IgM Antibodies Can Access Cryptic Antigens Denied to IgG: Hypothesis on Novel Binding Mechanism.

Antibodies are well-known protein mediators of immunity. IgM is the primordial member and the neglected sibling of the later-evolved and more proficient IgG in regard to their therapeutic and diagnostic use. Serendipitously, however, we found a paradox: While murine IgM antibodies specific for guanosine triphosphate (GTP) were able to recognize native guanylyl antigens found in primate or rat muscle tissues by immunofluorescence assays (which mimicked the auto-antibodies from autoimmune patients to skeletal or smooth muscle), the murine and human IgG counterparts failed.

Osteopontin Fragments with Intact Thrombin-Sensitive Site Circulate in Cervical Cancer Patients.

We investigated whether circulating osteopontin (OPN) could be used as a biomarker for cervical cancer. We employed a monoclonal antibody (mAb 659) specific for the unique and intact thrombin-sensitive site in OPN using an inhibition ELISA. We found significantly higher levels of OPN in 33 cervical cancer patients in both the plasma (mean +/- SD, 612 +/- 106 ng/mL) and serum (424 +/- 121 ng/mL) compared to healthy subjects [409 +/- 56 ng/mL, from 31 plasma samples (P < 0.

Microbiological culture simplified using anti-O12 monoclonal antibody in TUBEX test to detect Salmonella bacteria from blood culture broths of enteric fever patients.

Definitive diagnosis of infectious diseases, including food poisoning, requires culture and identification of the infectious agent. We described how antibodies could be used to shorten this cumbersome process. Specifically, we employed an anti-Salmonella lipopolysaccharide O12 monoclonal antibody in an epitope-inhibition 10-min test (TUBEX TP) to detect O12⁺Salmonella organisms directly from routine blood culture broths.

Combined rapid (TUBEX) test for typhoid-paratyphoid A fever based on strong anti-O12 response: design and critical assessment of sensitivity.

Rapid diagnostics can be accurate but, often, those based on antibody detection for infectious diseases are unwittingly underrated for various reasons. Herein, we described the development of a combined rapid test for two clinically-indistinguishable bacterial diseases, typhoid and paratyphoid A fever, the latter fast emerging as a global threat. By using monoclonal antibodies (mAbs) to bacterial antigens of known chemical structures as probes, we were able to dissect the antibody response in patients at the level of monosaccharides.

Modification of the TUBEX typhoid test to detect antibodies directly from haemolytic serum and whole blood.

The TUBEX test for typhoid fever detects serum antibodies in a simple and rapid assay system based on the inhibition of binding between two types of reagent particles - magnetic particles coated with an antigen (Salmonella O9 LPS) and coloured indicator particles coated with an anti-O9 mAb. A magnet is used to separate the colour indicator particles bound to the magnetic particles from the unbound indicator particles. Specific colour changes following magnetic separation are indicative of antibodies in the patient's serum; however, because results are interpreted based on changes in the colour red, haemolytic or icteric specimens cannot be used.

New rapid test for paratyphoid a fever: usefulness, cross-detection, and solution.

We described a 5-min colorimetric test for paratyphoid A fever, which detects anti-Salmonella O2 antibodies by inhibiting the binding between 2 types of reagent particles. This test (TUBEX-PA) is based on that (TUBEX-TF) used for typhoid fever, which detects anti-O9 antibodies. TUBEX-PA showed a sensitivity of 81.

The TUBEX test detects not only typhoid-specific antibodies but also soluble antigens and whole bacteria.

TUBEX (IDL Biotech) is a 5 min semiquantitative colorimetric test for typhoid fever, a widely endemic disease. TUBEX detects anti-Salmonella O9 antibodies from a patient's serum by the ability of these antibodies to inhibit the binding between an indicator antibody-bound particle and a magnetic antigen-bound particle. Herein, we report that TUBEX could also be used to specifically detect soluble O9 lipopolysaccharide in antigen-spiked buffer by the ability of the antigen to inhibit the same binding between the particles.

Rapid detection of early typhoid fever in endemic community children by the TUBEX O9-antibody test.

Typhoid remains a global public health problem, and quick accurate immunodiagnosis is needed. Here, we examined the performance of the 5-min TUBEX O9-antibody detection kit in 243 outpatients (mostly children and infants) in their first week of fever and 57 healthy subjects in the Bangladesh community. Based on culture results, TUBEX was 91.

