HABA biotin quantitation assay

The assay was performed followed by a commercial product protocol.

The HABA\avidin reagent was reconstituted by 100 μL DI water and mixed

HABA\avidin\sample. The absorbance of HABA\avidin\sample was measured at 500 nm (PBS-2 served as reference cell) and recorded as A500

HABA\avidin\sample. The mathematical equations described in section 5.2.3 were used to calculate the amount of biotin per mol of sample protein.⁶² [Chu, Y. W.; Wang, B. Y.; Lin, H.-S.; Lin, T.-Y.; Hung, Y.-J.; Engebretson, D. A.;

Lee, W.; Carey, J. R. Chem. Commun. 2013, in press, DOI:

10.1039/c2cc38233d] - Reproduced by permission of The Royal Society of Chemistry.

4.3.6 Measurement of color value

To investigate the SBPN method, we employ the measurement of color value from the image results using a desktop scanner. The color values (average of red, green, and blue values), tabulated in Tables 9, 11, 13, and 15, were estimated using ImageJ software. Using this software, the color values were selected based on a digital circular zone that was the size of the well.

The smaller the color values measured, the darker the spots and thus the stronger the signal.

The desk scanner suffers an inherent error caused by shadows during the scanning process. In order to minimize the scanning error, we measured the baseline color values from a BSA blocked well that contained biotinylated antibody and TMB. The results of each experiment were subtracted from the baseline color values to obtain relative color values. The relative color values are tabulated in Tables 10, 12, 14, and 16. The relative color values represent

the change of color values; the larger the relative color values the darker the spots.

For each experiment, the average of relative color values were calculated, called data(avg), and were plotted with error bars using one standard deviation (Figures 48, 54, 57, and 60). In addition, for each experiment, three background wells were performed. The average of the relative color values for the background experiments were calculated and are called background(avg). Finally, the background(avg) was subtracted from the data(avg) to give the normalized relative color values for each experiment.

The normalized relative color values for each experiment are plotted in Figures 49, 55, 58, and 61.⁶² [Chu, Y. W.; Wang, B. Y.; Lin, H.-S.; Lin, T.-Y.;

Hung, Y.-J.; Engebretson, D. A.; Lee, W.; Carey, J. R. Chem. Commun. 2013, in press, DOI: 10.1039/c2cc38233d] - Reproduced by permission of The

Royal Society of Chemistry.

4.4 Verification of the antigen binding ability of biotinylated capture antibody

PBS-7 and BSA-1 were used in the experiment. The plates were coated with IL-4 and IL-7 capture antibody by filling with 100 μL/well IL-4 capture antibody (0.5 μg/mL in PBS-7) and IL-7 capture antibody (5.0 μg/mL in

PBS-6. Plates were placed on the bench for 24 h after emptied by dumping solution and tapping on absorbent paper.

IL-4 and IL-7 were performed in different procedures due to the difference of the commercial ELISA protocol. For IL-4 based experiment, IL-4 antigens were reconstituted and diluted in various concentrations by PBS-10. 100 μL/well of IL-4 antigen were added into IL-4 plate at rt and incubated for 2 h. 50 μL/well of IL-4 detection antibody (1.0 μg/mL in PBS-10) and 50 μL/well of IL-4 CapA-PEG4-biotin (2.0 μg/mL in PBS-10) were introduced after washed 3 times with 400 μL/well of PBS-6. The plates were incubated at rt for 1 hour.

The microtiter plates were filled with 100 μL/well streptavidin-HRP (1.5×10^-4 mg/mL in PBS-5) and incubated at rt for 20 min after washed 3 times with 400 μL/well of PBS-6. The unreacted streptavidin-HRP was removed by washed 3 times with 400 μL/well of PBS-6. 100 μL/well ready-to-use TMB reagents were added in microtiter plates with dark surrounding and then incubated for 15 min in the dark. The absorbance was measured at 450 nm using plate reader after filling the microtiter plates with 100 μL/well 1 M H2SO4.

