Chapter 6. Advance research in PCBAM
6.3 Binding ability of reagents
6.3.2 Control experiment of SHRPSA with or without bBSA .71
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 Cycles
5 min 10 min 20 min
0 0.005 0.042 0.006 1 0.038 0.052 0.070 2 0.038 0.042 0.085 3 0.056 0.085 0.046 4 0.055 0.068 0.152 The absorbance is the average value of repeating the experiment 3 times.
In Table 15, although the absorbance at 5 min and 10 min incubation has rising trend, they still not work better than 20 min. Generally, keep incubating the reagents and stay over 20 min also can get higher absorbance. However, the weaknesses are, it will cost too much operation time and even make the background go higher. Hence, we still choose to incubate the reagent for 20 min.
6.4.4 Various concentration of reagents
When incubation time is establishing at 20 min, we start to focus on adjusted the concentrations of reagents. The purpose is to enhance the amount of DA binding with S-HRP and bBSA. If S-HRP contents more on the well surface, the absorbance will be higher. Herein we increase the reagents’ concentration from the original to 2 fold (2X, 3.0×10-4 mg/mL), 3 fold (3X, 4.5×10-4 mg/mL) and even 5 fold (5X, 7.5×10-4 mg/mL), the results shows in Table 16 and Table 17.
Table 16. The various ratios of reagents in SHRPSA method
Absorbance of various ratio of reagents (S-HRP:bBSA) Cycles The absorbance is the average value of repeating the experiment 3 times.
The first column of Table 16 which is adjust the reagents’ ratio makes the S-HRP to bBSA is 1:3, arise the concentration of bBSA to 3 fold. The purpose is to ensure the whole streptavidin which on the S-HRP reagents can all bonds to bBSA. However, the 1:3 ratios did not work better than original ratio, 1:1. Both are seems like the same. Use 3:3, so called 3X reagents, has more signal amplification than the original even though the blank also increase.
Table 17. The various concentration of reagents in SHRPSA method Absorbance of various concentration of reagents
Cycles a
a1X = Original reagents of bBSA and S-HRP which all at 1.5×10-4 mg/mL.
b2X = 2 fold reagents at 3.0×10-4 mg/mL. c5X = 5 fold reagents at 7.5×10-4 mg/mL. The absorbance is the average value of repeating the experiment 3 times.
The absorbance of using 5X reagents in Table 17 has about 5 times higher than 1X reagents. It implies that increasing the concentration of reagent can enhance the absorbance. Observe in Table 17, after four rounds operation by using original reagents, the absorbance of DA at 100 pg/mL without background is 0.178. If alter to employ 5X reagents, the absorbance would jump to 0.995. However, the background also increased, if we can reduce the background, then this method may be more competitive. Nevertheless, it still has an increasing trend in Figure 33 after subtract the background.
Therefore, from the results of Table 17, we will prefer choose the 2X reagents to do further study. For the reason that performance is better than the original and can get relative high absorbance after running four cycles.
Besides, the background is only a little more than the original. The most
important reason is use 2X reagents is cheaper than 5X. While the concentration increased to 5X also has its own favorable conditions, observe at third cycle, the absorbance is higher than four rounds of 2X and the background also relative lower than four rounds of 2X. The result suggests that we can use more concentrated reagents operate less cycle and reach the closed absorbance which gets from more running cycles in less concentration reagents, reduce the operation time.
1.0
Figure 33. The various concentration of reagents in SHRPSA. The absorbance was increased along with concentration of reagents and cycles of operation after remove the background. 5X (c) has higher absorbance than 2X () and original one () though the error bar and background is bigger than others.
6.5 Apply in detecting antigen
In final stage, we apply SHRPSA method in detecting the antigen, as shown in Figure 28. By means of the interaction between antigen and antibody, the DA bind to antigen and let the well surface contains biotin.
While utilizing the affinity between streptavidin and biotin, the S-HRP can be introduced to the antigen-DA complex and let the antigen can be detected. Subsequently, supplied bBSA to connect to S-HRP and makes signal more intense.
