Chapter 6. Advance research in PCBAM
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.
4
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.
84
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 HRP (2 biotins) is less than on the BSA (6 to 7 biotins).
Research demonstrates SHRPSA method can amplify the absorbance signal of IL-7 antigen. For an example, when the concentration of IL-7 antigen at 6.2 pg/mL, using SHRPSA method can enhance the ELISA absorbance from 0.075 to 0.220. In addition, the calibration curve shows linear plot and has good R square at 0.9979. The background issue in detecting antigen is small. The limit of detection (LOD) of antigen is 6.2 pg/mL whereas the LOD of DA is up to 10 pg/mL.
After a series of experiments, we confirmed that the absorbance is proportional to the incubation time, concentration, and running cycles. In the present study, the optimal conditions for this SHRPSA method are to
use 2 fold of concentration of the S-HRP and bBSA reagents (3.0×10-4 µg/µL), incubate them for 20 min in every step, and repeat operating up to 4 cycles. In shorten incubation time experiment, showing the time cannot be shortening. However, we believe that use more concentrated SHRPSA reagents can speed up the operating progresses. While the poly-SHRPSA method also can reduce the time which cost on the wash step.
The SHRPSA method still has room for improvement such as to reduce the background value to 0.100 or less. Although the absorbance can be raised to 1.000 or even over makes the background problem can be ignored, we still desire to reduce the background to make this method more attractive. The problem of background may cause by too often adding reagents into the well, let the S-HRP or bBSA adhesion on the well surface, then the following reagents would bonding together to make the signal getting bigger and bigger. Whereas in the poly-SHRPSA method, it shows low background result makes the poly-SHRPSA method more promising.
PCBAM proposal offers a new vision inspection system, and the experiments described advantages and disadvantages of this diagnostic system in the above. Currently, we prove the concept and demonstrate the SHRPSA method is feasible. However, it just the beginning of PCBAM proposal. How to make the system more complete and optimization or even built up a senor still has many challenges to overcome.
Chapter 8
Expansion of Currently Approach
8.1 Extension of SHRPSA method
We are dedicated to develop and study the PCBAM proposal.
Although the absorbance can be enhance to 1.000 and makes the problem of the background can be ignored, we still desire to reduce the background to make this method more attractive. By bringing in poly-HRP reagent to join the development of SHRPSA method may solve this problem and let the proposal step further.1 Additionally, the usage of poly-HRP is more matches with our original idea: To generate the polymer network on the analyte to detect the signal. In the future, we will focus on poly-HRP accordance with the same operating strategy which is aforementioned.
In addition, we can introduce tyramide signal amplification (TSA) 2-3 technique into our developed methods. TSA may be able to make the research go further. TSA method is employing the catalytic activity of HRP to produce high-density labeling of proteins. Through tyramide activated by the HRP, covalently binds to on the whole surface with electron rich moieties such as tyrosine residues in proteins. As shown in Figure 38, then we can add the reagent to stain it. If TSA can combine with our approach, perhaps our detection limit can go down to femtomolar.
Figure 38. The scheme of tyramide signal amplification. TSA method is utilizing tyramide activated by the HRP to covalently bind to proteins.
Through the catalytic activity of HRP, tyramide can high-density attach to the electron rich moieties such as tyrosine residues in proteins resulting in signal amplification.
The purpose of study PCBAM proposal is in order to achieve lower detection limits and build a more sensitive detection system to determine disease. Once the PCBAM was well-established, we will focus on apply into power-free microassay without any instrument. This will not only help for patients, but also for other resource-poor areas.
8.2 References
(1) Anderson, G. P.; Taitt, C.R. Biosens. Bioelectron. 2008, 24, 324-328.
(2) Bobrow, M. N.; Shaughnessy, K. J.; Litt, G. J. J. Immunol. Methods 1991, 137, 103-112.
(3) Bobrow, M. N.; Litt, G. J.; Shaughnessy, K. J.; Mayer, P. C.; Conlon, J.
J. Immunol. Methods 1992, 150, 145-149.
Biography
Bo-Shiuan Lin (林伯烜) was born in 1987 in Kaohsiung, Taiwan. He received his Bachelor degree from National University of Kaohsiung and now is study for M.S. degree in same university. His research field covers immunochemistry and bioinorganic chemistry. Now he is attempting to demonstrate the feasibility of SHRPSA method in PCBAM proposal and explored the detection limit. In future studies, he will try to solve the problem of background issues, introduce poly-HRP or tyramide signal amplification (TSA) technique to improve the SHRPSA method.