Expression and Functional Properties of an Anti-Triazophos High-Affinity Single-Chain Variable Fragment Antibody with Specific Lambda Light Chain.

Triazophos is a widely used organophosphorous insecticide that has potentially adverse effects to organisms. In the present study, a high-affinity single-chain variable fragment (scFv) antibody with specific lambda light chain was developed for residue monitoring. First, the specific variable regions were correctly amplified from a hybridoma cell line 8C10 that secreted monoclonal antibody (mAb) against triazophos. The regions were then assembled as scFv via splicing by overlap extension polymerase chain reaction. Subsequently, the recombinant anti-triazophos scFv-8C10 was successfully expressed in Escherichia coli strain HB2151 in soluble form, purified through immobilized metal ion affinity chromatography, and verified via Western blot and peptide mass fingerprinting analyses. Afterward, an indirect competitive enzyme-linked immunosorbent assay was established based on the purified anti-triazophos scFv-8C10 antibody. The assay exhibited properties similar to those based on the parent mAb, with a high sensitivity (IC50 of 1.73 ng/mL) to triazophos and no cross reaction for other organophosphorus pesticides; it was reliable in detecting triazophos residues in spiked water samples. Moreover, kinetic measurement using a surface plasmon resonance biosensor indicated that the purified scFv-8C10 antibody had a high affinity of 1.8 × 10(-10) M and exhibited good binding stability. Results indicated that the recombinant high-affinity scFv-8C10 antibody was an effective detection material that would be promising for monitoring triazophos residues in environment samples.

IgGs containing λ- and κ-type light chains and of all subclasses (IgG1-IgG4) from the sera of patients with autoimmune diseases and viral and bacterial infections hydrolyze DNA.

We present the first evidence demonstrating that small fractions of IgGs of all four subclasses (IgG1-IgG4) from patients with viral (tick-borne encephalitis), bacterial infections (streptococcal infection or erysipelas), and suppurative surgical infections caused by epidermal staphylococci as well as from patients with autoimmune diseases (systemic lupus erythematosus and multiple sclerosis) are catalytically active in the hydrolysis of supercoiled DNA. The hydrolysis of DNA was analyzed by agarose gel electrophoresis. The catalytic activities of nonfractionated IgGs increased in the following order: tick-borne encephalitis < suppurative surgical infection < streptococcal infection < multiple sclerosis < systemic lupus erythematosus, whereas IgGs of healthy donors were inactive. However, the pools of antibodies corresponding to any particular disease were characterized by a specific ratio of IgGs of all four subclasses (IgG1-IgG4) and IgGs containing λ- and κ-type light chains, and each of these subfractions of immunoglobulins demonstrated characteristic relative DNase activity. The relative activities of IgGs containing λ-type light chains may on average be higher, lower, or comparable with those for IgGs with κ-type light chains. The relative contributions of IgGs of different subclasses to the total activity of IgGs also varied widely in the case of various diseases: IgG1 (7%-45%), IgG2 (0.4%-73%), IgG3 (0%-12%), and IgG4 (9%-66%). Thus, immune systems of patients with different diseases can generate a variety of anti-DNA abzymes of different types and with different catalytic properties, which can play an important role in the pathogenesis or protection from the development of these diseases.

Lupus anticoagulant-like activity observed in a dimeric lambda protein produced by myeloma cells.