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The concentration range of IL-7 antigen is from 200 pg/mL to 6.2 pg/mL. In accordance with operating normal procedures of ELISA, adding different concentration of antigen into the well. Afterward we introduce our method in the procedures, operating 4 cycles and 2 cycles with alter adding reagents, respectively. The results as shown in Table 18. Due to absorbance of 200 and 100 pg/mL is out of detection range (>1.500) after four rounds operation, so we did not discuss in here.
Table 18. Apply SHRPSA method in detecting antigen
Absorbance of various operation cycles Conc.
The absorbance is the average value for repeating the experiment 4 times.
From Table 18, we can see that absorbance of antigen at 50.0 pg/mL is 0.247 and enhance to 0.922 after 4 cycles operation. The limit of detection (LOD) of IL-7 antigen is 6.2 pg/mL. While the signal of 6.2 pg/mL is from less than 0.100 rose to more than 0.100 by 4 operation cycles. Indicates the development of SHRPSA method has ability to improve the signal and work better than ELISA. The background at 4 cycles is close to 0.1, makes SHRPSA method even more attractive.
R2 = 0.9976
Figure 34. Apply SHRPSA method in detecting antigen. Various operation cycles in SHRPSA method to detect antigen. 0 cycle, or can consider as ELISA (), 2 cycles (), and 4 cycles (c). The amplified result depends on operation cycles.
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Making the plot from Table 18, the amplification effect has more clearly. Figure 34 shows this absorbance is increase by the running cycles.
Also, the signal of relatively flat slope (blue ) would turn into a sharp slope (green c) after 4 cycles operation. The r square still remained at about 0.9900, Showing this method can detect antigen and does work in amplified the signal. With these experiments describe above, the SHRPSA method was consider as established. Even though there still has improvement room, these are the best results in our current research.
However, we still not satisfied and wish that can shorten the incubation time, and solve the background issue.
6.6 Poly-SHRPSA method
Although these above experiments established SHRPSA method can be applied in detect antigen, but we still not satisfied. We hope we can get more amplification signal, solve the background problem and shorten the incubation time in the SHRPSA method. Back to the principle of SHRPSA method, it is using S-HRP as primary layer to link biotinylated protein. If the biotinylated analyte can bind to plenty of S-HRP at once, maybe it can enhance the signal and shorter the operation time.
Primary antibody Antigen Biotinylated antibody Poly-HRP Tyramide
-AlexaFluor 555
Figure 35. Illustration of using poly-HRP. Poly-HRP is a reagent that has lots of S-HRP conjugated on the polymer, allows a large number of biotinylated analytes attach to it at once.2
Poly-HRP is one of reagents to match our idea.2 As shown in Figure 35, this HRP reagent is let the S-HRP conjugate on the polymer, allows for connect a large number of analytes at once. The signal amplification may go further via this reagent employing in to our method. Furthermore, we modified the SHRPSA procedure as shown in Figure 36. Let the bBSA directly added into the well which has 20 min incubated S-HRP, through combining with each other between the two reagents and forming a polymer-like network structure. The concentration of poly-HRP is 5.0×10-1 mg/mL, so we diluted it for 5000 times (1.0×10-4 mg/mL) to make the concentration is similar to the reagents used in the previous concentration (1.5×10-4 mg/mL).
Figure 36. Procedure of poly-SHRPSA method. Poly-HRP was treated into well for 20 min and put the plate on the orbital shaker at 160 rpm in every step. Adds bBSA directly into the well for reacting another 20 min without wash. The total volume in the well would become 200 µL. Wash 3 times and repeat adding poly-HRP and bBSA for 3 cycles. Supply the TMB at last step.
In order to shorten the operation time, we compared the various time for incubation in the poly-SHRPSA method. We are expecting that the signal can still be amplified after the operation time was reduced. The conditions are combination of 10 min and 20 min with poly-HRP and bBSA. Various concentration of DA from 8.0×102 pg/mL to 2.5×10 pg/mL was coated on the plate as previous procedure and exercised the poly-SHRPSA method which is aforementioned. The result was shown in Table 19.
