Aim: Patient sera can contain multiple donor specific antibodies (DSAs) with different isotypes due to various sensitizing events. IgG and IgM antibody isotypes are associated with antibody mediated rejection (ABMR) and graft loss. One Lambda MagSort was developed and optimized to isolate IgG antibodies from patient sera. To further explore the utility of the MagSort product, we developed buffers that allow the isolation of IgM antibodies from patient sera.
Method: IgG elution buffer (MagSort MAGELBUF) and IgM elution buffer were developed. Sera and mouse monoclonal antibodies (mAbs) were screened to obtain IgM /IgG positive sera and IgM positive mouse mAbs, respectively. We performed serum treatment using MagSort A0201 and MagSort C1502 on Serum 1, and MagSort B0801 and MagSort B5701 on Serum 2; mAb treatment (IgM Ab 1 at 0.5 mg/ml and 0.05 mg/ml concentrations) was performed using MagSort A0201. Post-treatment sera and elution samples were tested on LABScreen Single Antigen (One Lambda, LS1A04) with anti-IgG or anti-IgM 2nd Ab. We compared elution signals for isolated antibodies using IgM elution buffer versus IgG elution buffer.
Results: IgG elution buffer does not elute either Class I or Class II specific IgM. IgM elution buffer effectively eluted Class I specific IgM. For IgM Ab 1, HLA-A*02:01 elution signals from IgM elution buffer are >100-fold compared to signals from IgG buffer. For Serum 2, HLA-B*08:01 elution signals from IgM elution buffer are >6-fold compared to IgG buffer. Additionally, IgM elution buffer isolated an HLA-B*08:01-specific IgM pattern while IgG elution buffer isolated HLA-B*08:01-specific IgG with a different pattern. For Serum 1, IgM elution buffer efficiently isolated HLA-A*02:01-specific IgM DSA targeting eplet 107W. IgG elution buffer isolated low levels of HLA-A*02:01-specific IgG DSA targeting eplet 79GT. Isolated antibody signals are specific as MagSort B5701 only eluted HLA-B*57:01-specific IgG but not HLA-B*57:01-specific IgM from Serum 2. Similarly, MagSort C1502 only eluted HLA-C*15:02-specific IgG targeting eplet 80K but not HLA-B*57:01-specific IgM from Serum 2.
Conclusion: We have demonstrated that it is feasible to isolate IgM and IgG HLA-specific antibodies from sera using individually optimized elution buffers in combination with the MagSort beads.
