Prove the concept of PCBAM step by step

On the purpose of confirming the idea of PCBAM is work, we monitor the signal change in every step by measuring the absorbance. The procedure is shown in Table 5. Step 1 was added IL-7 antigen and IL-7 DA for incubated 120 min and then treated with SA-DA₄ in step 2. ''-'' means did not add any reagents and directly operate the S-HRP and TMB steps. Step 3 was added different types of antigen and antibody, IL-4 antigen and DA₄. While step 4 and step 5 is repeated step 2 and step 3. In this experiment, with adding some other non-analyte antigen and antibody would help to generate the signal amplification. Due to SA-DA complex provides the biotin and epitope sites, the streptavidin and antigen would bind to the SA-DA to let the analyte complex grow bigger and bigger.

Moreover, using non-analyte antigen would not confuse the concentration of analyte antigen cause the concentration problem. Besides, in order to prevent the contamination, different array was used for different concentration.

Table 5. The absorbance of PCBAM in every step Step 1 antigen at 0.1 pg/mL. ^eDA₄ = IL-4 detection biotinylated antibody,

Observe in Table 5, the absorbance is increased after adding SA-DA₄ because of S-HRP can bind to the DA4. Through IL-4 antigen and DA4 was added in the next step, the absorbance was rose again. This result means the IL-4 antigen is connected to the antibody which on the SA-DA4. Keep adding SA-DA₄ again in step 4 would offer more DA₄ to bind with the S-HRP and makes the absorbance even higher. In step 5, the absorbance dramatically increase and cause off scale (>1.500). This consequence may be attributed to SA-DA4 has three binding sites which can be bind with other secondary antibodies. Follow the Table 5 to make an absorbance versus step plot in Figure 18. It has increasing trend which shows by

means of treating reagents continuously layer by layer would amplified the signal. So it can confirm the idea of PCBAM is work.

Absorbance (450 nm)

1.0 0.8 0.6 0.4 0.2 0.0

1 2 3 4

1.2

Operation step

Figure 18. Absorbance of IL-7 analyte for each step with PCBAM.

Step 1 treats with IL-7 antigen and DA7 whereas step 2 supplies the SA-DA4. Step 3 is adding different types of antigen and antibody, AG4

and DA4. While step 4 and step 5 is repeating step 2 and step 3. Step 5 is off scale and not shown in the figure. According to Table 5 directly operate the S-HRP and TMB steps after finish each step.

4.3.2 Detecting lower concentration of AG

with PCBAM

According to section 4.3.1, we had demonstrated the idea of PCBAM can be implemented. The signal can be amplified by utilizing SA as the strategy to offer extra biotin binding sites to grab DA4. In order to test PCBAM whether works in lower concentration or not, the experiment

was designed and shown in Table 6. The process presented as following, IL-7 capture antibody has been coated in the plate before use. Adding 100 μL of IL-7 antigen (AG₇) and the concentration is 6.2×10^-X pg/mL. X can be 3 to 8 which represented the different concentrations from 6.2×10^-3 pg/mL to 6.2×10^-8 pg/mL. Simultaneously, supplied 50 μL of IL-7 detection antibody (DA₇) in each well and co-incubated at rt for 120 min.

Table 6. The procedure of PCBAM detection biotinylated antibody. ^cSA-DA₄ = A complex which from by streptavidin binding to IL-4 antibody. ^dAG₄= IL-4 antigen at 1.8 pg/mL,

eDA₄ = IL-4 detection antibody. IL-7 capture antibody has been coated in the plate before use.

Pipette 75 μL of the SA-DA₄ complex solution, which is SA and DA₄ mixed with mole ratio at 1:3 together and added into well by incubating for 30 min. Supplied the 100 μL of IL-4 antigen (AG₄) and the concentration was at 1.8 pg/mL. Repeat adding SA-DA₄ and AG₄ again. Steps in accordance with Table 6, different concentration of samples was separated to independent array for prevent the cross-contamination. Treated with S-HRP for 20 min in step 6 which is not shown in the Table 6. The whole

complex was stained with TMB for developing 15 min in dark place in the end.

