Chapter 5. SDS-Polyacrylamide Gel Electrophoresis
5.4 Results and discussion
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 then 1:4, 1:3, 1:2, 1:1, 1:0.5, 1:0.25, separately. SA is in lane 8 and BSA is in lane 9. The gel was stain with coomassie blue at last.
5.5 Summary
The electrophoresis experiments at this stage are in order to prove the existence of SA-DA. From Figure 24, we can determine the SA and DA is forming complex because of SA band is vanish. In addition, the silver staining technique is more sensitive than coomassie blue, it helps for detecting lower amount of sample.
5.6 References
(1) Luo, Y.; Vassilev, P. M.; Li, X.; Kawanabe, Y.; Zhou, J. Mol. Cell.
Biol. 2003, 23, 2600-2607.
(2) Sano, T.; Cantor, C. R. J. Biol. Chem. 1990, 265, 3369-3373.
Chapter 6
Advance research in PCBAM
6.1 Foreword
Before going to discuss with the result, we would like to thank to Dr.
James R. Carey, Yuwei Chu and Boyao Wang first. The idea that introduces the biotinylated bovine serum albumin (bBSA) into our PCBAM proposal was propose by Dr. James R. Carey. By using bBSA makes the PCBAM having more flexible space and do not restrict by biotinylated antibodies. When we start to utilize bBSA as reagents, our creative research members, Yuwei Chu and Boyao Wang came out a new method can dramatically improve the PCBAM proposal. This method is called streptavidin biotinylated protein network (SBPN) method. This method inspires us to generate two another methods, SHRPSA and biHRP method. Without their constructive help, we cannot let the PCBAM proposal go further. We are very appreciating their contribution. Thank you, Dr. James R. Carey, Yuwei Chu and Boyao Wang.
To judge a method is working or not, it depends on its absorbance.
The instrument we use to measure the absorbance is ELISA reader. It can measure the signal from 0.000 to 4.000. However, as we known, the accuracy of detection would become an issue when the absorbance above 1.500. Hence, we set up the detection range from 0.000 to 1.500. Besides, the background of ELISA is always below 0.100. When absorbance is over
0.100, the solution has slightly color and can be tell from naked eye.
Therefore, when absorbance below 0.100 is an ideal background.
Another thing is the definition of cycle. In our methods, they all have two major reagents to alter using continuously until to final step. For instance, SHRPSA method is composed with streptavidin conjugated horseradish peroxidase (S-HRP) and bBSA. A two steps motion that added bBSA and then treated with S-HRP is called 1 cycle. As shown in Figure 25, just adding S-HRP is called 0 cycle and presented in light blue. Then added the bBSA (light orange) for incubating 20 min and added the S-HRP (blue) for another 20 min to become 1 cycle. Keep adding bBSA (orange) and S-HRP (purple) layer by layer to form 2 cycles complex and so on.
bBSA
S-HRP Detection antibody
0 cycle 1 cycle
2 cycles 3cycles
Figure 25. The definition of cycle operation. Just adding S-HRP which presents in light blue called 0 cycle. Then placed the bBSA (light orange) and S-HRP (blue) step by step on the 0 cycle is called 1 cycle. Keep adding bBSA (orange) and S-HRP (purple) would form 2 cycles complex.
6.2 Strategy of development
The strategy of the method development is including three stages:
Binding ability of reagents, interaction of reagents which on the antibody, and apply in detecting antigen. The detail is described as following: We begin with test the binding ability between reagents such as bBSA and streptavidin or S-HRP in various methods first, as shown in Figure 26.
ELISA ABC SHRPSA biHRP
bBSA Streptavidin
S-HRP B-HRP
Figure 26. The scheme of binding ability test in various methods.
Various methods from left to right are ELISA, ABC, SHRPSA and biHRP method. For detecting the bBSA, ELISA is just only adding the S-HRP one time. Whereas ABC method is treated with streptavidin first and then added B-HRP layer by layer. SHRPSA method is using the S-HRP binding to bBSA step by step. While the biHRP method is contained two different HRP reagents, S-HRP and B-HRP.
Initially, the bBSA is coated on the well surface, and then added the various reagents in different methods to grab bBSA. The left side of image
in Figure 26 is the original ELISA, just only adding the S-HRP one time.
