Anti-Mouse IgG2b polyclonal antibody [HRP]

The assembly between heavy and light chains is a critical step of immunoglobulin (Ig) and fragment antigen-binding (Fab) antibody expression and of their binding activity. The genes encoding Fab were obtained from hybridoma cells secreting monoclonal antibody (MAb, IgG2b) against adenylate cyclase activator forskolin (FOR). The subclass of the first constant domain of heavy chain (C(H)1) of IgG2b was modified to IgG1 via overlap extension polymerase chain reaction and expressed via Escherichia coli bacterial system. Since both Fabs (IgG2b and IgG1) were expressed as inclusion bodies, functional analysis was performed after in vitro refolding via stepwise dialysis. The result indicated that the folding efficiency between V-H-C(H)1 and V-L-C-L was improved by the C(H)1 modification from IgG2b to IgG1 subclass, although their specificity for FOR was not altered. Effective folding of IgG1 was also observed when they were expressed in the hemolymph of silkworm larvae using the Bombyx mori nuclear polyhedrosis virus bacmid system. An indirect competitive enzyme-linked immunosorbent assay (icELISA) was then developed for the determination of FOR using effectively prepared Fab IgG1. The sensitivity of FOR determination was in the range of 3.91-62.5 ng/mL with less than 9% relative standard deviation, implying the sensitive and reliable analysis of developed icELISA. In addition, high accuracy of the icELISA was supported by the results of spiked-and-recovery tests, ranging from 100.2 to 102.3%. Therefore, Fab could be utilized reliably for icELISA instead of the more expensive MAb. Collectively, this approach improved productivity of Fab and reduced the cost of antibody production.

Epidermolysis bullosa acquisita (EBA) is an autoimmune skin blistering disease characterized by IgG autoantibodies (aAb) against type VII collagen (COL7). The mechanisms controlling the formation of such aAbs and their effector functions in the skin tissue are incompletely understood. Here, we assessed whether the inhibitory IgG Fc receptor, Fc gamma RIIB, controls the development of autoimmune skin blistering disease in an active model of EBA. For this purpose, we immunized congenic EBA-susceptible B6.SJL-H2s (B6.s) and B6.s-Fcgr2b(-/-) mice with the immunodominant vWFA2 region of COL7. B6.s-Fcgr2b(-/-) mice developed a strong clinical phenotype with 15 +/- 3.3% of affected body surface area at week 4. In contrast, the body surface area in B6.s mice was affected to a maximum of 5% at week 6 with almost no disease signs at week 4. Surprisingly, we already found strong but similar COL7-specific serum IgG1 and IgG2b aAb production at week 2. Further, aAb and C3b deposition in the skin of B6.s and B6.s-Fcgr2b(-/-) mice increased between weeks 2 and 6 after vWFA2 immunization. Importantly, neutrophil skin infiltration and activation was much stronger in B6s-Fcgr2b(-/-) than in B6.s mice and already present at week 2. Also, the early aAb response in B6.s-Fcgr2b(-/-) mice was more diverse than in wt B6.s mice. Reactive oxygen species (ROS) release from infiltrating neutrophils play a crucial role as mediator of skin inflammation in EBA. In line, sera from B6.s and B6.s-Fcgr2b(-/-) mice induced strong ROS release from bone marrow-neutrophils in vitro. In contrast to the antibody-transfer-induced EBA model, individual targeting of Fc gamma RIII or Fc gamma RIV decreased ROS release to 50%. Combined Fc gamma R blocking abrogated ROS release from BM neutrophils. Also, ROS release induced by COL7-specific serum IgG aAbs was significantly higher using BM neutrophils from B6.s-Fcgr2b(-/-) than from B6.s mice. Together, our findings identified Fc gamma RIIB as a suppressor of skin inflammation in the active EBA model through inhibition of early epitope spreading, protection from strong early neutrophil infiltration to and activation of neutrophils in the skin and suppression of Fc gamma RIII activation by IgG1 aAbs which drive strong ROS release from neutrophils leading to tissue destruction at the dermal-epidermal junction.