cells transfected with human or mouse fur cDNA, a protein of about 100 kDa and a doublet of about 90 kDa could be detected with most of the monoclonal antibodies. Some of these antibodies appeared to be also reactive in immunofluorescence analysis of transfected COS-1 cells.

Splicing of the VASE exon of neural cell adhesion molecule (NCAM) in human small-cell lung carcinoma (SCLC).

Expression of the neural cell adhesion molecule (NCAM) on small-cell lung carcinoma (SCLC) cell lines and tumour tissue has been investigated. Cell lines were found to express highly sialylated NCAM. Neuraminidase treatment revealed the presence of the 140- and 120-kDa isoforms with differential expression of a 95-kDa protein. Similar data were obtained with SCLC tumour tissues. These results were corroborated by Northern blotting where mRNA of 6.7 and 5.5 kb coding for the 140- and 120-kDa isoforms, respectively, were identified. In a few tumours, a weaker band of 7.4-kb mRNA coding for the 180-kDa NCAM was also identified. This result could not be confirmed biochemically due to shortage of material. Finally, a 5-kb transcript was identified in all SCLC samples examined. The NCAM isoform coded by this mRNA remains unknown. Using the polymerase chain reaction (PCR), we have demonstrated the presence of the VASE mini-exon in some isoforms of SCLC NCAM. The VASE mini-exon sequence in human SCLC differs from the published murine sequence by only one base change. This substitution does not result in altered amino-acid sequence.

Application of recombinant DNA technology in epitope mapping and targeting. Development and characterization of a panel of monoclonal antibodies against the 7B2 neuroendocrine protein.

Three mouse monoclonal antibodies (Moabs) have been obtained with specificity for the 7B2 protein, a proposed member of the granin family of neuroendocrine proteins. Bacterially produced hybrid proteins of 7B2 were used as immunogens. The Moabs were designated MON-100, MON-101, and MON-102. Furthermore, we report the construction of 35 deletion mutants of the glutathione S-transferase-7B2 (GST-7B2) fusion-gene using recombinant DNA technology. The hybrid proteins encoded by eleven of these mutants were used in epitope mapping experiments and the results of these studies strongly suggested that recognition of 7B2 by all three Moabs involved the same 16 amino acid region of 7B2 (from amino acid residue 128-135). This was further substantiated by the observation that MON-101 and MON-102 specifically recognized a conjugate between bovine serum albumin and the synthetic peptide Phe-Glu-Pro-Glu-His-Asp-Tyr-Pro-Gly-Leu-Gly-Lys based upon the deduced amino acid sequence of the predicted epitope region in 7B2. In an approach to generate a series of 7B2-specific Moabs targeted against another epitope region in the 7B2 protein, the hybrid protein encoded by deletion mutant pPV32 was used as the immunogen. This protein lacked the epitope region recognized by the first series of Moabs. A second series of three Moabs, designated MON-142, MON-143, and MON-144, was obtained and, in all three cases, the region of 7B2 from amino acid residue 64-94 appeared to be involved in specific recognition by the Moabs. The whole panel of six anti-7B2 antibodies appeared to be useful in immunoprecipitation and Western blot analysis of the 7B2 protein and specifically stained neuroendocrine cells in immunohistochemical experiments. Using a double determinant sandwich enzyme immunoassay, 7B2 protein levels in rat pituitary were determined as 20 ng/mg tissue.

Coordinated expression of 7B2 and alpha MSH in the melanotrope cells of Xenopus laevis. An immunocytochemical and in situ hybridization study.

7B2 is a highly conserved protein present in many secretory cells. Using in situ hybridization techniques and immunocytochemistry, parameters concerning the biosynthesis and storage of the 7B2 protein were studied in the pituitary gland and median eminence of the clawed toad Xenopus laevis, in relation to the physiological process of background adaptation. 7B2-like immunoreactivity was present in the median eminence, in the neural and anterior pituitary lobes and, particularly, in the melanotrope cells of the intermediate pituitary lobe. In these cells, it coexisted with immunoreactivity to proopiomelanocortin (POMC)-derived alpha-melanocyte stimulating hormone (alpha MSH). The melanotropes of black-adapted animals had abundant 7B2-mRNA and POMC-mRNA; melanotropes of white-adapted toads had only low levels of these mRNAs. The presence of 7B2 in nerve terminals and endocrine cells supports the idea that the protein has a general function in the cellular secretory process. In X. laevis, 7B2 appears to be particularly associated with POMC and alpha MSH and, therefore, may play a role in the regulation of background adaptation.

Furin is a subtilisin-like proprotein processing enzyme in higher eukaryotes.

The human fur gene encodes a protein, designated furin, the C-terminal half of which contains a transmembrane and a cysteine-rich receptor-like domain. The N-terminal half of furin exhibits striking primary amino acid sequence similarity to the catalytic domains of members of the subtilisin family of serine proteases. We here report characteristics of the furin protein and propose a three-dimensional model for its presumptive catalytic domain with characteristics, that predict furin to exhibit an endoproteolytic cleavage selectivity at paired basic residues. This prediction is substantiated by transfection and cotransfection experiments, using COS-1 cells. Full length fur cDNA evokes the specific synthesis of two polypeptides of about 100 kDa and 90 kDa as appeared from Western blot analysis of transfected COS-1 cells using a polyclonal anti-furin antiserum. Functional analysis of furin was performed by cotransfection of fur cDNA with cDNA encoding the 'wild type' precursor of von Willebrand factor (pro-vWF) and revealed an increased proteolytic processing of provWF. In contrast, cotransfection of fur cDNA with a recombinant derivative (provWFgly763), having the arginine residue adjacent to the proteolytic cleavage site (arg-ser-lys-arg) replaced by glycine, revealed that provWFgly763 is not processed by the fur gene product. We conclude that in higher eukaryotes, furin is the prototype of a subtilisin-like class of proprotein processing enzymes with substrate specificity for paired basic residues.