For IL-7 based experiment, IL-7 antigens were reconstituted and diluted in various concentrations by PBS-10. 50 μL/well of IL-7 detection antibody (1.2 μg/mL in PBS-10) and IL-7 CapA-PEG4-biotin (10.0 μg/mL in PBS-10)

were incubated with 100 μL/well of IL-7 antigen at rt for 2 h, respectively.

The microtiter plates were filled with 100 μL/well streptavidin-HRP (1.5×10^-4 mg/mL in PBS-5) and incubated at rt for 20 min after washed with 400 μL/well of PBS-6 3 times. The unreacted streptavidin-HRP was removed by washed 3 times with 400 μL/well of PBS-6. 100 μL/well ready-to-use TMB reagents were added in microtiter plates with dark surrounding and then incubated for 15 min in the dark. The absorbance was measured at 450 nm using plate reader after filling the microtiter plates with 100 μL/well 1 M H2SO4.

4.5 Verification of the requirement of heterogeneous antibodies in sandwich ELISA

IL-7 ELISA kit, PBS-7, and BSA-1 were used in the experiment. The microtiter plates were coated with 100 μL/well of IL-7 capture antibody (5.0 μg/mL in PBS-7). The plates were put at 4 °C refrigerator overnight. The plates were blocked with by adding 250 μL/well of blocking solution (PBS-7 containing 5% BSA-1) at rt for 2 h after washed twice with 400 μL/well of PBS-6. Plates were placed on the bench for 24 h after emptied by dumping solution and tapping on absorbent paper.

In order to compare the difference between IL-7 capture antibody and

were incubated together with 50 μL/well of IL-7 detection antibody (1.2 μg/mL in PBS-10), IL-7 capture antibody (1.2 μg/mL in PBS-10), or PBS-10 at rt for 2 h.

The lanes in the microtiter plate were separated in two scaffolds after washed 3 times with 400 μL/well of PBS-6 because the number of steps was different among the conditions. One plate was added another desired reagents which were 50 μL/well of IL-7 detection antibody (1.2 μg/mL in PBS-10), IL-7 capture antibody (1.2 μg/mL in PBS-10), or PBS-10 at rt for 2 h. On the other hand, another plate was treated with the rest staining process.

The microtiter plates were filled with 100 μL/well of streptavidin-HRP (1.5×10^-4 mg/mL in PBS-5) and incubated at rt for 20 min after washed 3 times with 400 μL/well of PBS-6. The unreacted streptavidin-HRP was removed after washed 3 times with 400 μL/well of PBS-6. 100 μL/well TMB reagents were added in microtiter plates with dark surrounding and then incubated 15 min in the dark. The absorbance was measured at 450 nm using plate reader after filling the microtiter plates with 100 μL/well of 1 M H2SO4.

4.6 Verification of attachment of detection antibodies, streptavidin, and biotinylated BSA

PBS-8 and BSA-2 were used in the experiment. The microtiter plates were coated with IL-7 detection antibody and streptavidin by adding 100 μL/well IL-7 detection antibody (0.3 μg/mL in PBS), streptavidin (5.0 μg/mL

in PBS). The plates were incubated at 4 °C refrigerator overnight. The plates were blocked with by adding 250 μL/well of blocking solution (PBS-8 containing 5% BSA-2) at rt for 2 h after washed 3 times with 400 μL/well of PBS-6. Plates were placed on the bench for 24 h after emptied by dumping solution and tapping on absorbent paper.

The experiments were performed in two separated microtiter plates (plates A and B) since the total experimental steps were different. 100 μL/well streptavidin (10.0 μg/mL in PBS-5), PBS-5 and IL-7 detection antibody (0.6 μg/mL in PBS-5) was added to plate A. The plates were incubated with 160 rpm at rt for 1 h. 100 μL/well streptavidin (10.0 μg/mL in PBS-5) and PBS-5 was added to plate B. The plates were incubated by shook at 160 rpm at rt for 1 h. After washed 3 times with 400 μL/well of PBS-6, 100 μL/well streptavidin (10.0 μg/mL in PBS-5), PBS-5, and BSA-PEG4-biotin (6.2 μg/mL in PBS-5) was added. The plates were incubated with 160 rpm at rt for 1 h.

