31-OR: THE INFLUENCE OF ANTI-HLA ANTIBODY CONCENTRATION AND ISOTYPES ON SAB MFI

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• 作者：Steven Cate¹ ; Curtis P. McMurtrey¹ ; Ken Jackson¹ ; David Lowe² ; Rico Buchli³ ; Rod VanGundy³ ; Sean Osborn¹ ; Alexandar Mojsilovic¹ ; Daniel Zehnder² ; William Hildebrand¹
• 刊名：Human Immunology
• 出版年：2012
• 出版时间：October, 2012
• 年：2012
• 卷：73
• 期：supp_S
• 页码：27
• 全文大小：34 K

文摘

Aim

Single antigen beads (SAB) assays are routinely used to identify anti-HLA antibodies and the level of detection is commonly communicated as mean fluorescence intensity (MFI). Many factors may contribute to an MFI value, including antibody concentration and interference of non-IgG isotypes. Here we purified polyclonal anti-HLA antibodies from sensitized patients and tested the influence of antibody concentration and non-IgG isotypes on MFI.

Methods

Using an HLA-DR11 affinity column, we purified anti-HLA-DR11 antibodies from 13 sensitized human sera. The purified anti-HLA antibodies were then tested on a SAB assay using a secondary that only recognizes IgG isotypes. IgG isotypes were separated from IgM and IgA isotypes via size exclusion HPLC. Reduction of multimeric antibodies (IgM, IgA) to monomers was preformed with 100 mM DTT at 4 ¡ãC overnight with a total antibody concentration at 1 mg/ml.

Results

Serial dilution experiments with affinity purified anti-HLA antibodies demonstrated a strong correlation between antibody concentration and MFI within a single patient. However, between patients, the MFI value did not correlate to an antibody concentration - MFI was not comparable between patients. When IgM and IgA multimers were separated from IgG, a significant increase in MFI was observed compared to the original mix of isotypes. Furthermore, reduction of IgM and IgA into monomers with IgG did not impact MFI values as the monomeric mix provided an MFI matching the IgG monomer only.

Conclusions

Multiple factors influence SAB MFI. Here we tested the influence of both antibody concentration and the presence of IgM, and IgA on MFI. We show that MFI correlates with antibody concentration within a particular patient but not between patients. We also show that mixing IgM and IgA multimers (but not monomers) with IgG antibodies interferes with the assay by reducing MFI. Together these data exemplify the complexity of anti-HLA antibodies and factors that influence the SAB that is used to detect them.

Cate: Pure Protein: Employee. McMurtrey: Pure Protein: Consultant. Buchli: Pure Protein: Employee. VanGundy: Pure Protein: Employee. Osborn: Pure Protein: Employee. Mojsilovic: Pure Protein: Employee. Hildebrand: Pure Protein: Science Med Advisor.