Microscale screening of antibody libraries as maytansinoid antibody-drug conjugates.

Antibody-drug conjugates (ADCs) are of great interest as targeted cancer therapeutics. Preparation of ADCs for early stage screening is constrained by purification and biochemical analysis techniques that necessitate burdensome quantities of antibody. Here we describe a method, developed for the maytansinoid class of ADCs, enabling parallel conjugation of antibodies in 96-well format. The method utilizes ∼ 100 µg of antibody per well and requires <5 µg of ADC for characterization. We demonstrate the capabilities of this system using model antibodies. We also provide multiple examples applying this method to early-stage screening of maytansinoid ADCs. The method can greatly increase the throughput with which candidate ADCs can be screened in cell-based assays, and may be more generally applicable to high-throughput preparation and screening of different types of protein conjugates.

The CD200 receptor is a novel and potent regulator of murine and human mast cell function.

CD200R is a member of the Ig supergene family that is primarily expressed on myeloid cells. Recent in vivo studies have suggested that CD200R is an inhibitory receptor capable of regulating the activation threshold of inflammatory immune responses. Here we provide definitive evidence that CD200R is expressed on mouse and human mast cells and that engagement of CD200R by agonist Abs or ligand results in a potent inhibition of mast cell degranulation and cytokine secretion responses. CD200R-mediated inhibition of FcepsilonRI activation was observed both in vitro and in vivo and did not require the coligation of CD200R to FcepsilonRI. Unlike the majority of myeloid inhibitory receptors, CD200R does not contain a phosphatase recruiting inhibitory motif (ITIM); therefore, we conclude that CD200R represents a novel and potent inhibitory receptor that can be targeted in vivo to regulate mast cell-dependent pathologies.

High-resolution two-dimensional electrophoretic survey of serum protein genetic types in Schmiedeleut Hutterites.

High-resolution, two-dimensional electrophoresis (2DE) was used to examine allele frequencies in eight serum protein marker systems and to screen for rare or previously undescribed alleles in 152 members of the Schmiedeleut branch of the Hutterite Brethren. The results include the first report of α2 -HS glycoprotein, apolipoprotein E, and SPPM-158 frequencies and haptoglobin 1F and 2S subtype frequencies in the Hutterites. Designed as part of ongoing genetic studies in this reproductively isolated population, this study was done to determine which markers might correlate with medical features or could be useful in population studies. Prior studies of erythrocyte surface and enzymatic markers, lymphocyte surface and enzymatic markers, and serum proteins by other investigators have identified common, rare, and private markers in approximately 40 polymorphic systems. This study included five serum protein markers that were not examined in previous studies. The study identified a 2DE marker, SPPM-158, that was later confirmed in other populations to be a ubiquitous serum protein polymorphism. Allele frequencies for α2 -HS glycoprotein, apolipoprotein E, group-specific globulin, haptoglobin, SPPM-158, α1 -antitrypsin, apolipoprotein A-IV, and transferrin are presented. The first five of these had allele frequencies useful for population studies. We did not find any rare or private variants with 2DE in these systems. Overall, the 2DE data were in good agreement with prior studies in the Hutterites and relevant European populations.

Cell culture propagation of bovine coronavirus.

Although most field strains of bovine coronavirus (BCV) grow poorly in cell culture and fail to produce cytopathic effects (CPE) until after blind passage, primary calf kidney (PCK) and Vero cells have permitted primary isolation of virus. Cell culture-adapted strains of BCV replicate in PCK, bovine embryonic lung, bovine fetal thyroid, bovine fetal brain, bovine skin cells, ovine fetal kidney cells, and the cell lines pig kidney K3 and 15, Vero, human embryonic lung fibroblasts, HRT-18, MDBK and BEK-1, with trypsin useful for enhancing replication. Organ culture as well as suckling mouse, rat, and hamster brains also support the growth of cell culture-adapted BCV strains. Viral growth is most commonly detected by CPE, immunofluorescence, hemagglutination, and hemadsorption assays or electron microscopy of supernatants from infected cells. In this report, the optimal conditions for the growth and plaque assay of the NCDV strain of BCV in MDBK cells are described.