The neuroendocrine polypeptide 7B2 is a precursor protein.

The neuroendocrine protein 7B2 is highly conserved and widely present in neurons and endocrine cells. It is coexpressed with the prohormone proopiomelanocortin (POMC) in the intermediate lobe of the pituitary gland of Xenopus laevis. To study the biosynthesis of 7B2 in this amphibian, an anti-7B2 monoclonal antibody was used in immunoprecipitation analysis of newly synthesized radiolabeled proteins, produced by pulse and pulse-chase-incubated neurointermediate lobes. Following a 15-min pulse incubation, a single immunoprecipitable protein of 25 kDa was synthesized. During subsequent chase incubation, this newly synthesized 7B2 protein was processed to an 18-kDa immunoprecipitable form. Analysis of the chase incubation medium revealed that only the 18-kDa processed product of 7B2, and not 7B2 itself, had been secreted. This secretion is a regulated process because it was blocked completely by the dopamine receptor agonist apomorphine. A study of protein biosynthesis in lobes treated with tunicamycin to prevent N-linked glycosylation showed that in contrast to POMC and an 18-kDa derivative of POMC, neither 7B2 nor its 18-kDa derivative was glycosylated. Chemical and enzymatic peptide mapping showed that processing of 7B2 occurs in the carboxyl-terminal region. The function of the 7B2 protein is unknown; the present results show that 7B2 itself is a precursor molecule and can only have an intracellular function whereas an extracellular function can only be attributed to 7B2-derived peptides.

Development of a monoclonal antibody against recombinant neuroendocrine 7B2 protein.

Mouse monoclonal antibody MON-100 was raised against the neuroendocrine protein 7B2 using bacterially produced hybrid proteins. In Western blot analysis, MON-100 reacted with 3 different 7B2 hybrid proteins and not with the respective carrier proteins. Furthermore, MON-100 was reactive with recombinant 7B2 cleaved from a hybrid protein. In an immunohistochemical study, MON-100 exhibited strong reactivity with the intermediate lobe of the Xenopus pituitary gland, a tissue previously shown to contain 7B2 mRNA. Using MON-100, immunoprecipitation analysis of newly synthesized proteins produced by in vitro incubated Xenopus neurointermediate lobes revealed the biosynthesis of a single protein of Mr 24 kDa, the expected size of the 7B2 protein. It appears, therefore, that the anti-7B2 monoclonal antibody MON-100 can be successfully used for Western blot analysis and immunohistochemical analysis as well as for immunoprecipitation experiments.

Characteristics of a multicopy gene family predominantly consisting of processed pseudogenes.

The monoclonal antibody MOC-32 detected a 40 kDa protein in Western blot analysis. Immunological screening of an expression library of human SCLC cells with MOC-32 led to the isolation of overlapping cDNA clones. One of these, cHD4, was 1.0 kbp long and of about the same size as its corresponding mRNA. Preceded by an in phase stop codon, an open reading frame of 885 bp was present in cHD4 and a translational product of only 33 kDa could be calculated. Biochemical and immunological analysis established the relationship between the 40 kDa antigen and the isolated coding sequences and resolved the apparent discrepancy between the calculated molecular weight and the observed electrophoretic mobility. Nucleotide sequence comparison of cHD4 to the EMBL database revealed that cHD4 was nearly identical to a sequence claimed to encode a laminin binding protein. Southern blot and nucleotide sequence analysis indicated the presence of multiple copies of the gene in the human genome. At least five of these appeared to represent processed pseudogenes.

Selective modulation of two human monocyte Fc receptors for IgG by immobilized immune complexes.

Two types of IgG FcR, FcRI and FcRII, are constitutively expressed by human monocytes. FcRI (identified by mAb 32.2) binds human (h) IgG, FcRII (identified by mAb IV.3) has a low affinity for hIgG but interacts strongly with murine (m) IgG1. These receptors can be assayed by using indicator E sensitized by hIgG (EA-hIgG) or mIgG1 (EA-mIgG1), respectively. We further characterized these two FcR by modulation studies by using substrate-immobilized immune complexes containing rabbit IgG, goat IgG, or one of the mouse Ig classes or subclasses. After incubating monocytes in microtiter wells containing such immune complexes, binding of the two types of indicator red cells on the apical surface of the monocytes was quantitated using a photometric assay employing the pseudoperoxidase activity of E. No effect on the binding of sensitized E was observed after incubation of monocytes with immune complexes containing mouse IgE, IgA, or IgM, or F(ab')2 fragments of rabbit IgG. High concentrations of immune complexes containing IgG of mouse, rabbit, or goat, however, were able to induce a decrease in binding of both types of sensitized E, suggestive of modulation of both FcRI and FcRII. At lower concentrations of immune complexes, more selective patterns of modulation emerged. Under these conditions, immune complexes containing mIgG1 or mIgG2b, or, surprisingly, goat IgG induced a selective decrease in the binding of EA-mIgG1 (FcRII modulation), while immune complexes containing mIgG2a or rabbit IgG mainly affected the binding of EA-hIgG (FcRI modulation). By using anti-FcR mAb IV.3, it was confirmed that FcRII was modulated from the apical surface of monocytes after incubation on immune complex coated substrates. Selectivity of FcR-modulation was demonstrated by showing that under these conditions binding of anti-C receptor mAb, and several other anti-monocyte mAb did not decrease.