Application of In-Cell ELISA assay for hybridoma screening and selection of promising producers of monoclonal antibodies

I.I. Stepanova^*, K.A. Artemyeva^**, A.A. Stepanov^***, I.M. Bogdanova^****, E.A. Ponomarenko^*****, M.N. Boltovskaya^******

Avtsyn Research Institute of Human Morphology of FSBSI “Petrovsky National Research Center of Surgery”, Moscow, 119991 Russia

E-mail: ^*i-ste@yandex.ru, ^**artemjeva_ksenia@mail.ru, ^***9163407056@mail.ru,

​ ^****malaj43@mail.ru, ^*****maribolt@mail.ru, ^******ponomarenkoea75@mail.ru

Received May 27, 2022

ORIGINAL ARTICLE

Full text PDF

DOI: 10.26907/2542-064X.2022.4.535-550

For citation: Stepanova I.I., Artemyeva K.A., Stepanov A.A., Bogdanova I.M., Ponoma­renko E.A., Boltovskaya M.N. Application of In-Cell ELISA assay for hybridoma screening and selection of promising producers of monoclonal antibodies. Uchenye Zapiski Kazanskogo Universiteta. Seriya Estestvennye Nauki, 2022, vol. 164, no. 4, pp. 535–550. doi: 10.26907/2542-064X.2022.4.535-550. (In Russian)

Abstract

This study was motivated by the pressing need to obtain monoclonal antibodies (mAb) for research and medical purposes. Screening for mAb-producing hybridomas and evaluating various cell cultures requires a technique that combines the advantages of immunohistochemistry (IHC), heterogeneous enzyme immunoassay (ELISA), and Western blotting. Cellular ELISA (In-Cell ELISA, ICE) is valid for in situ measurement of target analytes in both attached and suspended cells with minimal time,  specificity, and

reproducibility of ELISA. This method can be successfully used to detect surface and intracellular antigens, as well as to assess phosphorylation, methylation, and acetylation. ICE is a simple, fast, inexpensive, and highly sensitive alternative to various common cyto- and histochemical methods. Here, we explored the potential of ICE for screening and selecting the most promising hybridomas and mAbs for IHC staining. The experiments were carried out with the help of the cultural method, the method of heterogeneous and cellular ELISA, and IHC staining.

Comparison of ICE and IHC showed a significant agreement in the intensity and localization of staining with the studied mAbs. The most suitable antibodies for subsequent IHC were selected, saving us much time of selection and the number of expensive mAbs. In heterogeneous ELISA, a negative reaction was noted in some cases. In ICE of whole cells, a pronounced response was detected microscopically. With ICE, it is possible to characterize cell cultures by markers that distinguish one cell line from another, show the stages of cell differentiation, and visualize their structures at different stages of cultivation. In some cases, ICE may be more informative than ELISA for selecting monoclones that produce specific mAbs. A preliminary ICE run can be used to determine the optimal concentration of working dilutions of primary antibodies in a subsequent IHC run.

Keywords: In-Cell ELISA, hybridoma, monoclonal antibodies, immunohistochemistry

Figure Captions

Fig. 1. Direct and indirect cellular ELISA for detection of cell surface antigens (а) and antibodies targeting the cell surface antigen (b), according to [16].

Fig. 2. Application of monoclonal antibody to Muc1 (IS32): a, b – moderate-to-strong ICE membrane and cytoplasmic staining, c, d – strong IHC staining; a – ZR-75-1, b – MCF-7, c, d – preparations of mammary adenocarcinoma. Scale bar, 100 μm.

Fig. 3. Application of monoclonal antibody to prostate-specific antigen (PS30): a, b – ICE cytoplasmic staining is strong (a) and moderate (b), c, d – IHC staining is strong (c) and moderate (d); a – ZR-75-1, b – MCF-7, c, d – preparations of mammary adenocarcinoma, ×200. Scale bar, 100 μm.

Fig. 4. Application of monoclonal antibody to carcinoembryonic antigen (PE274): a, b – ICE cytoplasmic staining is moderate (a) and weak (b), c, d – IHC cytoplasmic staining is moderate (c) and weak (d), a – ZR-75-1, b – MCF-7, c, d – preparations of mammary adenocarcinoma. Scale bar, 100 μm.

