An immunosensor for syphilis screening based on surface plasmon resonance.

In this paper the development of a surface plasmon resonance (SPR) immunosensor for syphilis screening is described. This immunosensor is based on the detection of antibodies in serum against the causative organism Treponema pallidum. In order to achieve selectivity a recombinant Treponema pallidum membrane protein A (TmpA) was used. This antigen can react with antibodies to T. pallidum, present in serum of syphilitic patients. Reproducible results have been obtained, using a 'sandwich SPR' method: binding of a sandwich antibody to the treponemal antibody after serum incubation was measured in real time while the binding was taking place. The SPR results obtained from ten blind-coded sera corresponded well with classical syphilis tests (Treponema pallidum haemagglutination assay (TPHA), fluorescent treponemal antibody-absorbed test (FTA-ABS), venereal diseases research laboratory flocculation test (VDRL) and TmpA-based enzyme-linked immunosorbent assay (TmpA-ELISA)). Preliminary experiments showed that direct measurement of serum (in the 'one step SPR') is not yet possible, probably as a result of non-uniformity of serum samples. The application of latex beads is considered to solve this problem.

Type-specific immunodetection of human heart fatty acid-binding protein with polyclonal anti-peptide antibodies.

In order to develop specific antibodies against human heart cytoplasmic fatty acid-binding protein (H-FABPc), four oligo-peptides of 15-20 amino-acids each and corresponding with different antigenic parts of the human H-FABPc molecule, were synthesized. Polyclonal antibodies against these synthetic peptides were raised in mice (Balb/C) and rabbits (Flemish giant). When tested in enzyme linked immunosorbent assays (ELISA, antibody-capture assay), antisera against three of the four peptides showed a high immunoreactivity with the synthetic peptide selected for immunization as well as with the native human H-FABPc. Some cross-reactivity with the other synthetic peptides was observed for the rabbit antisera but not for those from mice. Polyclonal antibodies against synthetic peptides can be applied for the specific detection of the native protein in biological preparations containing proteins that show a high degree of homology with the protein to be assayed.

Lack of immunological cross reactivity between the transport enzymes (Na+ + K+)-ATPase and (K+ + H+)-ATPase.

Goat antisera against (Na+ + K+)-ATPase and its isolated subunits and against (K+ + H+)-ATPase have been prepared in order to test for immune cross-reactivity between the two enzymes, whose catalytic subunits show great chemical similarity. None of the (Na+ + K+)-ATPase antisera cross-reacted with (K+ + H+)-ATPase or inhibited its enzyme activity. The same was true for the (K+ + H+)-ATPase antiserum with regard to (Na+ + K+)-ATPase and its subunits and its enzyme activity. So notwithstanding the chemical similarity of their subunits, there is no immunological cross-reactivity between these two plasma membrane ATPases.

Antibodies against distinct nuclear matrix proteins are characteristic for mixed connective tissue disease.

Specific nuclear proteins, separated according to their molecular weight (mol. wt) by polyacrylamide gel electrophoresis (PAGE) and subsequently transferred to nitrocellulose sheets, are able to bind antibodies in sera from patients suffering from different types of connective tissue diseases. Antibodies against a characteristic set of nuclear protein antigens are found in sera from patients with mixed connective tissue disease (MCTD). Screening of 21 MCTD sera revealed a typical immunoblot pattern with major protein antigens of mol. wt 70,000 (20/21) (not identical with the Scl-70 antigen characteristic for scleroderma), mol. wt 31,000 (17/21), two proteins around mol. wt 23,000 (15/21) and two around mol. wt 19,000 (10/21). The 70,000, 23,000 and 19,000 antigens appeared to be rather insoluble nuclear proteins (i.e. components of the nuclear matrix). On behalf of their structural character they were present in nuclei from several types of cells but only in low amounts detectable in salt extracts of thymus acetone powder. The presence of antibodies directed against the mol. wt 70,000 antigen correlated strongly with the diagnosis of MCTD. This 70,000 antigen is not identical with the RNP antigen, a soluble ribonuclease sensitive ribonucleoprotein, since antibodies against nuclear RNP can be separated from anti-nuclear matrix antibodies by affinity chromatography using immobilized thymus salt extract. The distinct character of soluble nuclear RNP and structural nuclear matrix antigens is further supported by the fact that from 14 other anti-RNP sera obtained from patients with systemic lupus erythematosus (SLE), only three contained antibodies against the mol. wt 70,000 protein. Since the immunoblot pattern obtained with MCTD sera mostly was clearly distinguishable from the patterns obtained with sera from patients with related connective tissue diseases our results suggest that the immunoblotting technique might be useful as a diagnostic tool and support the concept of MCTD as a distinct entity.

Anti-nuclear matrix antibodies in mixed connective tissue disease.

