Hybridoma technology is the most classic to produce high-quality monoclonal antibodies, which are the powerful therapeutic agents against various diseases. However, this technology is labor-intensive and time-consuming because it includes aspects of animal immunization, cell fusion, hybridomas screening and subcloning, as well as antibody characterization¹. The cell fusion is generally performed by either PEG- or electro-fusion, which produces heterogeneous cells including specific antibody-secreting cells, nonspecific antibody-secreting cells or nonsecretors². The key to improve the efficiency of hybridoma technology is to develop efficient screening methods for identifying and selecting specific antibody-secreting hybridoma cells. Although commonly used ELISA is mature and reliable for cell screening, its limited sensitivity requires the clonal expansion for 1-2 weeks to obtain enough secreted antibody for detection³. Droplet-based microfluidic techniques exhibit the capability of high throughput and efficient cell screening or sorting, but their popularization is hampered by the sophisticated and expensive equipment as well as the specialized operation⁴.

Aiming at developing an efficient screening system with simplicity, universality and low-cost, Prof. Huangxian Ju and his coworkers have designed a proximity-rolling circle activated enzymatic switch (P-RCAES) to develop a homogeneous chemiluminescent screening protocol. As shown in Fig. 1, the heterogeneous hybridoma cells are distributed on 96-well plate to allow single cell in a well by limiting dilution, which are then incubated for 1 day for secreting the target antibody and subjected to the chemiluminescent screening of the specific antibody-secreting hybridoma cells.

Fig. 1: Illustration of the chemiluminescent screening of specific hybridoma cells with proximity-rolling circle activated enzymatic switch.

In the chemiluminescent screening protocol, the secreted antibody was detected by its recognition to a pair of antigen-DNA probes to trigger the P-RCAES. In P-RCAES, a complex with a proximity-ligated oligonucleotide sequence was firstly formed by the antibody-induced proximity hybridization of two antigen-DNA probes. The formed sequence then hybridized with block to release the primer from block-primer, and thus the rolling circle amplification (RCA) could be initiated by the released primer. During the RCA process, long oligonucleotide amplicon was produced as well as a huge mass of PPi, which could take away Cu²⁺ from HRP-Cu²⁺ to switch on the enzymatic activity of HRP for catalysing the oxidation of luminol by H₂O₂. The produced strong chemiluminescent signal was thus used for the detection of secreted antibody and the identification of specific hybridoma cells.

As a proof-of-concept, the chemiluminescent response to PPi, primer and anti-PCSK9 antibody were firstly examined. The chemiluminescent signal increased logarithmically with the increasing concentration of PPi, primer and anti-PCSK9 antibody (Fig. 2), and the LOD of PCSK9-Ab was calculated to be 1 pg mL^-1 (equal to 18 fM) based on three times standard deviation, which was at least 3 orders of magnitude lower than those reported previously for antibody detection with various amplification strategies.

Fig. 2: Chemiluminescent responses to pyrophosphate anions, primer and PCSK9-Ab.

Owing to the ultrahigh sensitivity of this enzymatic switch, the screening of specific hybridoma cells could be completed within 1 day on the conventional 96-well plate (Fig. 3), which greatly shortened the period of the production of high-quality monoclonal antibodies with hybridoma technology. Comparing to common ELISA, the accuracy of the chemiluminescent assay proposed in this work exhibited good performance with the matching rate reached 95%.

Fig. 3: Screening of specific hybridoma cells.

The P-RCAES based chemiluminescent screening possesses attractive advantages of economy, simplicity, acceptable accuracy and good expansibility without need of sophisticated instruments. This work provides opportunities for hybridoma cell screening and the development of cellular secretion detection, and can promote the production of immunotherapeutic agents against various diseases.

More details on this work can be found in our manuscript “Chemiluminescent screening of specific hybridoma cells via a proximity-rolling circle activated enzymatic switch” published in Communications Biology. Link: https://www.nature.com/articles/s42003-022-03283-2

References

1. Alian, B., Wurch, T., Bailly, C. & Corvaia, N. Strategies and challenges for the next generation of therapeutic antibodies. Nat. Rev. Immunol. 10, 345–352 (2010).
2. Hnasko, R. M. & Stanker, L. H. Hybridoma technology. In Methods in Molecular Biology: ELISA (eds. Hansko, R.) vol. 1318, pp. 15–28. (Humana Press, 2015).
3. Chan, J. C. Y. et al. A proprotein convertase subtilisin/kexin type 9 neutralizing antibody reduce serum cholesterol in mice and nonhuman primates. Proc. Natl. Acad. Sci. USA 106, 9820–9825 (2009).
4. Gérard, A. et al. High-throughput single-cell activity-based screening and sequencing of antibodies using droplet microfluidics. Nat. Biotechnol. 38, 715–721 (2020).

Short biography of Prof. Huangxian Ju

Huangxian Ju received his BS, MS and PhD degrees from Nanjing University during 1982–1992, and was a postdoc in Montreal University (Canada) in 1996–1997. He became an associate and full professor of Nanjing University in 1993 and 1999. He is currently the director of State Key Laboratory of Analytical Chemistry for Life Science, fellow of the International Electrochemical Society and fellow of the Royal Society of Chemistry. His research interests focus on analytical biochemistry, biosensing and molecular diagnosis. He has authored 75 patents (39 approved), 6 English books, 7 Chinese books and 20 chapters, and published 786 papers in different journals with h-index of 100 (Google Scholar h-index 109 with more than 44000 citations). He was awarded with a National Science Fund for Distinguished Young Scholars in 2003, Changjiang Scholar Professor by the Ministry of Education in 2007, the National Key Talents in The New Century and the chief scientist of the national 973 project in 2009, the special government allowance of the State Council in 2011, the first Leici Outstanding Achievement Award of Chinese Chemical Sensors in 2019, and 2022 Advances in Measurement Science Lectureship Awards of American Chemical Society, as well as 18 natural science or science and technology prizes from Ministry of Education or Province Government.