Fig. 5. Application of monoclonal antibodies to Muc1 (IS32) and сarcinoembryonic antigen (PE 274): a, b – ICE staining in Caco-2 culture, c, d – IHC staining of colon adenocarcinoma preparations; a, c – IS32, moderate-to-strong staining, b, d – PE274, moderate staining. Scale bar, 100 μm.

Fig. 6. Application of monoclonal antibody to immunoglobulin G (G27): a, b – ICE, no staining, c, d – IHC, no staining; a – ZR-75-1, b – MCF-7, c, d – preparations of mammary adenocarcinoma. Scale bar, 100 μm.

Fig. 7. Application of monoclonal antibody to immunoglobulin G (G27): a – ICE, no staining, Caco-2, b – IHC, no staining, colon adenocarcinoma preparation. Scale bar, 100 μm.

Fig. 8. Comparison of ELISA results with glioblastoma U373 cell lysate and ICE with whole cells:      a–e – intensity of staining in ICE, f – optical density units (OD) in ELISA, white arrow – OD ≤ 0.3, a – negative staining, dotted arrow – OD ≤ 0.3, b – weak staining, thin arrow – OD  0.525, c – combination of weak, moderate, and negative staining, bold arrow – OD 1.1, d – strong staining, arrow head – OD ≤. 0.3, e – strong staining. Scale bar, 100 μm.

