Cloning, expression, and purification of an anti‐desipramine single chain antibody in NS/0 myeloma cells

Drug‐specific monoclonal antibodies and their antigen‐binding Fab fragments reverse acute desipramine toxicity in a rat experimental model by inducing a redistribution of drug from cardiac tissue into serum and extracellular fluid. In order to investigate the use of smaller recombinant antibody fragments such as single chain Fv (sFv) as an antidote, an efficient murine NS/0 myeloma expression system was developed. The variable light (V) and variable heavy (V) domains of a murine anti‐desipramine monoclonal antibody were cloned and sequenced. A 270 amino acid V‐(GlySer)‐V sFv was prepared by overlapping polymerase chain reaction (PCR) amplification of V with heavy chain leader peptide, V, and the linker. This construct was subcloned into a mammalian expression vector which utilizes the SRα promoter, a hybrid promoter consisting of the SV40 early promoter with portions of the human T‐cell leukemia virus type I long terminal repeat and also containing the Escherichia coli xanthine–guanine phosphoribo‐syltransferase gene for selection. NS/0 myeloma cells were transfected by electroporation. Stable recombinant NS/0 clones were screened for expression of sFv using reverse transcriptase‐PCR to detect mRNA and an enzyme‐linked immunosorbent assay (ELISA) to detect sFv. Secreted sFv from clones capable of growth to a cell density of 2–4 × 10 viable cells/mL was purified in a single step using a desipramine affinity column resulting in 12–39 mg/L of purified sFv. Affinity‐purified sFv had comparable desipramine binding activity to Fab when evaluated by competitive ELISA. Copyrigh

Monoclonal antibodies putatively identifying porcine B cells

Comparison was made of the binding of 38 test and three standard monoclonal antibodies (mAbs) to B cells from various pig lymphoid tissues by flow cytometry (FCM) and immunohistochemistry. Some mAbs were also tested on B cells from foetal pig tissues. Twenty of the new mAbs bound, though to variable degrees, to porcine B cells but only three were given cluster assignations: C35 (147) and BB6-11C9 (167) were assigned to wCD21 and 2F6/8 (057) was assigned to SWC7

Immune Response Against Porcine Reproductive and Respiratory Syndrome Virus During Acute and Chronic Infection

A significant obstacle to the prevention and control of porcine reproductive and respiratory syndrome virus (PRRSV) is the inability of current diagnostic tests to provide information concerning the stage of PRRSV infection. To explore possible prognostic combinations of cell-mediated and humoral immune responses, 3-week-old pigs (n = 10) were intramuscularly (IM) inoculated with PRRSV isolate VR-2332 and followed for 193 days post-inoculation (DPI). Negative control pigs (n = 10) were IM inoculated with minimum essential medium (MEM). At ∼2-week intervals, blood samples were collected from all animals and tested for the number of interferon (IFN)-γ-secreting peripheral blood mononuclear cells (enzyme-linked immunosorbent spot, Elispot), PRRSV viremia (quantitative reverse-transcriptase polymerase chain reaction, qRT-PCR), and serum antibodies using PRRSV protein ELISAs (N, GP5 3′, GP5 5′, M 5′, M 3′, GP5-M, and nsp2p) and a commercial PRRSV ELISA (IDEXX Laboratories Inc.). All pigs were viremic by 7 days post-inoculation, with 50% of the pigs resolving viremia by 56 DPI. A PRRSV-specific IFN-γ response was detected at DPI 28, reached a plateau at 42 DPI, declined slightly, and remained relatively stable from 56 to 193 DPI. On the basis of ROC area under the curve (AUC) analysis, the ELISAs that most reliably differentiated PRRSV-inoculated pigs from negative control pigs were the commercial ELISA (AUC = 0.97), the N ELISA (AUC = 0.96), and the M 3′ ELISA (AUC = 0.93). Multivariate analyses were performed to evaluate the relationship between the immune response and the duration and level of viremia. With all antibody assays and Elispot included in the models, the analysis determined that the serum-virus neutralizing antibody response was the best predictor of both level and duration of viremia. It was concluded that humoral antibody responses, particularly the commercial ELISA, N ELISA, and M 3′ ELISA were good predictors of prior exposure to PRRSV, but provided little information regarding the ontogeny of the protective immune response. Likewise, cell-mediated immunity based on the number of IFN-γ-secreting lymphocytes was a poor prognosticator of PRRSV infection status

Workshop studies with monoclonal antibodies identifying a novel porcine differentiation antigen, SWC9

Two monoclonal antibodies (mAb) within cluster M4 of the myeloid section of the Second International Swine CD Workshop, C4 (No. 144) and PM18-7 (No. 192), showed reactivity with thymocytes and among cells of myelomonocytic origin with mature macrophages but not with monocytes and granulocytes. Both mAb recognize a protein showing two bands of 205 kDa and 130 kDa under both reducing and non-reducing conditions. Although epitope mapping with these mAb could not be performed, this cluster received the SWC9 designation