1. OBJECTIVE CONCLUSIONS ABSTRACT Establishment of ELISA for AgI/II protein detection using monoclonal anti-AgI/II antibody Jae-Gon Kim, jeong-Yeol Park, Yeon-Mi Yang, Byeong-Ju Baik School of Dentistry, Chonbuk National University, JeonJu, Korea MATERIALS AND METHODS RESULTS Bacterial strains and culture conditions Serotype C S. mutans GS-5 was grown in BHI medium at 37 o C in an anaerobic chamber for 24 h then transferred into 5 ml of a fresh BHI broth. The optical density was measured at 540nm.. Streptococcus mutans has been known as a causative agent for dental caries. As well as acid production yielding the deminerali- zation of tooth enamel, adherence and colonization of S. mutans to the teeth are also important for their virulence. S. mutans attaches the dental pellicle by an adhesion from surface protein called as Antigen I/II. A secretory IgA for intact AgI/II or its saliva- ry-protein-binding segment has been found to block adherence of S. mutans. Therefore, it will be of most importance to detect the level of AgI/II molecules in saliva regarding dental caries and other systemic disease. Through the expression pattern of AgI/II of S. mutans GS-5, we suggest that AgI/II of S. mutans GS-5 file up in the culture media time dependently. We tested the reaction of between recombinant AgI/II-N, or native AgI/II and monoclonal anti-AgI/II antibody by ELISA. As a result, our monoclonal anti- AgI/II antibody can detect 1ng recombinant AgI/II-N and predict amount of the native AgI/II. In conclusion, through ELISA against AgI/II using our monoclonal anti-AgI/II antibody, we can detect the AgI/II in native condition. Also, we thought that analysis of secretory AgI/II can be utilized for prediction of disease associated S. mutans. The aim of this study was to evaluate the establishment of secretory AgI/II by ELISA using monoclonal anti-AgI/II antibody for prediction of dental caries. For the purpose, the expression pattern of AgI/II protein during the growth of S. mutans GS-5 was investigated, the optimal condition for AgI/II detection was searched using recombinant AgI/II protein and this ELISA condition was tested whether AgI/II protein in the supernatant of the bacterial culture was detected. We confirmed the presence of native AgI/II piling up in culture media. We established ELISA condition against recombinant AgI/II and native AgI/II We detected the 1 ng of recombinant AgI/II at 5 min. We detected native AgI/II in culture media containing others. Generation of monoclonal anti-AgI/II antibodies ELISA The 96 well plate were coated with 1, 10, 50, or 100 ng recombi- nant AgI/II-N, culture supernatant with or without concentrated by 50% ammonium sulfate at 4 o C, and blocked with 1% skim milk. Monoclonal anti-AgI/II-N antibody (1:100, 1:1000, 1:10000, 1:50000) was added in duplicate. After washing with PBS, AgI/II-N-specific antibody was detected using anti-mouse IgG conjugated alkaline phosphatase, and the absorbance was measured at 405nm. Expression and Purification of recombinant AgI/II Fig 1. SDS-PAGE analysis of S. mutans GS-5 culture supernatant. S. mutnas GS-5 culture supernatant (20ul) were collected at various time points during bacterial growth and separated in 8% SDS-PAGE gel. Fig 2. Western blot analysis of S. mutans GS-5 culture supernatant. S. mutans GS-5 culture supernatants (20ul) were collected at various time points of the bacterial growth and analyzed in Western blot using monoclonal anti-AgI/II antibodies. Fig 3. Time dependent ELISA against 1ng of recombinant AgI/II. Monoclonal anti-AgI/II antibody 1:100 (closed circle), 1:1000 (open circle), 1:10000 (closed triangle), and 1:50000 (open triangle) dilution was used to detect the 1 ng recombinant AgI/II protein. The results represent the mean values ± S.E.M. from two separate experiments. Mean antibody titers are given as –log 2 dilution. Fig 4. Time dependent ELISA against 10ng of recombinant AgI/II. Monoclonal anti-AgI/II antibody 1:100 (closed circle), 1:1000 (open circle), 1:10000 (closed triangle), and 1:50000 (open triangle) dilution was used to detect the 1 ng recombinant AgI/II protein. The results represent the mean values ± S.E.M. from two separate experiments. Mean antibody titers are given as –log 2 dilution. Fig 5. Time dependent ELISA against 50ng of recombinant AgI/II. Monoclonal anti-AgI/II antibody 1:100 (closed circle), 1:1000 (open circle), 1:10000 (closed triangle), and 1:50000 (open triangle) dilution was used to detect the 1 ng recombinant AgI/II protein. The results represent the mean values ± S.E.M. from two separate experiments. Mean antibody titers are given as –log 2 dilution. Fig 6. Time dependent ELISA against 100ng of recombinant AgI/II. Monoclonal anti-AgI/II antibody 1:100 (closed circle), 1:1000 (open circle), 1:10000 (closed triangle), and 1:50000 (open triangle) dilution was used to detect the 1 ng recombinant AgI/II protein. The results represent the mean values ± S.E.M. from two separate experiments. Mean antibody titers are given as –log 2 dilution. Fig 7. Comparison of ELISA titer by 1:1000 dilution of monoclonal anti AgI/II antibody. Titer of recombinant AgI/II 1, 10, 50, and 100 ng was compared with time dependently color density by monoclonal anti AgI/II antibody (1:1000). Fig 9 Time dependent ELISA against concentrated protein of culture supernatant. Concentrated protein S. mutans GS-5 culture supernatant (100 ng) was detected using monoclonal anti-AgI/II antibody 1:100 (circle) and 1:1000 (triangle) dilution. Fig 8. Comparison of ELISA titer by 1:100 dilution of monoclonal anti AgI/II antibody. Titer of recombinant AgI/II 1, 10, 50, and 100 ng was compared with time dependently color density by monoclonal anti AgI/II antibody (1:100). To obtain recombinant AgI/II-N protein, induced its expression using isopropyl-  -D-thiogalactopyranoside(IPTG) and purified the protein with nickel chelated agarose column. To quantify purified AgI/II-N, carried out SDS-PAGE and transferred it to nitrocellulose paper for western blot analysis using monoclonal anti-AgI/II-N. Figure 10. Comparison of ELISA titer between concentrated protein and culture supernatant. Titer of concentrated protein of S. mutans GS-5 culture supernatant, 50 ng and 100 ng, and culture supernatant of S. mutans GS-5, 100 ul was compared by 1: 100 dilution of monoclonal anti AgI/II antibody.