Purified serum antibodies of patients suffering from mixed connective tissue disease were tested for their immunological specificity against nuclear constituents of HeLa S3 cells. In the indirect immunofluorescent staining technique, using cells and nuclei as targets, a typical speckled intranuclear staining pattern was obtained, that persisted after degradation and extraction of all nucleic acids and their associated proteins. This treatment of nuclei with detergents, DNase, RNase and high salt concentrations leave intact only the so-called nuclear matrix which is an intranuclear proteinaceous network. Further proof that nuclear matrix proteins were targets of the autoimmune reaction was obtained after separation of these proteins by sodium dodecyl sulfate polyacrylamide gel electrophoresis and electrophoretic transfer to nitrocellulose (blotting). A specific number of blot-transferred matrix proteins reacted with purified serum antibodies of 10 patients with mixed connective tissue disease, whereas this reaction was negative with normal healthy individuals. IgG preparations of 7 patients with systemic lupus erythematosus showed a weak, if any, reaction with matrix constituents. Obviously, in some connective tissue diseases serum antibodies are expressed which are directed to specific nuclear matrix antigens.

Incorporation of nucleic acid and protein precursors in enriched populations of proliferating normal and leukemic cells.

Nucleic acid and protein synthesis was determined in immature myeloid cells isolated from seven normal bone marrow samples and compared to blasts from nine patients with fullblown acute non-lymphocytic leukemia. Samples obtained from normal bone marrow contained a mean of 82% immature myeloid cells with 29.9% S-phase cells. These cells incorporated [3H]thymidine with a mean of 17,500 counts/min [3H]uridine with a mean of 24,000 counts/min and [3H]leucine with a mean of 2100 counts/min per 0.2 x 10(6) cells. Leukemic bone marrow cells could be separated in fractions with different proliferative activities. Leukemic samples with a mean of 3.6% S-phase cells incorporated [3H]thymidine with a mean of 1100 counts/min, [3H]uridine with a mean of 15,700 counts/min and ]3H]leucine with a mean of 2600 counts/min per 0.2 x 10(6) cells. For leukemic samples with a mean of 30.6% S-phase cells these values were: [3H]thymidine 22,200 counts/min, [3H]uridine 49,700 counts/min and [3H]leucine 6700 counts min per 0.2 x 10(6) cells. The incorporation studies were carried out for the first time in normal and leukemic cells with a comparable proliferative activity. Non-lymphocytic leukemic blastic cells showed a two-fold increase in RNA synthesis and a three-fold increase in protein synthesis compared to enriched samples of normal early myeloid cells with the same proliferative activity.

Kinetics of synthesis and processing of precursor polypeptides of murine leukemia virus in frog oocytes following microinjection of viral RNA.

Microinjection of Rauscher murine leukemia viral RNA into living oocytes from Xenopus laevis, in contrast to cell-free systems, allowed detailed studies on the processing of newly synthesized viral precursor polypeptides. The viral messenger appeared to be stable for at least 5 days. Maximal rate of translation of 70-S virion RNA was observed 10 h after injection. The predominant translation products after a 1-h labeling period were three precursor polypeptides of Mr 77000, 75000 and 65000. Following longer labeling periods the most stable precursor polypeptide of Mr 65000 was most prominent. In addition, several intermediates of Mr 35000--60000 were observed. After about 24 h, mature viral core proteins appeared. The rate of synthesis of the 75000-Mr and 77000-Mr viral proteins decreased gradually after injection, suggesting that viral core polypeptides somehow regulated processing or synthesis of the group-specific antigen precursors. A heterogeneous group of 90000--95000-Mr polypeptides seemed to be post-translationally modified products of the 75000-Mr and 77000-Mr proteins. However, in this study no envelope-related polypeptides were synthesized, when viral RNA (70-S or 35-S) was injected into the cytoplasm or the nucleus of Xenopus oocytes.

Translation of Rauscher leukemia virus RNA in heterologous cell-free systems.

Rauscher leukemia virus RNA (RLV RNA) is translated in mammalian cell-free systems into distinct polypeptides which are immunoprecipitable by an antiserum directed against RLV proteins. These polypeptides partially comigrate electrophoretically with native viral proteins synthesized in vivo in JLS-V9 cells. Besides 72000-, 65000- and 50000-dalton polypeptides a 15000-dalton polypeptide is also synthesized in vitro. Analysis of incubations of RLV RNA in different cell-free systems reveals that no virus-specific factors are required in the translation of RLV RNA in vitro.

Virus-specific messenger RNA on free and membrane-bound polyribosomes from cells infected with Rauscher leukemia virus.

Cells infected by Rauscher leukemia virus synthesize virus-specific RNA which can be detected by hybridization to the single-stranded DNA copy of the viral RNA. Evidence is provided that virus-specific RNA is present in free and membrane-bound polyribosomes of these cells. The relative content of virus-specific RNA, as measured by hybridization, is 6-10 times less on free polyribosomes than on membrane-bound polyribosomes. The messenger RNA associated with both classes of polyribosomes was characterized by density gradient centrifugation. In addition to a major RNA species identified as 36S RNA, at least 2 minor components in the 14S and 21S region have also been found. There is a striking difference in the distribution of these RNA species between free and membrane-bound polyribosomes.