Repeat Chain of Protein G immunoglobulin Binding Domain. Domain III of the immunoglobulin binding domains of streptococcal protein G was used to construct tandem repeat (TR) chain. It is known that they bind to Fc and to Fab domain of the antibody. The domain III of the three Ig binding domains from protein G was used to construct up to 10 repeat (TR10) (Supplementary Fig.1) 5. As expected from its antibody binding characteristics, we found that TR10 could bind up to three Fab containing molecules. (Supplementary Fig.2). In this study, it is expected that increased structural flexibility between the binding domains and increased number of the binding domain in the repeat chain will increase the number of antibodies bound to the repeat chain. As an antibody molecule has flexible bending, free rotating Fab domain, we predicted that TR10 could bind three whole antibody molecules. The whole antibody has two Fab domains and one Fc domain, and it is possible for an antibody to bind two repeat chain simultaneously. This simultaneously binding results in cross-binding between multiple antibody-single repeat chain complex. The cross-binding between the complexes can produce supercomplexes, a large group of cross bound complexes.
Supercomplex. The antibody binding domain of protein G is known to bind to Fc and Fab domain of an antibody with different affinity 6. There are 10 antibody binding domains in TR10. It is possible to imagine that the cross-binding between complexes can make super-molecular supercomplex. We hypothesized that the supercomplex may have a snowball like gigantic congregate shape (Fig. 1b). To check our assumption for the supercomplex formation and antibody signal amplification by the TR10, we tested the effect of TR10 on immunological assays. We made the supercomplexes of TR10 and anti-β-actin monoclonal antibody (Pri. Ab). Three different molar ratios of antibody to TR10, 1:1/10, 1:1/5, and 1:1, were used to check the difference of the effect of supercomplex formation depending on the molar ratio. The supercomplex was freshly made simply by mixing TR10 and antibody and incubating at 37°C for an hour, and kept on ice until use.
Signal Amplification by Supercomplex; western blotting. Adding TR10 to the western blot assay increased signal more than 15-fold than the conventional way. Clear lysate of A431 human cancer cells and primary and secondary antibody (Santa Cruz, USA) were used. The supercomplex was made freshly each time and at the same concentration of the primary antibody, 1 in 1000 dilution. When we used conventional ECL reagent on nitrocellulose membrane, it was only possible to detect at very high concentration of the lysate. (Data not shown) We tested sensitive Femto luminescence reagent at low lysate concentration (Supplementary Fig.3). It was also tested whether the Supercomplexes give different signal amplification depending on their Ab:TR10 molar ratio. No strong dependency of the signal on molar ratio was observed within the tested.
We used PVDF membrane to compare with nitrocellulose membrane. On nitrocellulose membrane, we could see about 17-fold increase of signal (Fig.2a). With PVDF membrane, we probed first with conventional reagent ECL. We instantly washed the membrane with 1X TBST and the Super signal Femto luminescence reagent(Thermo scientific, USA) was added to get the second western signal. For the third analysis on the same PVDF membrane, the antibodies were stripped out from the membrane and it was reprobed with supercomplex (Ab:TR10=10:1) and ECL reagent (Fig.2b). The supercomplex amplified signal with ECL reagent was comparable to that with Femto reagent. However, huge background noise was observed with femto reagent due to nonspecific adsorption of secondary antibody on PVDF membrane and highly sensitive femto reagent. Supercomplex can increase the sensitivity of the conventional reagent on PVDF about 15-fold and it is similar to the increase observed on NC membrane. With the conventional ECL reagent, we could see high performance of PVDF membrane compared to NC membrane with Femto reagent. In the densitometry analysis, the correlation between the signal amplification and molar ratio was not clearly observed because the band intensity on the film got saturated. Because of the saturation of band intensity on film, there was inherent limit of precise measurement of signal in the densitometry analysis (Supplementary Fig.4).
ELISA; Quantitative analysis of signal amplification of the Supercomplex. The effect of supercomplex was tested on ELISA. We set up an indirect ELISA that detects β-actin coated on the 96-well plate. The spectroscopic measurement gave very accurate quantitative result (Fig.3a). With 1:60 dilution of primary antibody, the supercomplex was prepared at primary antibody to TR10 molar ratio of 1:1/10, 1:1/5 and 1:1, and gave 5.3, 13.3, and 21.9fold increase over the conventional method.
The binding of the secondary antibody is through primary antibody, and the intensity of detected signal is dependent on how many primary antibodies are bound. At the very high dilution (1:3000) of primary antibody, the increase of secondary antibody did not give differences (Supplementary Fig.5). With the dilution of primary antibody, the signal amplification decreased regardless of the molar ratio (Fig.3b). It seems that the amplification effect by supercomplex disappeared when the amount of primary antibody is far less than that of antigen (Fig.3b, 1:3840 dilution). The increase of A450 was higher at high primary antibody concentration at all molar ratio. The formation of supercomplex is dependent on the primary antibody concentration and the molar ratio of antibody and TR10. At the 1:30 dilution of primary antibody, the supercomplex formed at 1:1/10 molar ratio showed 10.2-fold increase, but the supercomplex at 1:1/5 molar ratio gave too large signal over the limit of ELISA reader (Supplementary Fig.6). With 1:120 dilution of primary antibody, the antigen was serially diluted to see the signals at low antigen concentration (Fig.3c). The addition of TR10 gave higher signal level at all tested antigen concentration than without TR10, and amplified signals depending on the antigen concentration showed large differences whereas the conventional method gave marginal differences. The signal increases of 1:1 molar ratio was highest at all antigen concentration and it gave the highest increase at high antigen concentration (Fig.3d). The average of the signal increase at molar ratio of 1:1/10, 1:1/5, and 1:1 are 3.983, 6.950, and 14.48 fold, respectively. We think that they are dependent on the number of primary antibody on the surface of supercomplex that is accessible to the secondary antibody. The formation of supercomplex is assumed to be dependent on the molar ratio, affinity between the antibody and TR10, cross-binding between complexes, steric hindrance and preparation condition. The patent has been applied.7