Carrier-specificity of a phosphorylcholine-binding antibody requires the presence of the constant domains and is not dependent on the unique VH49 glycine or VH30 threonine residues.

Bacterially-produced antibody fragments, such as single-chain Fv (scFv) which comprises the variable regions of the light (VL) and heavy (VH) chains joined together by a short flexible linker, are useful as diagnostic and therapeutic agents. We previously constructed a scFv fragment from a hybridoma antibody (Mab2) but it unexpectedly lacked the unique carrier specificity of the native antibody. Thus, it bound indiscriminately to various phosphorylcholine (PC)-associated antigens, whereas the hybridoma antibody recognized the PC epitope only in the context of the immunizing antigen.

Pathogenic autoantibodies: emerging insights into tissue injury.

Accumulating evidence is emerging that B lymphocytes and autoantibodies are critical in the development of autoimmune disease. Even in certain disorders initially thought to be T cell-mediated, these immune components are now considered key players in the disease pathogenesis, and new autoantibody specificities have been added to the growing list of targets including cell surface receptors and ion channels that may be involved in a variety of neuropsychiatric and cardiovascular disorders. Studies of autoantibodies penetrating living cells suggest a dosage effect in generating a biological outcome in vivo.

Antibody avidity maturation during severe acute respiratory syndrome-associated coronavirus infection.

The maturation of virus-specific immunoglobulin G avidity during severe acute respiratory syndrome-associated coronavirus infection was examined. The avidity indices were low (mean +/- SD, 30.8% +/- 11.

Evaluation of a recombinant nucleocapsid protein-based assay for anti-SARS-CoV IgG detection.

A high throughput accurate assay for anti-SARS-CoV IgG detection is needed for large-scale epidemiological studies. The evaluation of a commercial recombinant nucleocapsid protein-based microtitre plate enzyme immunoassay, ELISARS is described. The results on 150 sera from SARS patients and 450 sera from non-SARS controls showed that this assay had a high level of sensitivity (96.

BRE enhances in vivo growth of tumor cells.

Human BRE, a death receptor-associating intracellular protein, attenuates apoptotic response of human and mouse tumor cell lines to death receptor stimuli in vitro. In this report, we addressed whether the in vitro antiapoptotic effect of BRE could impact on tumor growth in vivo. We have shown that the mouse Lewis lung carcinoma D122 stable transfectants of human BRE expression vector developed into local tumor significantly faster than the stable transfectants of empty vector and parental D122, in both the syngeneic C57BL/6 host and nude mice.

Nuclear telomerase is less accessible to antibody probing than known nuclear antigens: retrieval with new immunostaining buffer.

Telomerase is an important tumor marker but few antibodies to the enzyme have been described or used without difficulty in histochemical detection. Here we report specific detection of the enzyme in cell and tissue preparations using a new monoclonal antibody (mAb 476) and a new antigen-retrieval buffer (Enhancing buffer). When used to detect telomerase under normal immunostaining conditions in HL-60 cells or tissue sections of hepatocellular carcinoma or metastatic choriocarcinoma, unexpectedly, the antibody stained the cytoplasm rather than the nucleus.

A death receptor-associated anti-apoptotic protein, BRE, inhibits mitochondrial apoptotic pathway.

BRE, brain and reproductive organ-expressed protein, was found previously to bind the intracellular juxtamembrane domain of a ubiquitous death receptor, tumor necrosis factor receptor 1 (TNF-R1), and to down-regulate TNF-alpha-induced activation of NF-kappaB. Here we show that BRE also binds to another death receptor, Fas, and upon overexpression conferred resistance to apoptosis induced by TNF-alpha, anti-Fas agonist antibody, cycloheximide, and a variety of stress-related stimuli. However, down-regulation of the endogenous BRE by small interfering RNA increased apoptosis to TNF-alpha, but nottoetoposide, indicating that the physiological antiapoptotic role of this protein is specific to death receptor-mediated apoptosis.

Antibody response of patients with severe acute respiratory syndrome (SARS) targets the viral nucleocapsid.