The data was averaged for 16 independent wells and removed the background 0.760 and then organized in Table 7. Since specific antigen is bind to specific antibody, IL-4 antigen and IL-7 antibody will not bind to each other. It can be regarded as a fixed concentration of IL-7. Absorbance is increased with the concentration from 0.710 to 1.311. The coefficients of variation (C.V.) for the IL-7 antigen concentration were lower than 10% in all cases.

Table 7. The coefficients of variation in various concentration Absorbance

Concentration

(pg/mL) Mean Standard deviation C.V. (%)

6.2×10^-3 1.311 0.14 5.24

6.2×10^-5 1.060 0.13 6.39

6.2×10^-6 0.992 0.18 9.86

6.2×10^-7 0.856 0.14 8.31

6.2×10^-8 0.710 0.15 9.95

However, the background was obtained 0.760 which is more than the background of control experiment. Control experiments are detected the streptavidin, SA-DA4 and DA4 in coated-well separately. They all had less absorbance which is lower than 0.100, as shown in Table 8. Implied that the reagent would not affect the experiment by itself.

Table 8. Background of various reagents in control experiment

Reagent Absorbance

IL-4 antibody 0.059

Streptavidin 0.055

aSA-DA₄ 0.060

aSA-DA₄ = A complex which form by streptavidin and IL-4 antibody.

As shown in Figure 19, the plot is take log to concentration and versus with absorbance. The range of concentration of IL-7 antigen is from 6.25×10^-3 pg/mL to 6.25×10^-8 pg/mL. The result achieved linear calibration curve and shown a regular increasing tendency. The correlation coefficient was 0.9916, proved that the amplification principle is feasible.

The relationship between the absorbance and the concentration is more obvious. Presented that using the higher concentration would obtain the greater absorbance.

Since this experiment just repeat adding SA-DA₄ one time, we tried to repeat three times with added SA-DA₄ to make more signal amplification. As we expect, the absorbance becomes more intense. Those absorbance can reach 3.000 or even 4.000 approximately. Nevertheless, the background also increased and became more complicated in the solution. Furthermore, long operation time also cause the cost increased.

1.4

Figure 19. PCBAM linear dilution profiles. Normalize the curve on log-concentration and versus the absorbance. The range of IL-7 antigen concentration is from 6.2×10^-3 pg/mL to 6.2×10^-8 pg/mL. Absorbance is increased with the concentration.

-2

4.4 Summary

We have demonstrated that utilize of protein conglomeration based amplification method (PCBAM) significantly increased the sensitivity of the ELISA in current stage. The performance of the PCBAM detection system is enhanced by using SA-DA₄ complex chiefly. However, when the concentration is lower, the wells were in the higher risk of contamination. Moreover, it is hard to reduce the PCBAM background

below to 0.100. It may because of SA-DA₄ involved in too many and too complex reagents or even stick on the well bring about increasing background result. In addition, repeat adding SA-DA₄ and antigen more often, the control factors would become more complicated.

If the mixture which adds into well have more ingredients, it will cause the background increased. In this situation, the result in measurement of absorbance would be difficult to approach the true value.

In recent study of PCBAM, the best conditions we know currently are used SA and DA at ratio of 1 to 3. Incubated for 30 min and repeat the operation of SA-DA cycle only once to reduce the time issue which based on the time cost considerations. Nonetheless, to discuss how to simplify and shorten the process, prove SA-DA₄ exist and reduce the background signal is our urgent goal.

4.5 References

(1) Gonzalez, M.; Bagatolli, L. A.; Echabe, I.; Arrondo, J. L. R.; Argarana, C. E.; Cantor, C. R.; Fidelio, G. D. J. Biol. Chem. 1997, 272, 11288-11294.

(2)⁰Bontempo, D.; Maynard, H. D. J. Am. Chem. Soc. 2005, 127, 6508-6509.

