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Updated: Jun 27, 2026
Generation of Discriminative Human Monoclonal Antibodies from Rare Antigen-specific B Cells Circulating in Blood
Published on: February 6, 2018
[Eukaryotic expression and determination of ZCH-7-2F9 single chain antibody against human CD14]
Bo-tao Ning1, Yong-min Tang, Jiang Cao
1Department of Hematology and Oncology, the Children's Hospital of Zhejiang University School of Medicine, Hangzhou, China.
Objective:
Acute monocytic leukemia (AML)-M5 is the common type of acute myeloid leukemias in children. Studies have shown that there are abundant lipopolysaccharide (LPS) receptor (designated as CD14) molecules on the cell membrane of M5 cells and they play an important role in the diagnosis of M5, since they can be recognized and bound by mouse-anti-human CD14 monoclonal antibody (McAb). However, mouse-originated antibodies are largely not suitable for clinical application due to the severe side effects, thus "humanized antibody" is desired. As the first step for developing humanized antibody, the construction and expression of single chain antibody (scFv) with functional protein are necessary. The present study aimed to express ZCH-7-2F9 ScFv (scFv(2F9)) in eukaryotic cells and obtain the scFv(2F9) protein with a high biological activity.
Methods:
Four primers were synthesized to construct the eukaryotic expressional vector, which included SfiI and EcoRI enzyme cleaving site, 6 x His and stop code TGA sequences. scFv(2F9) gene was amplified through splicing by overlap extension (SOE) using the high fidelity Taq polymerase. Positive recombinants (pSectag2A/scFv(2F9)) were identified through enzyme cleaving and sequenced before expression and were transformed into Chinese hamster ovary (CHO) cells for expression. Western-Blot and flow cytometry (FCM) were carried out to determine the relative molecular mass (Mr) and binding activity of scFv(2F9).
Results:
The cloned scFv(2F9) gene was identified to be functional by sequencing and expressing. The interested protein could be detected in the culture supernatant of transformed CHO cells with an Mr of 31 000. The blocking test showed that the positive cell percentages, the mean fluorescence intensity (MFI) and the peak of channel (peak Ch) were reduced by 90.02%, 63.30% and 63.38%, respectively after blocking with scFv(2F9), while those were 4.55%, 10.09% and 5.02% after blockage using the supernatant from the CHO cells transfected with blanked vector pSectag2A.
Conclusions:
The scFv(2F9) against human CD14 antigen was successfully expressed in eukaryotic cells and showed a high biological activity, which may be useful for the further studies on its humanized antibodies.
Insights
The study successfully expressed a functional single chain variable fragment (scFv) protein targeting CD14 in eukaryotic cells. This highly active scFv(2F9) is a crucial step toward developing humanized antibodies for acute monocytic leukemia (AML)-M5 diagnosis.
Area of Science:
- Biotechnology
- Immunology
- Oncology
Background:
- Acute monocytic leukemia (AML)-M5 is a common childhood leukemia.
- CD14, the lipopolysaccharide (LPS) receptor, is abundant on M5 cells and a diagnostic target.
- Mouse anti-human CD14 monoclonal antibodies (McAbs) show clinical limitations due to side effects, necessitating humanized antibody development.
Purpose of the Study:
- To construct and express the ZCH-7-2F9 single chain variable fragment (scFv(2F9)) in eukaryotic cells.
- To obtain a biologically active scFv(2F9) protein as a precursor for humanized antibody development.
Main Methods:
- Constructed an eukaryotic expressional vector with specific enzyme sites and tags.
- Amplified the scFv(2F9) gene using splicing by overlap extension (SOE) PCR.
- Expressed the recombinant protein in Chinese Hamster Ovary (CHO) cells and analyzed its molecular mass and binding activity using Western-Blot and flow cytometry (FCM).
Main Results:
- Sequencing and expression confirmed a functional scFv(2F9) gene.
- The expressed protein, with a molecular mass of approximately 31,000 Da, was detected in the culture supernatant of transformed CHO cells.
- Blocking assays demonstrated significant reduction (up to 90%) in CD14-positive cell detection by scFv(2F9), indicating high biological activity.
Conclusions:
- Successfully expressed functional scFv(2F9) targeting human CD14 in eukaryotic cells.
- The expressed scFv(2F9) protein exhibits high biological activity.
- This achievement is a significant step towards developing humanized antibodies for AML-M5 diagnosis and treatment.
