Research Article: Immuno-biosensor for Detection of CD20-Positive Cells Using Surface Plasmon Resonance

Date Published: June 30, 2017

Publisher: Tabriz University of Medical Sciences

Author(s): Dariush Shanehbandi, Jafar Majidi, Tohid Kazemi, Behzad Baradaran, Leili Aghebati-Maleki, Farzaneh Fathi, Jafar Ezzati Nazhad Dolatabadi.


Purpose: Surface plasmon resonance (SPR) sensing confers a real-time assessment of molecular interactions between biomolecules and their ligands. This approach is highly sensitive and reproducible and could be employed to confirm the successful binding of drugs to cell surface targets. The specific affinity of monoclonal antibodies (MAb) for their target antigens is being utilized for development of immuno-sensors and therapeutic agents. CD20 is a surface protein of B lymphocytes which has been widely employed for immuno-targeting of B-cell related disorders. In the present study, binding ability of an anti-CD20 MAb to surface antigens of intact target cells was investigated by SPR technique.

Partial Text

Surface plasmon resonance (SPR) technology is widely used for the study of the interactions between a variety of chemical compounds and various biomolecules such as proteins, peptides and nucleic acids.1 Assessment of the interactions between analytes and immobilized ligands such as antibody/antigen and complementary nucleic acids is possible using this technology.2,3 Membrane proteins as important targets for drug discovery have recently attracted a great deal of interest for binding studies by this system.4 SPR based assessments are highly reproducible and permit the real-time investigation of probable interactions in label free form.4 CD20 is a surface protein which has been extensively utilized for targeted therapy of hematologic malignancies and autoimmune disorders.5 Rituximab was the first FDA approved anti-CD20 monoclonal antibody (MAb) for the targeted therapy of non-Hodgkin’s lymphoma and chronic lymphocytic leukemia.6 However, Rituximab and current therapeutics have not associated with complete remission in a considerable portion of the patients.7 Hence there is an urgent need for development and assessment of more efficient therapeutics. Targeted therapy, due to the reduced drug dosage and minimized harmful effects on unintended tissues has been considered as a more tolerable treatment approach.8 In addition to MAbs with native format, antibody derivatives have been also introduced for targeting studies.5 Bispecific antibodies (which simultaneously target two different antigens) and CAR T cells (engineered T cells with surface expression of chimeric antigen receptors) are examples of novel targeting agents.9 These agents acquire their antigen binding parts from the single-chain variable fragments (scFvs) of the input MAbs. Consequently, efficient binding of the utilized MAb is a prerequisite for engineering of this type therapeutics.

N-hydroxysuccinimide (NHS), 11-mercaptoundecanoic acid (MUA), Bovine serum albumin (BSA) and PBS 10X were purchased from Sigma–Aldrich (Steinheim, Germany). Fetal bovine serum, penicillin and streptomycin were obtained from Gibco (Thermo Fisher Scientific, USA). Pure gold (Au) chips were acquired from bionavis company (Tampere, Finland). The recombinant Staphylococcus aureus protein A (SpA) was kindly provided by Dr. Gholamreza‏ Ahmadian and Dr. Garshasb‏ Rigi (Department of Molecular Genetics, National Institute of Genetic Engineering and Biotechnology, NIGEB).‏ The murine IgG2a anti-CD20 MAb was acquired from our previous works.9,10 Raji (a Burkitt’s lymphoma cell line) and MOLT-4 (human T lymphoblast related to acute lymphoblastic leukemia) were purchased from the National Cell Bank of Iran (Pasteur Institute, Tehran, Iran).

Several SPR-based experiments are designed for detection of isolated or recombinant antigens. However, detached antigens or recombinant proteins may fail to reflect the exact interactions of the in vivo interactions. CD20 is a surface protein which spans the cell membrane 4 times. Most of anti-CD20 MAbs bind to discontinuous epitopes on this molecule.9 It is expected that, the antigens on the cell surfaces which retain their natural three dimensional (3D) conformations, produce more factual binding values. In this regard, we tried to assess the interaction of a MAb with CD20 antigen on the intact cells. A number of studies have employed SPR for investigating ligand interactions with bacteria and mammalian intact cells.16-19 It should be noticed that, because of having evanescent filed near 400 nm on the Au surface, the size of the ligands to be immobilized is limited in SPR method.18 Alternatively, the interaction partner with applicable dimensions should be fixed on the sensor chip. In the present study, the antibody of interest was fixed on the Au chips and the investigated cells were injected as analyte.1 By running the apparatus, sensorgrams indicating the cell binding/detection rates were obtained for the two studied strategies. In the first approach, SpA was used for antibody fixation on Au chips. SpA, a surface protein of Staphylococcus aureus, is widely used for antibody purification in laboratory. This molecule has five Fc-binding domains and shows acceptable binding affinities to different immunoglobulins, especially human IgG1 and mouse IgG2a molecules.12

In this study, “11-mercaptoundecanoic acid” and “Staphylococcus aureus protein A” were utilized for immobilization of an anti-CD20 antibody on gold surface of SPR chips. According to the results, both strategies were applicable for this purpose and the created sensors were able to detect target cells. Furthermore, the investigated monoclonal antibody had acceptable binding specificity to CD20-positive Raji cells, whereas its interaction with CD20-negative MOLT-4 cells was negligible. Therefore, the introduced systems could be employed for immuno-detection of intact CD20-positive cells by SPR.

This work was supported by a grant from Immunology Research Center, Tabriz University of Medical Sciences, Tabriz, Iran. Here, we wish to express our sincere gratitude and appreciation to Dr. Gholamreza Ahmadian and Dr. Garshasb Rigi for providing recombinant protein A.

Not applicable.

The authors declare no conflict of interests.




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