Anti-Mouse IgG2b polyclonal antibody [DyLight® 550]

Apolipoprotein E (APOE) alleles are strongly related to the risk of Alzheimer's disease (AD). APOE genotype also affects inflammatory processes in response to damage. We tested whether APOE genotype affected the levels of specific immunoglobulins in healthy, uninfected APOE knock-in mice. We measured specific immunoglobulins in brain, spleen, and plasma. Levels of total IgG in brain and spleen were highest in APOE-epsilon 3 mice, significantly higher than in APOE-epsilon 2 and APOE-epsilon 4 mice; no differences were observed for levels of total IgG in plasma. We also measured specific subtypes of IgG. IgG1 was only detectable in plasma and did not differ by APOE genotype. IgG3 was detectable in plasma and spleen, and also did not differ by APOE genotype. IgG2b showed the same pattern as levels of total IgG by APOE genotype, with the highest levels of IgG2b in brain, spleen, and plasma of APOE-epsilon 3 mice. IgG2a showed an entirely different pattern, with significantly higher levels in spleen and plasma of APOE-epsilon 4 mice compared to APOE-epsilon 2 and APOE-epsilon 3 mice. We also measured IgM and IgA in spleens and plasma of these mice. In spleen, APOE-epsilon 4 mice had the lowest IgA levels and the highest levels of IgM; both being significantly different from APOE-epsilon 2 mice. In total, murine IgG2a and IgM were highest in APOE-epsilon 4 mice, while total IgG and Ig2b were highest in APOE-epsilon 3 mice. These dramatically different distributions of immunoglobulins could allow for human AD risk biomarkers based on specific immunoglobulin subtypes.

Formation of disulfide bonds between heavy and light chains is challenge for production of recombinant immunoglobulin G (IgG) and fragment antigen-binding (Fab). Insufficient formation of the bond influences productive yield and quality of recombinant IgG and Fab. To investigate how amino acid sequences of the first heavy chain constant region (CH1) effect on assembly between heavy chain (VH-CH1 or Fd) and light chain (VL-CL), the CH1 gene sequence of an anti-paclitaxel fragment antigen-binding (anti-PT Fab IgG2a) was modified using the splicing by overlap extension-polymerase chain reaction (SOE-PCR) to be gene sequences encoding amino acid sequence of IgG1, IgG2b, and IgG3 subtypes. These anti-PT Fabs were expressed in Escherichia coli and silkworm larva expression systems. The efficiency of the assembly between VH-CH1 and VL-CL was evaluated. Based on sodium dodecyl sulfate polyacrylamide gel electrophoresis and Western blot analysis, assembly between heavy of IgG1 subtype and light chain was found to be superior over other subtypes using all tested expression systems. Furthermore, reactivity analysis using an enzyme-linked immunosorbent assay (ELISA) revealed that anti-PT Fab IgG1 subtype showed the highest reactivity against target compound paclitaxel. The folding efficiency and reactivity of the anti-PT Fab was influenced by CH1 amino acid sequence, which raises the possibility that this modification can be applied to improve recombinant Fab production.