Cancers, Vol. 14, Pages 5829: A Novel High-Throughput Screening Method for a Human Multicentric Osteosarcoma-Specific Antibody and Biomarker Using a Phage Display-Derived Monoclonal Antibody

Conceptualization, T.H., N.Y., H.Y. (Harumoto Yamada) and N.F.; methodology, N.Y., G.K., M.K., N.H., Y.K. (Yu Kato), M.T., H.Y. (Hisateru Yamaguchi) and Y.K. (Yoshikazu Kurosawa); software, H.Y. (Hisateru Yamaguchi); validation, N.Y. and N.F.; formal analysis, N.Y., H.Y. (Hisateru Yamaguchi) and N.F.; investigation, T.H., N.Y., G.K., K.T., M.K., N.H., Y.K. (Yu Kato), Y.K. (Yoshikazu Kurosawa) and H.Y. (Harumoto Yamada); resources, N.Y.; data curation, N.Y.; writing—original draft preparation, T.H., N.Y. and N.F.; writing—review and editing, T.H., N.Y., G.K., K.T., M.K., N.H., Y.K. (Yu Kato), M.T., H.Y. (Hisateru Yamaguchi), Y.K. (Yoshikazu Kurosawa), H.Y. (Harumoto Yamada) and N.F.; visualization, N.Y.; supervision, T.H., N.Y., Y.K. (Yoshikazu Kurosawa) and N.F.; project administration, N.Y. and N.F.; funding acquisition, N.F. All authors have read and agreed to the published version of the manuscript.

Figure 1. Measurement of fluorescence intensity of the 95 clones prepared by the fourth screening of the secondary human multicentric osteosarcoma (HMOS-P) using fluorescence probe-based enzyme-linked immunosorbent assay (ELISA). Among these, six clones showed a fluorescence intensity ≥ 10,000 arbitrary units (AU).

Figure 1. Measurement of fluorescence intensity of the 95 clones prepared by the fourth screening of the secondary human multicentric osteosarcoma (HMOS-P) using fluorescence probe-based enzyme-linked immunosorbent assay (ELISA). Among these, six clones showed a fluorescence intensity ≥ 10,000 arbitrary units (AU).

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Figure 2. Comparison of antibody reactivities toward various cell lines. The six clones were also incubated with HMOS-P, the primary human multicentric osteosarcoma (HMOS-A), MNNG-HOS, MG-63, and Saos2, and the fluorescence intensity was measured using ELISA. All antibodies showed stronger fluorescence intensity, indicating greater reactivity with HMOS-A than with HMOS-P. Antibody numbers 10 and 43 were positive for MNNG-HOS. In MG63 cells, only antigen number 10 was positive, whereas only antigen number 43 was positive in Saos2 cells.

Figure 2. Comparison of antibody reactivities toward various cell lines. The six clones were also incubated with HMOS-P, the primary human multicentric osteosarcoma (HMOS-A), MNNG-HOS, MG-63, and Saos2, and the fluorescence intensity was measured using ELISA. All antibodies showed stronger fluorescence intensity, indicating greater reactivity with HMOS-A than with HMOS-P. Antibody numbers 10 and 43 were positive for MNNG-HOS. In MG63 cells, only antigen number 10 was positive, whereas only antigen number 43 was positive in Saos2 cells.

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Figure 3. Correlation between ELISA-based and FCM fluorescence intensities at HMOS-P. FCM histograms of antibody numbers 10 (a), 12 (b), 17 (c), 19 (d), 43 (e), and 77 (f) incubated with HMOS-P. The FCM fluorescence intensity (GeoMean) for all cell lines was investigated. According to the results of flow cytometry of the HOS cell lines, most antibodies against antibody numbers 12, 17, 19, and 77 were negative (the values highlighted in blue were judged to be negative). In the HMOS cell line, most cells of antibody numbers 12, 43, and 77 were positive (g). The correlation between ELISA fluorescence intensity (median) and FCM fluorescence intensity (GeoMean) for all cell lines was strong. The correlation coefficient was r = 0.805 (h).

Figure 3. Correlation between ELISA-based and FCM fluorescence intensities at HMOS-P. FCM histograms of antibody numbers 10 (a), 12 (b), 17 (c), 19 (d), 43 (e), and 77 (f) incubated with HMOS-P. The FCM fluorescence intensity (GeoMean) for all cell lines was investigated. According to the results of flow cytometry of the HOS cell lines, most antibodies against antibody numbers 12, 17, 19, and 77 were negative (the values highlighted in blue were judged to be negative). In the HMOS cell line, most cells of antibody numbers 12, 43, and 77 were positive (g). The correlation between ELISA fluorescence intensity (median) and FCM fluorescence intensity (GeoMean) for all cell lines was strong. The correlation coefficient was r = 0.805 (h).