Chapter 5

SDS-Polyacrylamide Gel Electrophoresis

5.1 Research intention

Since the molecular weight of antibody (150 kDa) is larger than streptavidin (SA, 60 kDa), so based on three-dimensional hindrance, may hard to make three DA on the same SA. The reaction may not match the ratios of SA:DA at 1:3 which we expected. The possible solution is to employ the cross-linking agent, For example, NHS-PEG4-biotin, NHS-PEG12-biotin, or SAMA.¹ The cross-linking agent can make a long chain arms to increase the distance between the SA and DA so that can avoid steric hindrance which caused by the directly linkage. Take PEG₄ for an example, it has a long-chain polyethylene glycol (PEG). With this long-chain, we may improve the steric obstacles between the DA and SA.

In order to ensure the 1:3 ratio of SA-DA complex can be made, we introduce the electrophoresis technique to allow us understand the molecular weight of complex. Also, electrophoresis can provide some evidence of bonding in SA-DA complex.

5.2 SDS-PAGE technique

In electrophoresis system, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) is using surfactants SDS (sodium dodecyl sulfate) attached to the hydrophobic surface of protein. Due to the negative

charge was carried by SDS, it would lead the protein become mobile.

Since the charge which carried by the protein is much smaller than the charge which attached on SDS molecules, so the charge which on the protein did not affect the mobility rate. Mobility rate only depends on the molecular weight of protein. In the sample handling process, the tertiary structure of protein was destroyed by using the heat. The hydrophobic intramolecular would hence be exposured and combined with SDS. While the reducing agent, β-mercaptoethanol, helped to destroy the disulfur bond.

Therefore, the SDS-PAGE technique is widely used in determines the molecular weight on protein subunits.

5.3 Materials and methods

5.3.1 Materials

30% acrylamide/bis solution was purchased from Bio-Rad.

Separation buffer was mixed with 180 μL of N,N,N',N'-tetra -methyl-ethylenediamine (TEMED, Bio-Rad) and 90.80 g of Tris(hydroxymethyl)aminomethane (Tris, Bio-Rad) and dissolved in 300 mL of deionized water (DI water) first. After adjusted to pH 8.8 with HCl, add DI water again to reach 500 mL. TEMED is a catalyst to help free radical transfer. Stacking buffer is dissolved 6.00 g of Tris and 400 μL of TEMED in 40 mL of DI water. Use HCl to adjust the pH to 6.8 and added DI water to reach 100 mL. 10% (w/v) SDS solution was added 10.00 g SDS in DI water to reach 100 mL. 10.0 mg of Ammonium persulfate

(APS) was dissolved in 100 μL of DI water. APS solution has to use immediately or distributed into the required volume and stored at -20 °C in the frozen refrigerator. SDS-PAGE sample buffer (2X) is included Tris 3.00 g (0.125 M), EDTA-2Na 14.80 g (0.002 M), SDS 4.00 g (2%) and 10 mL of β-mercaptoethanol (5%). Dissolved above chemical in 80 mL of DI water first and then adjusted with HCl to reach pH 6.8. Filled the DI water to reach 100 mL afterward. SDS electrophoresis buffer (5X) is contained Tris 54.50 g (0.090 mM), boric acid 24.80 g (0.080 M) and EDTA-2Na 4.70 g (0.025 mM). Dissolve these chemical in 800 mL of DI water and adjusted the pH to 8.4 with HCl. Filled the water to 1000 mL afterward while SDS was added in dilution to become 0.1% SDS.

Tracking dye solution is dissolved 1.0 mg of bromophenol blue in 5 mL of DI water, and then added 5 mL of glycerol. Staining dye is dissolved 1.00 g of coomassie brilliant blue R-250 in 250 mL water, and then added 250 mL of methanol and 50 mL acetic acid. Washing solution is consisted of 20% methanol and 10% acetic acid. 200 mL of MeOH mixed with 100 mL of acetic acid and then filled the water to reach 1000 mL.

5.3.2 Procedure of SDS-PAGE

In the beginning, the glass molds and plastic equipment was set up and put up vertically. Avoid leakage when fill in gel solution. The needed concentration was prepared in SDS gel solution according to Table 9. Two

pieces of gels requires 10 mL resolving buffer and 5 mL stacking solution.