References

1. Taylor C.R., Burns J. The demonstration of plasma cells and other immunoglobulin-containing cells in formalin-fixed, paraffin-embedded tissues using peroxidase-labelled antibody. J. Clin. Pathol., 1974, vol. 27, no. 1, pp. 14–20. doi: 10.1136/jcp.27.1.14.
2. Boveia V., Schutz-Geschwender A. Quantitative analysis of signal transduction with in-cell    western immunofluorescence assays. In: Kurien B., Scofield R. (Eds.) Detection of Blotted Proteins. Methods in Molecular Biology. Vol. 1314. New York, Humana Press, 2015, pp. 115–130. doi: 10.1007/978-1-4939-2718-0_13.
3. Chen H., Kovar J., Sissons S., Cox K., Matter W., Chadwell F., Luan P., Vlahos C.J., Schutz-Geschwender A., Olive D.M. A cell-based immunocytochemical assay for monitoring kinase signaling pathways and drug efficacy. Anal. Biochem., 2005, vol. 338, no. 1, pp. 136–142. doi: 10.1016/j.ab.2004.11.015.
4. Pandre M.K., Shaik Sh., Pratap V.V.V.S., Yadlapalli P., Yanamandra M., Mitra S. A novel in-cell ELISA method for screening of compounds inhibiting TRKA phosphorylation, using KM12 cell line harboring TRKA rearrangement. Anal. Biochem., 2018, vol. 545, pp. 78–83. doi: 10.1016/j.ab.2018.01.014.
5. Bishop G.A., Hwang J. Use of a cellular ELISA for the detection of cell surface antigens. Biotechniques, 1992, vol. 12, no. 3, pp. 326–330.
6. Moerke N.J., Hoffman G.R. Development of in-cell Western assays using far-red fluorophores. Curr. Protoc. Chem. Biol., 2011, vol. 3, no. 1, pp. 39–52. doi: 10.1002/9780470559277.ch100153.
7. Renukaradhya J.G., Sriram V., Polakova K., Russ G., Brutkiewicz R.R. Development of a quantitative cell-based intracellular ELISA for the screening of B cell hybridoma supernatants: A novel rapid assay to detect positive clones. Hybrid Hybridomics, 2004, vol. 23, no. 6, pp. 373–379. doi: 10.1089/hyb.2004.23.373.
8. Grunow R., D'Apuzzo M., Wyss-Coray T., Frutig K., Pichler W.J. A cell surface ELISA for the screening of monoclonal antibodies to antigens on viable cells in suspension. J. Immunol. Methods, 1994, vol. 171, no. 1, pp. 93–102. doi: 10.1016/0022-1759(94)90232-1.
9. Ogino T., Wang X., Ferrone S. Modified flow cytometry and cell-ELISA methodology to detect HLA class I antigen processing machinery components in cytoplasm and endoplasmic reticulum. J. Immunol. Methods, 2003, vol. 278, nos. 1–2, pp. 33–44. doi: 10.1016/s0022-1759(03)00224-2.
10. Kashyap R.S., Kainthla R.P., Satpute R.M., Agarwal N.P., Chandak N.H., Purohit H.J., Taori G.M., Daginawala H.F. Differential diagnosis of tuberculous meningitis from partially-treated pyogenic meningitis by cell ELISA. BMC Neurol., 2004, vol. 4, art. 16, pp. 1–6. doi: 10.1186/1471-2377-4-16.
11. Shan Z., Yamasaki T., Suzuki A., Hasebe R., Horiuchi M. Establishment of a simple cell-based ELISA for the direct detection of abnormal isoform of prion protein from prion-infected cells  without cell lysis and proteinase K treatment. Prion, 2016, vol. 10, no. 4, pp. 305–318. doi: 10.1080/19336896.2016.1189053.
12. Fuhrmann S., Kirsch M., Wewetzer K., Hofmann H.-D. Use of cell ELISA for the screening of neurotrophic activities on minor cell populations in retinal monolayer cultures. J. Neurosci. Methods, 1997, vol. 75, no. 2, pp. 199–205. doi: 10.1016/s0165-0270(97)00073-3.
13. Falahat R., Wiranowska M., Gallant N.D., Toomey R., Hill R., Alcantar N. A Cell ELISA for the quantification of MUC1 mucin (CD227) expressed by cancer cells of epithelial and neuroectodermal origin. Cell Immunol., 2015, vol. 298, nos. 1–2, pp. 96–103. doi: 10.1016/j.cellimm.2015.09.009.
14. Schöler L., Le-Trilling V.T.K., Eilbrecht M., Mennerich D., Anastasiou O.E., Krawczyk A., Herrmann A., Dittmer U., Trilling M. A novel In-Cell ELISA assay allows rapid and automated quantification of SARS-CoV-2 to analyze neutralizing antibodies and antiviral compounds. Front. Immunol., 2020, vol. 11, art. 573526, pp. 1–11. doi: 10.3389/fimmu.2020.573526.
15. Filardo S., Di Pietro M., Pasqualetti P., Manera M., Diaco F., Sessa R. In-cell western assay as a high-throughput approach for Chlamydia trachomatis quantification and susceptibility testing to antimicrobials. PLoS ONE, 2021, vol. 6, no. 5, art. e0251075, pp. 1–13. doi: 10.1371/journal.pone.0251075.
16. Kohl T.O., Ascoli C.A. Direct and indirect cell-based enzyme-linked immunosorbent assay. Cold Spring Harbor Protoc., 2017, vol. 2017, no. 5. doi: 10.1101/pdb.prot093732.
17. Kudel'kina V.V., Khalanskii A.S., Makarova O.V., Tsvetkov I.S., Kosyreva A.M., Alekseeva A.I., Shelkov A.Yu., Maksimenko O.О., Razzhivina V.A., Gel'perina S.E. Comparative morphological and biochemical characteristics of the toxic effects of doxorubicin and nanosomal PLGA-doxorubicin form in the experimental glioblastoma treatment. Klin. Eksp. Morfol., 2021, vol. 10, no. 1, pp. 58–65. doi: 10.31088/CEM2021.10.1.58-65. (In Russian)
18. Crowther J.R. ELISA. Theory and practice. Methods Mol. Biol., 1995, vol. 42, pp. 1–218. doi: 10.1385/0-89603-279-5:1.
19. Lourenço E.V., Roque-Barreira M.-C. Immunoenzymatic quantitative analysis of antigens expressed on the cell surface (cell-ELISA). Methods Mol. Biol., 2010, vol. 588, pp. 301–309. doi: 10.1007/978-1-59745-324-0_29.
20. Wang X., Campoli M., Cho H.S., Ogino T., Bandoh N., Shen J., Hur S.Y., Kageshita T., Ferrone S. A method to generate antigen-specific mAb capable of staining formalin-fixed, paraffin-embedded tissue sections. J. Immunol. Methods, 2005, vol. 299, nos. 1–2, pp. 139–151. doi: 10.1016/j.jim.2005.02.006.

The content is available under the license Creative Commons Attribution 4.0 License.