Ann Lab Med.  2023 Jan;43(1):86-91. 10.3343/alm.2023.43.1.86.

Improvement of Anti-CD36 Antibody Detection via Monoclonal Antibody Immobilization of Platelet Antigens Assay by Using Selected Monoclonal Antibodies

• Xu X
• Chen D
• Ye X
• Xia W
• Shao Y
• Deng J
• Chen Y
• Ding H
• Liu J
• Xu Y
• Santoso S
• Fu Y

• KMID: 2551607
• DOI: http://doi.org/10.3343/alm.2023.43.1.86

Abstract

Antibodies against human CD36 are responsible for several immune-mediated disorders. The detection of anti-CD36 antibodies using the standard monoclonal antibody (mAb) immobilization of platelet antigens (MAIPA) assay is hampered by a high frequency of false-negative results, most likely due to competitive inhibition of the mAb used as the capture antibody. We generated a panel of mouse mAbs against CD36 and seven hybridomas (GZ-3, GZ-13, GZ-70, GZ-143, GZ-413, GZ-507, and GZ-608), which were selected for MAIPA assays, as they reacted with mouse and human CD36. Fourteen anti-CD36 sera were assayed; all of which showed a positive reaction in a PakPlus (Immucor GTI Diagnostics, Inc., Waukesha, WI, USA) ELISA-based screening (optical density: 0.257–2.292). When the reference anti-CD36 mAb FA6-152 was used in the MAIPA assay, only 6/14 (42.9%) sera displayed a positive reaction. In contrast, anti-CD36 antibodies were detected in 13/14 (92.9%) sera when GZ-70 and GZ-608 mAbs were used. This significant improvement resulted in the identification of anti-CD36 antibodies by an antigen capture assay. Since patient’s platelets possibly carrying rare native antigens are used, this method will facilitate the identification of new platelet antibodies against CD36 that are involved in immune-mediated thrombocytopenia and other diseases, such as transfusion-related acute lung injury.

Keyword

CD36; Monoclonal antibodies; Antigen capture assays

Figure

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  Fig. 1 Flow cytometry analysis of monoclonal antibodies (mAbs) against CD36 using transfected HEK293T cells and platelets. Upper panels: mAbs (GZ-608 and GZ-70) were incubated with mock, hCD36, and mCD36. Bound antibodies were detected using fluorescence-conjugated goat anti-mouse IgG and analyzed using flow cytometry. Lower panels: CD36+ platelets from a healthy blood donor and CD36− platelets from a CD36-deficient donor were incubated with GZ-608 or GZ-70 mAb. “Mock” stands for C220T variant CD36 construct-transfected HEK293T cells, “hCD36” for human CD36-transfected HEK293T cells, “mCD36” for mouse CD36-transfected HEK293T cells, and “Isotype” for mouse IgG1.

• 

  Fig. 2 Characteristics of anti-CD36 serum in MAIPA and binding assays using different capture mAbs. (A) Platelets were first incubated with anti-CD36 serum (serum number 8, gray columns) and AB serum (white columns) and then with eight anti-CD36 mAbs (20 μg/mL) as indicated and analyzed using the MAIPA assay. (B) Platelets were incubated with anti-CD36 serum at different dilutions (neat, 1:2, 1:4, and 1:8) and the anti-CD36 mAbs FA6-152, GZ-70, and GZ-608 (20 μg/mL), as indicated. The cut-off for each assay was determined by analyzing AB sera from healthy blood donors (N=8, white columns). The reaction was considered positive when the result was >0.200 (cut-off; mean value±3 SDs; N=8; dotted line). (C, D) Effects of the mAbs FA6-152 and GZ-608 on the binding of serum number 14 to CD36+ platelets as determined using flow cytometry. “Isotype” stands for mouse IgG1, “Ctrl” for AB serum+mAb, and “GAM” for fluorescence-labeled goat anti-mouse IgG antibody. Abbreviations: OD, optical density; MAIPA, monoclonal antibody immobilization of platelet antigens; mAbs, monoclonal antibodies.

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