Before add into the glass molds, the gel was mixed with APS solution which is fresh prepared. Once APS is introduced in stacking gel solution, the solution has to fill into glass molds immediately or it would be solidified. After mixed APS and all the components which in Table 9, the resolving gel solution has been made. Cast into the molds along with glass side. Added the gel solution slowly to avoid producing the air bubbles during the operation. When gel was coagulated, supply the stacking gel solution. The preparation is the same way in accordance with Table 9 for required concentration and with APS in it.

In the section of analysis, to mix with the protein sample and the same volume of sample buffer and 100 μL of track dyes. Heat at 95 °C for 5 min to unfold the protein. In the meantime, filled running buffer about 750 mL into the tank. After cooling the sample, pipette into the gel slot and set up working voltage at about 100 V~150 V for running 100 min.

When the gel sample is near bottom, then terminate the electrophoresis process. Removed the glass and took out the gel. Stained with coomassie blue dye and put it on a shaker for staining about 30 min. After staining step, the gel is soaked into the washing solution for wash several times.

When the background of gel is completely transparent, the wash step was completed.

Table 9. Various conditions in various percentage of SDS-PAGE gel

a Gel Buffer has two different concentration of Tris-HCl as following:

Resolving Gel Buffer: 1.5 M Tris-HCl, pH 8.8 Stacking Gel Buffer: 0.5 M Tris-HCl, pH 6.8

5.4 Results and discussion

5.4.1 The demonstration of sample marker

To realize the molecular weight of various reagents in PCBAM can correctly infer the PCBAM theory in the actual experiment to understand the difficulties or binding evidence. Therefore, the electrophoresis test is the integral part of the experiment. On the purpose of clearly understand

the working procedures of electrophoresis, Figure 20 demonstrated the electrophoresis analysis by usage of HiMark^TM Pre-stained gel. Each piece of gel has 10 wells and each well can load the different samples. Add in the middle can avoid tailing and gets the better results.

In addition, samples at molecular weight 460 kDa requiring a more dilute concentration of gel components. The concentration of the gel is 5%.

The reason why using more dilute concentrations is because of the composition of gel has big gaps in this concentration. So as to make 460 kDa macromolecules go through the big gaps. The electrophoresis gel in Figure 20 did not show all the bands. This is because in order to achieve better separation, thus extend the operation time to 2 h and abandon the smaller weight of marker which are below 71 kDa. This electrophoresis gel is using 100 V as working voltage. If increased working voltage, the time can be shortened. However, it will bring in incomplete separation or the risk of uneven band if the voltage is too large.

Figure 20. The demonstration of HiMark^TM Pre-stained marker

5.4.2 Electrophoresis analysis of proteins

In Figure 21, shows the various samples in the gel. Lane 1 and lane 2 are SA, lane 3 and 4 are DA₄, lane 5 and 6 are DA₇, lane 7 is IL-7 capture antibody (CA₇), and lane 8 and lane 9 are the SA-DA₄. The gel is overload due to the sample amount (about 50 µg) is too high. SA is hard to observe may cause by the amount is too little (less than 5 µg) to display on this gel.

DA₄ and DA₇ has huge bands at about 60 kDa, it is likely that the antibody was fall apart because of a single antibody molecular weight of 75 kDa.

Figure 21. Electrophoresis analysis of antibodies, SA and SA-DA4. The sample at lane 1 and lane 2 are SA, lane 3 and 4 are DA₄, lane 5 and 6 are DA₇, lane 7 is CA₇, and lane 8 and lane 9 are the SA-DA₄.

5.4.3 The effect of dithiothreitol

Figure 22 presents the effect of dithiothreitol (DTT) in the sample.

Owing to DTT is a reducing agent can destroy the protein structure to unfold the protein, with or without DTT can affect the result. Lane 1 to lane 4 is adding DTT in the sample, and respectively loading the SA, DA₄, DA₇, SA-DA₇. Whereas lane 5 to 8 is without DTT, only adds SA, DA₄, DA₇, SA-DA₇, individually. After diluted ten times (about 5 µg), this gel gets a cleaner band. SA is hard to observe because it is too little content displayed on this gel.