After washed 3 times with 400 μL/well of PBS-6, the microtiter plates were filled with 100 μL/well of streptavidin-HRP (1.5×10^-4 mg/mL in PBS-5) and biotinylated HRP (1.5×10^-4 mg/mL in PBS-5). The microtiter plates were incubated at rt for 30 min with 160 rpm. The unreacted streptavidin-HRP was

scanner (Epson Perfection V33, Epson).

4.7 Comparison of the various cycles of the SBPN method (0-10 cycles)

In order to determine the number of cycles for the SBPN method that provides the largest amplification, we compared several SBPN cycles that were performed on a biotinylated antibody coated microtiter plate. First, we compared 0, 1, 3, 5, 7, and 10 SBPN cycles. The SBPN method with various cycles were performed side by side and in triplicate using 3 different technicians. We refer to the procedure that directly adds S-HRP to the biotinylated antibody coated microtiter plate as the direct linked amplification (DLA) method which is equal to the 0 SBPN cycle.

The experiments were performed as follows. Streptavidin and BSA-PEG4-biotin were diluted with PBS-5. The SBPN method was initiated by the addition of 100 μL/well streptavidin (7.5 μg/mL in PBS-5). The solution was emptied out immediately after the addition and the plates were washed 3 times with PBS-6 (400 μL/well). The addition of 100 μL/well BSA-PEG4-biotin (1.5 μg/mL in PBS-5) was introduced and the first SBPN cycle was completed after the plates were washed 3 times with PBS-6 (400 μL/well).

The various SBPN cycles were performed by following the same steps described above. S-HRP was introduced by addition of 100 μL/well S-HRP

(1.5×10^-4 mg/mL in PBS-5) after each specific SBPN cycle was completed.

The plates were incubated on a 160 rpm shaker at 30 °C for 30 min. The plates were washed 3 times with PBS-6 (400 μL/well), followed by the addition of 100 μL/well ready-to-use TMB. The plates were placed in the dark at rt for 15 min and images of each experiment were recorded using a desktop scanner. The color values (average of red, green, and blue values) were estimated using ImageJ software. Using this software, the color values were selected based on a digital circular zone that was the size of the well method described in section 4.3.6).⁶² [Chu, Y. W.; Wang, B. Y.; Lin, H.-S.; Lin, T.-Y.; Hung, Y.-J.; Engebretson, D. A.; Lee, W.; Carey, J. R. Chem. Commun.

2013, in press, DOI: 10.1039/c2cc38233d] - Reproduced by permission of The Royal Society of Chemistry.

4.8 Comparison of the various cycles of the SBPN (5-30 cycles) method

In order to determine the optimal number of cycles for the SBPN method, we increased the number of SBPN cycles to 30 cycles. Studies were run in triplicate, and were performed using three technicians for each specific SBPN cycle. Each operator performed the DLA method as a positive control to ensure that the biotinylated antibody was coated on the microtiter plates and

Lee, W.; Carey, J. R. Chem. Commun. 2013, in press, DOI:

10.1039/c2cc38233d] - Reproduced by permission of The Royal Society of Chemistry.

4.9 Comparison of the ABC, DLA, and SBPN (20 cycles) methods performed on a biotinylated antibody coated surface

Figure 28 graphically displays the procedural differences of the ABC, DLA, and SBPN (20 cycles) amplification methods. The microtiter plates were coated with various concentrations of biotinylated antibodies by the addition of 100 μL/well of biotinylated antibody at various concentrations (0 to 3000 pg/mL in PBS-1). Controls were generated by the addition of 100 μL/well PBS-1 to wells without antibody. After coating specific wells with antibody, the plates were incubated at 4 °C overnight. After incubation, the plates were washed twice with PBS-6 (400 μL/well) to remove unbound biotinylated antibodies. The plates were then blocked at 30 °C by the addition of 250 μL/well blocking solution (PBS-1 containing 5% BSA) and incubated on a 160 rpm shaker for 2 h. After being emptied by dumping the solution and tapping the plates upside down on an absorbent paper, the plates were placed on the bench at rt for 24 h.