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Figure 4. Immunohistochemistry of cancer tissues. Staining of similar paraffin sections of HMOS (upper row) and HOS (lower row). The cell lines exhibited strong positive staining with antibody numbers 10, 12, 17, and 77. The staining was also positive for antibody numbers 19 and 43 but relatively weaker. Immunohistochemical staining shows positive cells (yellow arrowheads). These results were comparable to those of the ELISA fluorescence intensity. Scale bar, 50 μm.

Figure 4. Immunohistochemistry of cancer tissues. Staining of similar paraffin sections of HMOS (upper row) and HOS (lower row). The cell lines exhibited strong positive staining with antibody numbers 10, 12, 17, and 77. The staining was also positive for antibody numbers 19 and 43 but relatively weaker. Immunohistochemical staining shows positive cells (yellow arrowheads). These results were comparable to those of the ELISA fluorescence intensity. Scale bar, 50 μm.

Cancers 14 05829 g004 Figure 5. SDS-PAGE of the antigen–antibody complex and confirmation analysis with purified protein. Using immunoprecipitation through magnetic beads, the band incubated separately with the antibody was detected. Number 12 (a); M, marker; L, cell lysate solution; N, negative; IP, positive samples recovered by immunoprecipitation. Figure 5a is inverted in black and white and shows an enlarged view of more specific bands. This band (yellow arrowhead) was analyzed by Liquid chromatography-mass spectrometry (LC/MS) (b). The reactivity of commercially available CAVIN1/PTRF with antibody number 12 was confirmed. The protein had a stronger reactivity with the purified protein compared to that with the negative control, suggesting the occurrence of an antigen–antibody reaction (* pc). Figure 5. SDS-PAGE of the antigen–antibody complex and confirmation analysis with purified protein. Using immunoprecipitation through magnetic beads, the band incubated separately with the antibody was detected. Number 12 (a); M, marker; L, cell lysate solution; N, negative; IP, positive samples recovered by immunoprecipitation. Figure 5a is inverted in black and white and shows an enlarged view of more specific bands. This band (yellow arrowhead) was analyzed by Liquid chromatography-mass spectrometry (LC/MS) (b). The reactivity of commercially available CAVIN1/PTRF with antibody number 12 was confirmed. The protein had a stronger reactivity with the purified protein compared to that with the negative control, suggesting the occurrence of an antigen–antibody reaction (* pc). Cancers 14 05829 g005

Figure 6. Immunostaining, FCM, and Western blot (WB) as a validation experiment for CAVIN1/PTRF. Tissue immunostaining of HMOS and HOS (upper row, red/yellow arrowheads indicate positive cells) and FCM of HMOS-P and NOS-1 cells (lower row) using the commercial antibody for CAVIN1/PTRF. The WB analysis result showed a specific and strong band (red arrowhead) of CAVIN1/PTRF. A very weak band at approximately 43 kDa, as calculated molecular weight, was also detected (yellow arrowhead). M, marker; 1, cell lysate of HMOS-P solution; 2, cell lysate of HOS solution. Scale bar = 50 μm.

Figure 6. Immunostaining, FCM, and Western blot (WB) as a validation experiment for CAVIN1/PTRF. Tissue immunostaining of HMOS and HOS (upper row, red/yellow arrowheads indicate positive cells) and FCM of HMOS-P and NOS-1 cells (lower row) using the commercial antibody for CAVIN1/PTRF. The WB analysis result showed a specific and strong band (red arrowhead) of CAVIN1/PTRF. A very weak band at approximately 43 kDa, as calculated molecular weight, was also detected (yellow arrowhead). M, marker; 1, cell lysate of HMOS-P solution; 2, cell lysate of HOS solution. Scale bar = 50 μm.

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Table 1. Positivity rate of flow cytometry (FCM) for HMOS-A, HMOS-P, MNNG-HOS, MG63, and Saos2 cells.

Table 1. Positivity rate of flow cytometry (FCM) for HMOS-A, HMOS-P, MNNG-HOS, MG63, and Saos2 cells.

Cell LineNo. 10No. 12No. 17No. 19No. 43No. 77HMOS-A90.3 ± 1.188.9 ± 1.588.8 ± 1.576.1 ± 1.492.1 ± 1.092.9 ± 1.1HMOS-P77.5 ± 2.178.8 ± 2.377.1 ± 1.963.2 ± 2.277.8 ± 2.082.9 ± 1.7MNNG-HOS96.8 ± 0.93.4 ± 0.20.3 ± 0.10.2 ± 0.191.1 ± 1.445.4 ± 5.1MG6376.4 ± 1.51.0 ± 0.10.2 ± 0.10.2 ± 0.141.5 ± 6.324.7 ± 2.1Saos222.3 ± 1.90.6 ± 0.268.2 ± 3.60.7 ± 0.296.1 ± 0.714.4 ± 1.7

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