We report here a lupus anticoagulant (LA)-like activity observed in a 45-year-old man with Bence-Jones protein (BJP) lambda-type multiple myeloma. This patient showed no clinical symptoms of thrombosis or bleeding diathesis. Laboratory examination on admission showed mild anemia, prolongation of activated partial thromboplastin time (APTT) (APTT, 56.2 seconds; control, 29.1 seconds), normal prothrombin time, normal thrombin time, and massive proteinuria (2.3 g/d). The mix test with normal plasma showed the presence of circulating anticoagulant. Based on the assumption that the lambda-type BJP may have been responsible for the prolongation of APTT, we purified the BJP from the patient's urine using column works. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting showed that the purified protein was a 48-kd homodimer of immunoglobulin lambda-chains. Addition of the purified dimeric lambda-type BJP to the normal plasma prolonged both APTT and dilute Russell's viper venom time (DRVVT) in a dose-dependent manner, and the negatively charged phospholipid-dependent prothrombinase activity was significantly inhibited in the presence of this protein. Furthermore, both the prolongation of DRVVT and the inhibition of the prothrombinase activity were almost completely abrogated under the condition of high ionic strength. These findings collectively suggest that the dimeric lambda-type BJP showed LA-like activity via the mechanism of ionic charge.

In vitro AL-amyloid formation by rat and human mesangial cells.

AA-amyloid has been produced experimentally in animal models, allowing the study of mechanisms involved in AA-amyloidogenesis, but those involved in renal AL-amyloidogenesis have not been adequately investigated due, in part, to lack of appropriate in vitro models. Rat and human mesangial cells were grown on a human extracellular matrix (Amgel) derived from normal tissues and on coverslips in the presence of 10 microliters of amyloid enhancing factor (AEF) per milliliter of media and 10 micrograms/ml monoclonal lambda light chains (LCs) obtained from two patients with AL-amyloidosis. Two additional lambda LCs derived from the urine of patients with myeloma and tubulointerstitial renal disease were used as controls. To verify amyloid deposition, light and electron microscopic examination, as well as Congo red and thioflavin T staining, were performed on samples incubated under different experimental conditions. Intracellular and extracellular amyloid was identified in samples incubated for 24 hours with human mesangial cells (for 48 hours with rat mesangial cells), amyloidogenic monoclonal LCs, and AEF. The amount of amyloid detected, which increased with longer incubation times, was found to be most abundant at 14 days. Amyloid was not present in cultures of mesangial cells incubated with amyloidogenic LCs alone or in the absence of mesangial cells. Likewise, incubation of mesangial cells with amyloidogenic LC or AEF separately or amyloidogenic LC in the presence of AEF but without mesangial cells did not result in amyloid formation. Amyloid was not seen when LCs obtained from the urine of patients with tubulointerstitial renal disease were incubated with AEF and mesangial cells. AL-amyloid production requires all three components--mesangial cells, amyloidogenic LCs, and AEF. In addition, amyloid was detected intracellular in mesangial cells, supporting the hypothesis that the production of AL-amyloid in the kidney requires intracellular processing by these cells. This system provides a unique experimental model to study renal AL-amyloidogenesis and a platform to explore mesangial cell-matrix interactions.

Effect of myeloma light chains on phosphate and glucose transport in renal proximal tubule cells.

Primary cultures of cells derived from the rat proximal tubule were exposed to up to 200 microM lambda- or kappa-light chain obtained from myeloma patients. Light chains inhibited the uptake of both phosphate and glucose by the cells while albumin had no effect. The half-maximal inhibitory concentration (IC50) of both the lambda- and kappa-light chains on phosphate transport were similar, 34 and 35 microM respectively. The IC50 of the kappa-light chain on glucose transport was 360 microM. The inhibitory effect of light chains was dose-dependent (r = 0.90, p < 0.01 for the lambda-light chain and r = 0.93, p < 0.001 for the kappa-light chain, on phosphate transport; and r = 0.93, p < 0.001 for glucose transport). Dixon and Line-weaver-Burk plot analyses were characteristic for noncompetitive inhibition. The inhibition constant 89 microM for phosphate uptake derived from the Dixon plot was similar to the IC50 calculated from the dose-response curves. These findings indicate that light chains, at concentrations found in the tubule fluid of a typical myeloma patient, are potent inhibitors of phosphate and glucose transport in proximal tubular cells, and that direct cell toxicity is a major mechanism of light chain nephrotoxicity.