Figure 28. Experimental procedure for the ABC, DLA, and SBPN methods.

(a) ABC method: The avidin-biotin-peroxidase complex (ABC) was prepared 30 min before direct addition to the biotinylated antibody coated surface. (b) DLA method: S-HRP is directly added to the biotinylated antibody coated surface. (c) SBPN method: Streptavidins and biotinylated proteins were added in a layer by layer fashion to complete 20 SBPN cycles followed by using S-HRP as the SGM. [Chu, Y. W.; Wang, B. Y.; Lin, H.-S.; Lin, T.-Y.;

Hung, Y.-J.; Engebretson, D. A.; Lee, W.; Carey, J. R. Chem. Commun. 2013, in press, DOI: 10.1039/c2cc38233d] - Reproduced by permission of The

When performing the SBPN method, streptavidin and BSA-PEG4-biotin were diluted to 7.5 μg/mL and 1.5 μg/mL with PBS-5, respectively. The SBPN method was initiated by the addition of 100 μL/well streptavidin (7.5 μg/mL in PBS-5). The solution was emptied out immediately after the addition and the plates were washed 3 times with PBS-6 (400 μL/well). The addition of 100 μL/well BSA-PEG4-biotin (1.5 μg/mL in PBS-5), followed by washing 3 times with PBS-6 (400 μL/well) completes the first SBPN cycle.

20 SBPN cycles were performed by following the same steps described above.

S-HRP was introduced by the addition of 100 μL/well S-HRP (1.5×10^-4 mg/mL in PBS-5) after the 20 SBPN cycles were completed. The plates were incubated on a 160 rpm shaker at 30 °C for 30 min. The plates were washed 3 times with PBS-6 (400 μL/well), followed by the addition of 100 μL/well ready-to-use TMB. The plates were placed in the dark at rt for 15 min and images of the microtiter wells were recorded using a desktop scanner.

The ABC method (Pierce, Rockford, IL) was performed according to the product protocol sheet. Briefly, 1 drop of avidin and 1 drop of biotinylated HRP were mixed together in 5 mL of PBS-3. The mixture was incubated at rt for 30 min. The biotinylated antibody coated plates were washed 3 times with PBS-6 (400 μL/well) before the addition of 100 μL/well of the ABC complex.

The plates were incubated at 30 °C for 30 min while shaking at 160 rpm. 100 μL/well of ready-to-use TMB was added to the wells after washing each well 3 times with PBS-6 (400 μL/well). The plates were placed in the dark at rt for

15 min and scanned as above.

For the DLA method, the biotinylated antibody coated plates were washed 3 times with PBS-6 (400 μL/well) before the addition of 100 μL/well S-HRP (1.5×10^-4 mg/mL in PBS-5). The plates were incubated at 160 rpm at 30 °C for 30 min. 100 μL/well of ready-to-use TMB was added to the wells after washing each well 3 times with PBS-6 (400 μL/well). The plates were placed in the dark at rt for 15 min and imaged as above.⁶² [Chu, Y. W.; Wang, B. Y.; Lin, H.-S.; Lin, T.-Y.; Hung, Y.-J.; Engebretson, D. A.; Lee, W.; Carey, J. R. Chem. Commun. 2013, in press, DOI: 10.1039/c2cc38233d] - Reproduced by permission of The Royal Society of Chemistry.

4.10 Comparison of the ABC, DLA, and SBPN (5 cycles) methods performed on a biotinylated antibody coated surface

In order to verify that the SBPN amplification is not reagent source dependent, we purchased our reagents from different sources and made comparisons. The following reagent changes were made for experiments described in this section. PBS-7 (product number UR-PBS001, UniRegion Bio-Tech, Taiwan) was used instead of PBS-1 (homemade). PBS-5 and PBS-6 were prepared from PBS-7 instead of using PBS-1. Streptavidin was

number 32050). Detailed reagent information is described in Materials section. For these comparison tests, 5 SBPN cycles were performed in order to reduce the reagents cost and time. The operation time of 5 SBPN cycles is approximately the same time that is needed to perform the ABC method. Each experimental condition was performed side by side in three separate wells at the same time and all experiments were performed by 4 different people on 4 different days.