Figure 22. Various samples with or without dithiothreitol (DTT). Lane 1 to lane 4 is adding DTT in the sample, and respectively adding SA, DA₄, DA₇, SA-DA₇. Whereas lane 5 to 8 is without treating DTT, only adds SA, DA₄, DA₇, SA-DA₇.

DA₄ and DA₇ have band below 84 kDa, there are likely that antibody fracture to from a single part, due to the molecular weight of single part of antibody is 75 kDa. Compare to with or without treating DTT, the lane 2 to lane 4 has additional fragments under 60 kDa while lane 6 to lane 8 just has shows bigger molecular weight above 60 kDa. Therefore, utilizing DTT is warrant and helpful for analysis components of sample.

5.4.4 Analysis of various antibodies and SA-DA complex

Figure 23 shows various antibodies and binding with SA at ratio of one SA to three antibodies in the gel. Lane 1 is DA₄, and lane 2 is SA-DA₄. Lane 3 is loading DA₇, while loading SA-DA₇ at lane 4. Lane 5 is PEG₄ linked IL-7 capture antibody (PC₇), lane 6 is streptavidin-PC₇ complex (SA-PC₇), lane 7 is PEG₄ linked IL-7 detection antibody (PD₇), lane 8 is streptavidin-PD₇ complex (SA-PD₇), and lane 9 is SA in BSA buffer.

In Figure 23A, compare lane 1 (DA₄) and lane 2 (SA-DA₄), we find that lane 2 has a band at 210 kDa and more intense than lane 1. This result implies some of SA fragment is attach to the antibody, thus cause the band is denser than without SA. The similar situation is happen in other lanes, such as the bands in lane 4 (SA-DA₇) is more intense than in lane 3 (DA₇). Lane 5 and lane 7 did not shows any band may due to the concentration of PC₇ and PD₇ is too low.

(B)

Figure 23. Analysis of various antibodies and SA-DA complex. From left to right (1 to 9) is loading DA4, SA-DA4, DA7, SA-DA7, PC7, SA-PC7, PD7, SA-PD7 and SA, independently. Stain with different dyes would obtain (A) coomassie blue staining gel and (B) silver stain gel.

kDa (A)

To further analysis the gel, we employee the silver stain to detect low amount samples which on the gel. The result is shown in Figure 23B.

The silver stain is more sensitive than coomassie blue dye, the difference

can be distinguish between Figure 23A and 23B. The gel also shows the bands of lane with adding SA are more intense than just adding antibody.

5.4.5 Analysis of SA-DA7 complex in various ratios

On the purpose of confirming the SA-DA₇ complex is formed or not, we loaded various ratios of SA:DA₇ to monitor the interaction. In Figure 24, each lane represent as following, lane 1 is DA₇, while from lane 2 to lane 7 are SA-DA₇ complex with various ratios. The ratios of SA:DA₇ are 1:4, 1:3, 1:2, 1:1, 1:0.5, 1:0.25, respectively. Lane 8 is SA and lane 9 is BSA, use for internal standard. In lane 8, SA has a band under 18 kDa suggests that SA is fall apart to four subunits and this band is regard as one subunit.² Observe in lane 8 to lane 5, the band of SA subunit is vanishing, support the SA-DA₇ complex is formed. The SA subunit is disappear at lane 4 to lane 2 shows it is possible that all the SA is conjugate with DA₇. These ratios are SA:DA₇ at 1:2, 1:3, 1:4. Moreover, lane 4 to lane 2 has bands on the top, it is consider as SA-DA₇ complex due to the bands in lane 1 (DA₇) is more dilute than lane 4 to lane 2.

Figure 24. Analysis of SA-DA7 complex in various ratios. From left to right (1 to 9) is loading various SA:DA7 ratios. Loading DA7 at first and

Figure 24. Analysis of SA-DA7 complex in various ratios. From left to right (1 to 9) is loading various SA:DA7 ratios. Loading DA7 at first and