Table 19. Various incubation time conditions in poly-SHRPSA method Absorbance of various incubation time
Conc.
800 -0.268 -0.275 0.613 0.523 -0.083 -0.200
400 -0.165 -0.175 0.430 0.393 -0.048 -0.108
200 -0.060 -0.063 0.049 0.158 -0.021 -0.041
100 -0.032 -0.029 0.053 0.083 -0.002 -0.007
050 -0.011 -0.006 0.013 0.029 -0.001 -0.003
025 -0.003 -0.000 0.004 0.032 -0.005 -0.001
aP10 = Poly-HRP incubate for 10 min. bb10 = bBSA incubate for 10 min.
cb20 = bBSA incubate for 20 min. dP20 = Poly-HRP incubate for 20 min.
The absorbance is the average value of repeating the experiment 3 times.
In Table 19, P10 means poly-HRP incubates for 10 min whereas b20 means bBSA incubate for 20 min and so on. Although the result is not very attractive, we still can observe that it has increase tendency with concentration. The reason why cause the amplification is not huge may due to the concentration is less than before. The concentration of poly-HRP is 1.0×10-4 mg/mL while previous concentration is 1.5×10-4 mg/mL. Besides, this method just running 3 cycles is one of possible reasons. Also, the incubation time is less than 20 min blocks the performance of amplification.
Generally, the signal of 20 min incubation is better than 10 min, while the incubation time of bBSA has less affect then poly-HRP as the result of first two columns is almost the same. The background is under 0.100, presents very significant results. In Figure 37, the data was deducted the background. Compare poly-SHRPSA method to ELISA, as shown in Figure 37, no matter how long incubation time is, ELISA signal still cannot perform as good as poly-SHRPSA method.
0.8
200
100 300 Poly-HRP 10 min, bBSA 20 min
ELISA 40 min
Poly-HRP 10 min, bBSA 10 min
Poly-HRP 20 min, bBSA 10 min Poly-HRP 20 min, bBSA 20 min
ELISA 20 min
600 700 800
Detection antibody (pg/mL)
Figure 37. Various incubation time conditions in poly-SHRPSA.
Normally, the more incubation time has more absorbance. While employing ELISA and prolong the incubation time to 40 min, the signal still cannot perform as good as poly-SHRPSA method.
The 40 min incubation of ELISA (yellow ¾) is better than the 20 min incubation (black ×) but they cannot work as well as poly-HRPSA method.
Even just incubate for 10 min (blue ) in poly-HRPSA method can work better than 40 min incubation of ELISA. Indicates we can use 10 min incubation as quick testing. By manipulate this new procedure, the operation become more simple and diminish the time consuming because the reagents would mix together and reduce the time of washing step. This advantage makes the poly-HRPSA method more competitive.
6.7 References
(1) Hsu, S. M.; Raine, L; Fanger, H. J. Histochem. Cytochem. 1981, 29, 577-580.
(2) Anderson, G. P., Taitt, C.R. Biosens. Bioelectron. 2008, 24, 324-328.
Chapter 7 Conclusion
At current stage of this study, we have demonstrated the protein conglomeration based amplification proposal such as SHRPSA method can significantly improve the sensitivity of ELISA. By operating SHRPSA method, the signal of ELISA can be increase dramatically. This is because the SHRPSA method introduces the bBSA as a core reagent and connecting the S-HRP molecules as a bridge to form a polymer-like network. When bBSA has more biotin, it can grab more S-HRP to let the signal more intense. That is also a reason why the biHRP method did not work better than SHRPSA method because of the amount of biotin on the
At current stage of this study, we have demonstrated the protein conglomeration based amplification proposal such as SHRPSA method can significantly improve the sensitivity of ELISA. By operating SHRPSA method, the signal of ELISA can be increase dramatically. This is because the SHRPSA method introduces the bBSA as a core reagent and connecting the S-HRP molecules as a bridge to form a polymer-like network. When bBSA has more biotin, it can grab more S-HRP to let the signal more intense. That is also a reason why the biHRP method did not work better than SHRPSA method because of the amount of biotin on the