Microtiter plates were coated with 100 μL/well of biotinylated antibody at various concentrations (0 to 3000 pg/mL in PBS-7). Controls were generated by the addition of 100 μL/well PBS-7 to wells without antibody.

After coating, the plates were incubated at 4 °C overnight. Plates were washed twice with PBS-6 (400 μL/well) to remove unbound biotinylated antibodies. Plates were blocked at 30 °C by the addition of 250 μL/well blocking solution (PBS-7 containing 5% BSA) and incubated on a 160 rpm shaker for 2 h. After being emptied by dumping the solution and tapping the plate upside down on an absorbent paper, the plates were placed on the bench at rt for 24 h.

When performing the SBPN method, streptavidin and BSA-PEG4-biotin were diluted with PBS-5. The SBPN method was initiated by the addition of 100 μL/well streptavidin (7.5 μg/mL in PBS-5). The solution was emptied out immediately after the addition and the plates were washed 3 times with PBS-6 (400 μL/well). 100 μL/well BSA-PEG4-biotin (1.5 μg/mL in PBS-5)

was introduced and the first SBPN cycle was completed after the plates were washed 3 times with PBS-6 (400 μL/well). 5 SBPN cycles were performed by following the same steps described above. S-HRP was introduced by addition of 100 μL/well S-HRP (1.5×10^-4 mg/mL in PBS-5) after 5 SBPN cycles were completed. The plates were incubated on a 160 rpm shaker at 30 °C for 30 min. The plates were washed 3 times with PBS-6 (400 μL/well), followed by the addition of 100 μL/well ready-to-use TMB. The plates were placed in the dark at rt for 15 min and images of the experiments were recorded using a desktop scanner.

The ABC method was performed according to the protocol sheet supplied with the ABC kit. Briefly, 1 drop of avidin and 1 drop biotinylated HRP were added together in 2.5 mL PBS-4 containing 0.05% Tween 20. The mixture was incubated at rt for 30 min. The biotinylated antibody coated plates were washed 3 times with PBS-6 (400 μL/well) before the addition of 100 μL/well of each ABC reagents. The plates were incubated at 30 °C for 30 min while shaking at 160 rpm. 100 μL/well of ready-to-use TMB was added to the wells after washing each well 3 times with PBS-6 (400 μL/well). The plates were placed in the dark at rt for 15 min and imaged as above.

For the DLA method, the biotinylated antibody coated plates were

after washing each well 3 times with PBS-6 (400 μL/well). The plates were placed in the dark at rt for 15 min and imaged as above.⁶² [Chu, Y. W.; Wang, B. Y.; Lin, H.-S.; Lin, T.-Y.; Hung, Y.-J.; Engebretson, D. A.; Lee, W.; Carey, J. R. Chem. Commun. 2013, in press, DOI: 10.1039/c2cc38233d] - Reproduced by permission of The Royal Society of Chemistry.

4.11 Verification of attachment of streptavidin, biotinylated BSA, and streptavidin coated microspheres

PBS-8 and BSA-2 were used in the experiment. The microtiter plates were coated with BSA-PEG4-biotin, streptavidin, and BSA by adding 100 μL/well BSA-PEG4-biotin (3.1 μg/mL in PBS), streptavidin (5.0 μg/mL in PBS), and BSA (PBS containing 1% BSA), respectively. The plates were incubated at rt for 2 h and SμS was washed 3 times during the incubation.

After 3 washes with PBS-6, the microtiter plates were added 100 μL/well streptavidin (5.0 μg/mL in PBS-5), PBS-5, and SμS (0.03% in PBS-5). The plates were incubated by shacked with 160 rpm at rt for 1 h. After washing each well 3 times with PBS-6 (400 μL/well), the microtiter plated were added 100 μL/well streptavidin-HRP (1.5×10^-4 mg/mL in PBS-5) and biotinylated HRP (1.5×10^-4 mg/mL in PBS-5). The plates were incubated with 160 rpm at rt for 30 min. After washing each well 3 times with PBS-6 (400 μL/well), the microtiter plated were added 100 μL/well ready-to-use TMB in the dark place.

