Quantitative measurement of bitagged recombinant proteins using an immunometric assay: application to an anti-substance P recombinant antibody.

We have developed two different immunometric assays to directly quantify both the total and the active fractions of a recombinant antibody (single chain fragment variable, or ScFv) as obtained in a crude extract from an Escherichia coli expression system. For total determination, the assay is based on the simultaneous recognition of two different peptide Tag sequences (Ha-Tag and Myc-Tag) at each of the N- and C-terminal extremities of the recombinant protein. A monoclonal antibody (mAb 12CA5, directed against Ha-Tag), coated on microtiter plates, is used for capture, and the mAb 9E10 (directed against Myc-Tag), labeled with acetylcholinesterase (AChE, EC 3.1.1.7), acts as tracer. In parallel, for the determination of the active fraction, the capture is performed using microtiter plates coated with the antigen, while solid-phase-immobilized ScFv is measured using the same 9E10 tracer mAb. A synthetic peptide in which the two Tag sequences were joined was used as a standard, thus avoiding the laborious purification of a recombinant protein as reference. The method was applied to the direct measurement, in periplasmic extracts, of the total and active fractions of an ScFv produced at different induction temperatures.

Differential modulation of alpha, beta and gamma enolase isoforms in regenerating mouse skeletal muscle.

Nothing is known about the expression of the glycolytic enzyme enolase in skeletal muscle alterations such as myofiber degeneration and regeneration. Enolase is a dimeric enzyme which exhibits cell type specific isoforms. The embryonic form, alphaalpha, remains expressed in most adult tissues, whereas a transition towards specific isoforms occurs during ontogenesis in two cell types with high energy requirements: alphagamma and gammagamma in neurons, alphabeta and betabeta in striated muscle cells. During murine myogenesis, beta enolase transcripts are detected early in the forming muscles, and the beta gene is further upregulated at specific stages of muscle development. The alpha and beta subunits exhibit characteristic developmental microheterogeneity patterns. High levels of beta enolase subunits characterize the glycolytic fast-twitch fibers of adult muscles. We have investigated the expression of enolase subunits in a mouse experimental model of muscle regeneration. Following a single intramuscular injection of the necrotic agent cardiotoxin, we observed a rapid decrease in the level of the major muscle enolase subunit beta, accounting for the drop in total enolase activity that correlated with the degeneration of myofibers. Concomitant with the regeneration of new fibers, beta subunit levels began to increase, reaching normal values by 30 days after injury. Changes in the embryonic and ubiquitous subunit, alpha, mimicked those occurring during development by two aspects: modifications in electrophoretic variants and redistribution between soluble and insoluble compartments of muscle extracts. Imunocytochemical analyses of alpha and beta enolase subunits first revealed a homogeneous labeling within myofibers. Striations characteristic of normal adult muscle tissue were visible again by day 14 after injury. A perinuclear alpha and beta immunoreactivity was often observed in regenerating myofibers but its functional significance remains to be elucidated. Double labeling experiments with anti-gamma enolase and FITC-alpha bungarotoxin allowed us to follow the neuromuscular junction remodeling that occurs during muscle regeneration despite the absence of nerve injury.

Quantification of lamivudine (3TC) by competitive immunoassay.

Lamivudine or 3TC, the (-) eniantiomer of 2'-deoxy-3'-thiacytidine, is a prototype of a novel class of levogyre dideoxynucleosides analogues used in treatment of HIV and HBV infection. We describe a method corresponding to the first enzyme immunoassay for quantifying this antiviral drug. This technique use an enzyme conjugate that not require the use of radioactive labelling. In this study, anti-3TC antibodies were raised in rabbits by immunising with 3TC-HS-kelhoyle limpet hemocyanin (KLH) conjugate.

Synthesis of analogues of 5'-mono-, 5'-di-, and 5'-triphosphate-AZT for the development of specific enzyme immunoassay for monitoring of intracellular levels of AZT-MP, AZT-DP, and AZT-TP.

The synthesis of analogue compounds of polyphosphorylated AZT is described. The compounds are designed to raise specific antibodies against AZT-MP, -DP and -TP in rabbits.

A sensitive sandwich enzyme immunoassay for calcitonin gene-related peptide (CGRP): characterization and application.

Thirty mouse monoclonal antibodies (mAbs) directed against rat calcitonin gene-related peptide-alpha (CGRP-alpha) have been obtained. These mAbs are classified in 2 groups, one recognizing the peptide N-terminus and the other binding the C-terminus. A two-site immunometric assay was developed using mAb CGRP-83 as capture antibody, whereas mAb CGRP-72 acts as tracer, covalently labeled with enzyme acetylcholinesterase. This assay appeared sensitive (limit of detection: 2 pg/ml) and precise, allowing quantitative measurement of all human and murine CGRP isoforms. The assay was used to determine specific concentrations of CGRP in different rat, mice and guinea pig samples. The validity of the test was demonstrated by HPLC fractionation experiments.

Modulation by IL-10 of antigen-induced IL-5 generation, and CD4+ T lymphocyte and eosinophil infiltration into the mouse peritoneal cavity.

Sensitized BALB/c mice challenged i.p. with 1 microgram of OVA showed IL-5 release in the peritoneal lavage fluid, which peaked at 6 h and decreased thereafter. This was followed by a massive eosinophil accumulation, which started at 6 h and reached a plateau between 24 and 48 h. The i.p. injection of recombinant murine (rm) IL-10 (0.01-0.1 microgram/cavity) along with OVA reduced IL-5 release at 6 h and allergic eosinophilia at 6, 24, and 48 h. rmIL-10 also blocked in vitro IL-5 generation by sensitized peritoneal cells cultured in the presence of OVA. The inhibitory effect of rmIL-10 on Ag-induced eosinophilia and IL-5 release was suppressed by pretreatment of the animals with 1 mg/mouse of a neutralizing anti-mIL-10 mAb. Flow cytometric analysis revealed an increase in the number of CD4+ and CD8+ T lymphocytes and in the number of CD25+/CD4+ cells in the peritoneal lavage fluid collected 24 and 48 h after challenge, respectively; these numbers were reduced significantly by the administration of 0.1 microgram of rmIL-10. Finally, rmIL-10 failed to modify the anti-CD3-induced IL-5 release in vivo in the peritoneal cavity and in vitro from purified spleen CD4+ T lymphocytes. This suggests that rmIL-10 acts indirectly, by deactivating APC, rather than directly on T cell activation. These findings indicate that rmIL-10 displays anti-allergic activity in sensitized BALB/c mice by preventing Ag-induced CD4+ T lymphocyte and eosinophil accumulation as well as IL-5 release in the peritoneal cavity.