The second image from the left is stands for ABC method,1 based on biotinylated horseradish peroxidase (B-HRP) binding with the streptavidin layer by layer. While SHRPSA method is to use the S-HRP binding with bBSA layer by layer to form a complex. The right side is biHRP method compose with two different HRP reagents, B-HRP and S-HRP. Except the ELISA, other three methods are using streptavidin-biotin interaction to form a polymer-like network complex. Through the polymer-like complex, the absorbance signal of analytes can be amplified.
When the most effective method is verified, then we can move on to next stage: Investigate with bonding capacity of reagents which on the detection antibody (DA). As shown in Figure 27, take SHRPSA method for an example, DA was coated in the well surface first, and then added the S-HRP to detect the DA. If DA can be detected, it implies that S-HRP can bind to the DA. It is significant that S-HRP can bind to the DA because of DA can capture the antigen and we need to applied the method in antigen detection in the further study.
The binding capacity can be determined by controversially adding reagents and observing the difference on the cycles of operation. When the absorbance is increasing with running cycles, indicates S-HRP and bBSA can binding together. As shown in Figure 27, the amount of HRP would rise through the bBSA function as a bridge to link two S-HRP molecules and also gets higher absorbance.
bBSA S-HRP Detection antibody
Figure 27. The scheme of interaction of reagents on the antibody. Take SHRPSA method for an example, the DA can be detected by S-HRP. Next, the bBSA was added to connect the S-HRP. With layer by layer adding, the signal can be enhance and verify the binding capacity among DA, S-HRP and bBSA.
Subsequently, when these two stages can confirm the DA and reagents has ability to bind together, we would try to apply in detecting antigen which shows in Figure 28. Through the binding affinity between antigen and antibody, DA would let the well surface contains biotin. At this moment, employed the streptavidin to seize biotin with its own property.
After a series of steps, the antigen can be detected by measuring the absorbance. The first operation in accordance with normal procedures of ELISA, which shows in left side of Figure 28. After brings in our approach which is adding the reagents layer by layer on the ELISA, we assume the result would become polymer-like complex, as shown in right side of Figure 28.
bBSA S-HRP
Detection antibody Antigen
Capture antibody
Figure 28. The scheme of apply method in detecting antigen. Apply the developing method in detect the antigen is the final stage of development.
Take SHRPSA method for an example, the signal should be increased from the left to the right. After a series of steps, the result would become polymer-like complex and produced signal amplification.
6.3 Binding ability of reagents
6.3.1 Various reagents in various methods
According to aforementioned experimental methods and strategy of operation, we start at examined the bonding ability of bBSA and S-HRP or streptavidin. Before running the experiment, the plate has to pre-coated 100 pg/mL of bBSA on the well surface. Subsequently, treated with different reagents in different manners by means of adding reagents layer by layer. Our presumption is the stronger binding ability would get the higher absorbance. The results as shown in Table 10.
Table 10. The binding ability of various reagents in various methods Various methods in 100 pg/mL bBSA
Cycles
The absorbance is the average value of repeating the experiment 2 times.
Overall, the SHRPSA method is work better than other methods.
While using the combination of streptavidin and B-HRP which is similar to ABC method has less performance then we expected. It may due to the amount of B-HRP which binds to streptavidin on the well was not enough to generate high absorbance. Take note that biHRP method has higher absorbance than SHRPSA method at first cycle. It may result from biHRP method has contain more HRP (B-HRP and S-HRP) at beginning, so it has higher absorbance. Nevertheless, after running a few cycles of operation, SHRPSA method has much better performance than biHRP method.
Theoretically, B-HRP is a small molecular (~44 kDa) which has advantage in avoid the steric hindrance which from binding to S-HRP.
Nonetheless, the biHRP method did not work better than SHRPSA method.
The possible reason why the larger size of bBSA (~60 kDa) binds to S-HRP is more effective than B-HRP is the amount of biotin on the BSA (6 or 7 biotins) is more than HRP (2 biotins). Hence, bBSA can binds more S-HRP than B-HRP dose and gets more intense results.