The recent outbreak of severe acute respiratory syndrome (SARS) provided an opportunity to study the antibody response of infected individuals to the causative virus, SARS coronavirus. We examined serum samples obtained from 46 patients with SARS, 40 patients with non-SARS pneumonia, and 38 healthy individuals, by use of Western blotting (WB), enzyme-linked immunoassay (ELISA), and immunofluorescence assay, using both native and bacterially produced antigens of the virus. We found a highly restricted, immunoglobulin G-dominated antibody response in patients with SARS, directed most frequently (89% by ELISA) and predominantly at the nucleocapsid.

Antibodies to guanosine triphosphate misidentified as anti-double-stranded DNA antibodies in a patient with antinuclear antibody-negative lupus, due to buckling of insolubilized assay DNA.

Objective: To investigate why the serum of a pediatric patient with systemic lupus erythematosus was persistently (>30 months) and strongly positive for antibodies to double-stranded DNA (dsDNA) as revealed by enzyme-linked immunosorbent assay (ELISA), but yielded negative results on the antinuclear antibody test (HEp-2 immunofluorescence [IF]).

Methods: The patient's antibodies were isolated on dsDNA and single-stranded DNA (ssDNA) supports, which were then examined by dsDNA ELISA and HEp-2 IF. Tests included the use of various inhibitors to determine the fine specificity of the antibodies.

Osteoporosis and transforming growth factor-beta-1 gene polymorphism in Chinese men and women.

Transforming growth factor-beta-1 (TGF-Beta(1)) has been implicated in bone mineral density (BMD) determination. We investigated the relationship between the TGF polymorphism, BMD, and vertebral fractures in 588 Chinese men and women. No association between TGF polymorphism and BMD was observed in postmenopausal women (aged 55-59 years), elderly men (aged 70-79 years), or elderly women (aged 70-79 years) at the hip, spine, or total body ( P >> 0.

The TUBEX typhoid test based on particle-inhibition immunoassay detects IgM but not IgG anti-O9 antibodies.

A serological test kit (TUBEX, IDL Biotech, Sweden) developed recently for the diagnosis of typhoid fever detects antibodies to the Salmonella enterica serovar Typhi lipopolysaccharide (LPS) O9 antigen. The antibodies are detected by their ability to inhibit the interaction between two types of reagent particles: (a). indicator latex microspheres sensitized with an anti-O9 monoclonal antibody, and (b).

Expression of a conserved mouse stress-modulating gene, Bre: comparison with the human ortholog.

Mouse Bre, an evolutionarily conserved stress-modulating gene, like its human counterpart, is expressed in multiple alternative transcripts. The main transcript, which is ubiquitously expressed, encodes a protein that binds tumor necrosis factor receptor 1 (TNF-R1) and downregulates TNF-induced activation of NF-kappaB. Alternative splicing of mouse Bre occurs only at the 5' region of the gene, generating either nonfunctional transcripts or transcripts that can encode putative protein isoforms differ at the N-terminal sequence.

Generation of monoclonal antibodies against Hong Kong nasopharyngeal carcinoma-associated Epstein-Barr virus latent membrane protein 1 (LMP1).

A panel of monoclonal antibodies specific to Hong Kong Chinese nasopharyngeal carcinoma (NPC)-associated Epstein-Barr virus (EBV) latent membrane protein 1 (LMP1) variants has been generated. These monoclonal antibodies not only differentiate the Hong Kong Chinese NPC-associated LMP1 variants from the prototype B95-8 LMP1, derived from Caucasian infectious mononucleosis, but also differentiate the 2 highly homologous LMP1 deletion variants commonly found in Hong Kong primary NPC. The predominant deletion type variant, DV-Asp335, is characterized by an aspartic acid at residue 335 located in the cytoplasmic C-terminal region, whereas the other minor deletion variant, DV-Gly335, has a glycine in the same residue position.

Single-chain Fv fragment lacks carrier specificity of the native antibody.

A single-chain antibody fragment (scFv) was constructed from a hybridoma antibody that binds to phosphorylcholine (PC) only when this hapten is presented in the form of the immunizing antigen (derived from Trichinella) but not when it is presented on other carriers (as found, for example, in pneumococcal capsules). The scFv derivative was found to lack this carrier specificity as it bound indiscriminately, but specifically, to the various PC-associated antigens, and exhibits a two-fold lower affinity (3.5x10(5)M(-1)) for nitrophenyl-PC than the native antibody.