The plates were placed in the dark for 15 min and results were recorded by scanner.

4.12 Visual detection of streptavidin coated microspheres using the SBPN method

PBS-8 and BSA-2 were used in the experiment. The microtiter plate was coated with BSA-PEG4-biotin. A PBS blank control was created by adding 100 μL/well BSA-PEG4-biotin (3.1 μg/mL in PBS) in PBS. The microtiter plate was incubated 4 °C overnight. Washing each well 3 times with PBS-6 (400 μL/well) before the blocking step. The plate was blocked with 250 μL/well of blocking solution (PBS-8 containing 5 % BSA-2) at rt for 2 h with 160 rpm. Plates were placed on the bench for 1 h after emptying the solution and tapping the plate upside down on the absorbent paper.

The microtiter plate was added 100 μL/well PBS-5 and SμS (0.03% in PBS-5) and incubated at rt for 1 h in 160 rpm shacking. After washing each well 3 times with PBS-6 (400 μL/well), the microtiter plates were filled with 100 μL/well streptavidin-HRP (1.5×10^-4 mg/mL in PBS-5) and biotinylated HRP (7.5×10^-4 mg/mL in PBS-5). The microtiter plates were incubated at rt for 30 min with 160 rpm shacking. The unreacted streptavidin-HRP was removed after washing each well 3 times with PBS-6 (400 μL/well). 100 μL/well ready-to-use TMB reagents were added in microtiter plates with dark surrounding and incubated 15 min in the dark. The results were recorded by a

4.13 Streptavidin coated microsphere bind to biotinylated BSA which was gradient coated on the microtiter surface

PBS-8 and BSA-2 were used in the experiment. The microtiter plates were coated with BSA-PEG4-biotin and PBS blank control by adding 100 μL/well BSA-PEG4-biotin and PBS-8. The sequential concentration BSA-PEG4-biotin was used (staring from 0.6 μg/mL). The microtiter plate was incubated in 4 °C refrigerator overnight. The plate was washed 3 times with PBS-6 (400 μL/well) before blocking step. The plate was blocked with 250 μL/well of blocking solution (PBS-8 containing 5 % BSA-2) at rt for 2 h with 160 rpm. Plates were placed on the bench for 1 h after emptying the solution and tapping the plate upside down on the absorbent paper.

The microtiter plate was added 100 μL/well streptavidin-HRP (1.5×10^-4 mg/mL in PBS-5) and SμS (0.03% in PBS-5). The plate was incubated at rt for 1 hour in 160 rpm shacking. The unreacted reagents were removed after washing each well 3 times with PBS-6 (400 μL/well). 100 μL/well ready-to-use TMB reagents were added in streptavidin-HRP binding parts of microtiter plates with dark surrounding and incubated 15 min in the dark. The results were recorded by scanner. The experiment related to SμS was observed by optical microscopy and recorded by digital camera.

4.14 Synthesis of IL-7 capture antibody-PEG

-biotin

4.14.1 Synthetic method of IL-7 capture antibody-PEG

-biotin

Buffer of 100 μL IL-7 capture antibody was changed to PBS-2 using

centrifuge filter (MWCO 50 kDa). 1 vials of NHS-PEG4-biotin was reconstituted by adding 170 μL DI water when it got equilibrium with rt. All the amount of IL-7 capture antibody was mixed with prepared 170 μL NHS-PEG4-biotin (20 mM in DI water). The reaction mixture was placed in 4

centrifuge filter (MWCO 50 kDa). 1 vials of NHS-PEG4-biotin was reconstituted by adding 170 μL DI water when it got equilibrium with rt. All the amount of IL-7 capture antibody was mixed with prepared 170 μL NHS-PEG4-biotin (20 mM in DI water). The reaction mixture was placed in 4