When running more than 2 cycles of operation, the difference between these two methods will even more significant. Indicates bonding ability of bBSA and S-HRP is better than B-HRP and S-HRP. Therefore, keep adding reagents makes SHRPSA method has more advantages than biHRP method. By using SHRPSA method to detect the bBSA, the absorbance would over 1.000 after running 4 cycles amplification. Besides, the absorbance is twice higher than the biHRP method.
1.4
Figure 29. Running cycles of various methods. The binding ability test in various reagents with various methods, test B-HRP and S-HRP in biHRP (), test bBSA and S-HRP in SHRPSA (), and test B-HRP and streptavidin in ABC (c).
SHRPSA method and biHRP method are shown increasing trend in Figure 29. While the ABC method did not work in this experiment.
Though biHRP method can amplify the signal, the SHRPSA method gives more sharp amplification implies this method has more effective and competitive. In the background section, the background of SHRPSA method has a little bit higher, at 0.174. Nonetheless, since the signal can be amplified after 4 rounds operation, the background is relative low enough to be ignored. As shown in Figure 29, after subtract the background still SHRPSA method presents increasing tendency although the background also has an increasing trend.
6.3.2 Control experiment of SHRPSA with or without bBSA
The experiment which is aforementioned presents the binding ability of reagents in various methods. The higher absorbance implies the binding ability is stronger. The SHRPSA method proves bBSA and S-HRP can bind to each other. In addition, bBSA can be detecting by bind to the S-HRP while S-HRP is bonding with bBSA through layer by layer treatment to make the signal more intense. The background value is relative low indicating the contribution of absorbance is small, however, it just a piece of evidence to support our method. In order to determine the bonding ability between the bBSA and S-HRP again, we devise another way to investigate.
bBSA S-HRP BSA With bBSA Without bBSA
Figure 30. The SHRPSA method with or without bBSA. 5% BSA was blocked on the well to avoid other reagents to affect the test. Treating with bBSA is called experimental group; whereas without adding bBSA and use HRP buffer instead is called control group. Comparing the difference after running 5 cycles operation.
This experiment is comparison between the S-HRP with or without adding bBSA. In the beginning, 5% BSA was blocked for 2 h on the empty well. This blocking step is preventing others reagent stick on the well surface to affect the experiment. Washed and added the S-HRP for incubating 20 min. As shown is Figure 30, there are two groups contain experimental group and control group. Experimental group is treating with bBSA; while the control group did not adding bBSA and use HRP buffer instead. Repeated adding reagents after 5 cycles would get the Table 11.
From Table 11, it shows the absorbance of just adding S-HRP is less than 0.100. The result suggests that S-HRP itself will not aggregate,
adsorption or binding together. While in experimental group, adding the bBSA making the absorbance slight rising, proved the S-HRP and bBSA indeed can bonding together. With this experiment, our idea was validate again and the SHRPSA method is indeed feasible.
Table 11. Control experiment of S-HRP with or without bBSA
Cycles With bBSA Without bBSA
0 0.062 0.068
1 0.098 0.080
2 0.104 0.080
3 0.117 0.083
4 0.213 0.079
5 0.231 0.063
The absorbance is the average value of repeating the experiment 4 times.
6.4 Interaction of reagents on the antibody 6.4.1 Comparison with SHRPSA and biHRP
After reveal the binding ability of SHRPSA method in detecting bBSA is perform better than other methods, we then go to next stage to test the bonding capacity between S-HRP and bBSA on the DA. Moreover, in order to prove SHRPSA method has more advantage than biHRP method again, we are coating different concentrations of DA in the well surface to observe the difference. The range of concentrations is from 1000 to 10 pg/mL. The results are shown in Table 12 and Table 13.
Table 12. BiHRP method introduced to DA coated array
Concentration of detection antibody (pg/mL) Cycles
The absorbance is the average value of repeating the experiment 4 times.
Table 12 is the result of biHRP method. It can work well in 1000 pg/mL of DA, whereas the absorbance is only a little higher than the background at 100 pg/mL of DA. Remove the background afterwards, 0 and 1 cycle operation which in 100 pg/mL even become minus. It reveals the detection limit has been reached.
Table 13. SHRPSA method introduced to DA coated array
Concentration of detection antibody (pg/mL) Cycles
The absorbance is the average value of repeating the experiment 4 times.
