# Generation of Monoclonal Antibodies for Sensitive Detection of Pro-Inflammatory Protein S100A9
## 3 Results
### Identification of Variable Domain Sequences  
We cultured the five hybridoma clones and identified the subtypes of the IgG heavy (H) and light (L) chains by isotyping. We confirmed that the H chains of 5B3, 7C8, and 9D9 were IgG2b, and the L chains were kappa. Meanwhile, the H chains of 4C4 and 22A10 were IgG1, and the L chains were kappa. Next, we cultured three hybridoma clones: 5B3 and 9D9, which had high effective concentration (EC\({}_{50}\)) and low limit of detection (LOD) values, and 14C4, which had a low EC\({}_{50}\) and high LOD. The cDNA encoding the variable domains  
\begin{table}
\begin{tabular}{c c c c c c} \hline \hline  & **5B3** & **7C8** & **9D9** & **14C4** & **22A10** \\ \hline Absorbance (a.u.) & 2.9 \(\pm\) 0.1 & 2.9 \(\pm\) 0.5 & 3.1 \(\pm\) 0.2 & 3.0 \(\pm\) 0.1 & 3.2 \(\pm\) 0.5 \\ EC\({}_{50}\) (ng) & 28 \(\pm\) 1.4 & 60 \(\pm\) 15 & 30 \(\pm\) 2.3 & 75 \(\pm\) 1.5 & 61 \(\pm\) 12 \\ LOD (ng) & 5.6 & 17 & 4.7 & 20 & 1.5 \\ \hline \hline \end{tabular}
\end{table}
Table 1: Absorbance values of monoclonal antibodies with 300 ng of rhS100A9, EC\({}_{50}\) of the antibodies, and limit of detection (LOD) values of the assay.  
Figure 1: (**a**) SDS-PAGE analysis of Ni-NTA purified rhS100A9. T, S, FT, W, and E indicate total protein, soluble protein, flow through fraction, wash fraction, and elution fraction, respectively. (**b**) Schematic representation of the indirect ELISA for confirming the antibody-binding activity of purified rhS100A9. (**c**) ELISA titer with different concentrations of rhS100A9 or commercial hS100A9.  
of the heavy chain (VH) and light chain (VL) was isolated and sequenced using three-step 5'-RACE technology [14]. We prepared the mRNA of each hybridoma sample using RT-PCR with 5' phosphorylation gene-specific primers and performed cDNA circularization using T4 RNA ligase 1. Inverse PCR amplification was performed using inverted primers against a known constant region sequence. Finally, we cloned the PCR product into the T vector and obtained the VH and VL sequences (Figure 3 and Supplementary Table S1).  
Figure 2: (**a**) Schematic representation of the indirect ELISA for confirming the dose-dependent antigen binding efficiency of selected five monoclonal antibodies. (**b–f**) ELISA signals of monoclonal antibodies with various concentrations of hS100A9. The points represent absorbances at the indicated antigen concentrations. The lines indicate dose–response curves, which were fitted to a four-parameter equation, y = a + (b - a)/(1 + (c/x)’d), using GraphPad Prism software, where a, b, c, and d indicate ABS in the absence of antigen, ABS in the presence of 300 ng antigen, EC50, and hill slope, respectively. The EC50 values were estimated based on the parameter c in the formula. The LOD was obtained as the estimated antigen concentration that shows the mean blank value +3 SD. SD = standard deviation.  
sequence information is a good example of applied research and will be useful for the development of recombinant antibodies or antibody fragments.  
The following are available online at [[https://www.mdpi.com/article/10.3390/app11104659/s1](https://www.mdpi.com/article/10.3390/app11104659/s1)]([https://www.mdpi.com/article/10.3390/app11104659/s1](https://www.mdpi.com/article/10.3390/app11104659/s1)), Table S1: Sequences of VH and VL of anti-S100A9 monoclonal antibodies.  
Conceptualization, C.-S.L., Y.-G.K., B.J.K., H.-J.J., and B.-G.K.; methodology, E.-J.K. and G.-M.I.; writing--original draft preparation, E.-J.K., G.-M.I., and H.-J.J.; writing--review and editing, C.-S.L., Y.-G.K., B.J.K., and B.-G.K.; visualization, H.-J.J.; supervision, B.-G.K.; funding acquisition, C.-S.L., Y.-G.K., B.J.K., H.-J.J., and B.-G.K. All authors have read and agreed to the published version of the manuscript.  
Not applicable.  
We thank A-Frontier Co Ltd. for providing hybridoma-based monoclonal antibodies.  
The authors declare no conflict of interest.