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

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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.

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