On the contrary, SHRPSA method in Table 13 shows a gap between blank and 100 pg/mL of DA. While the absorbance of 1000 pg/mL of DA even beyond the detection range (>1.500) after third round of operation. This result provides evidence that SHRPSA method is more competitive than biHRP method. To combine with Table 12 and Table 13 can make the Figure 31. We can easy to compare the difference between biHRP method and SHRPSA method in Figure 31.
Absorbance (450 nm)
Cycles of SHRPSA and biHRP method
0 -0.1
Figure 31. Comparison with SHRPSA method and biHRP method.
SHRPSA method has an increasing tendency along with operation cycles in detecting 100 pg/mL of DA (). While use biHRP method for detecting 100 pg/mL of DA (c) looks like a horizon line after subtract the background. Same thing happened to SHRPSA at detecting 10 pg/mL of DA (). The absorbance is closed to background and become minus after deduct the background.
We can see the absorbance of DA at 100 pg/mL with SHRPSA method is increasing and shows a linear tendency obviously. While biHRP method in same concentration is close to background so it looks like a horizon line after subtract the background. The detection limit of DA with SHRPSA might at 10 pg/mL owing to it is close to background and become minus after deduct the background. Once operating a several cycles, the signal can be amplified. However, operating too much cycles result in prolongs the total experimental time and also cause the background will increase. Therefore, we consider that operating 3 or 4 cycles is fitting with profits of SHRPSA method. It not only has higher amplification signal than 2 cycles but also has relative low background and less operation time than 5 or 6 cycles.
6.4.2 Different cycles of operation
Since we had roughly compared with the binding ability between two methods on DA, then we go for further studied in detecting the DA. The concentration range reduced from 4.0×102 pg/mL to 3.0×10 pg/mL. As mention above, operating too much cycles cause long detection time and also increasing the background. Therefore, we will compare with the differences among running 0, 3 and 4 rounds of operation. The results are shown in Table 14.
In Table 14, the 0 cycle is similar to initial detection method, ELISA.
Owing to the process just only adds S-HRP, so it can be regard as ELISA.
The absorbance at 0 cycle is very low, even the highest signal just reach 0.105. Also, the absorbance signals of each concentration are closed and make the difference not obvious. Whereas, the difference of absorbance has more distinguishable than 0 cycle at running 3 and 4 cycles of operation afterwards.
Table 14. Different cycles of operation
Absorbance of operation cycles
The absorbance is the average value of repeating the experiment 4 times.
According to Table 14, we plot the Figure 32. It can clearly observe in the 0 cycle, or called ELISA, the absorbance is below 0.100 after deducting the background and get a relative flat curve. However, with SHRPSA method running after 4 cycles, the signal deducting the background still can maintain at high value, 0.849, and has a great r square value, 0.9991.
The result implies that development of this method is quite promising.
Before apply in detecting the antigen, we still need to try many different conditions, such as modifying the ratio of reagents or adjusting incubation time, to make this method to achieve the optimal results.
4 cycles
Figure 32. Different cycles of operation in detecting DA. 0 cycle (c) can regard as ELISA and gets a relative flat curve. Nonetheless, after running 3 cycles () or 4 cycles () of operation with SHRPSA method would generate the amplified signal and let the slope become sharper.
Besides, the absorbance became distinguishable in various concentration and obtained good r square value at 0.9991 after 4 cycles of operation.
6.4.3 Incubation time
Time is a very vital factor in the experiment, especially in diagnose disease. When some of detection has to against the clock, makes it even more important. Therefore, if SHRPSA method can shorten the operating
time, this method will be more competitive. First, we try to cut down the incubation time from 20 min to 5 min, experimental operation was same as before except the incubation time, and results are present in Table 15.
According to Table 15, reduce incubation time cannot get good results, because of the S-HRP does not have enough time to bond with DA. Even though the streptavidin and biotin are binding together rapidly, the factors of concentration of reagents, steric hindrance and other factors make binding still needs longer time for incubation.
Table 15. The various incubation time in SHRPSA method Absorbance of various incubation time
Table 15. The various incubation time in SHRPSA method Absorbance of various incubation time