Chapter 3. Materials and Methods
3.2 Methods for development of PCBAM
3.2.8 Spectrophometeric analysis
After added colorless TMB substrate into the well and incubated for 15 min in dark place or wrap with aluminum foil, the color of TMB reagent would turn into blue. The degree of blue color depends on how many HRP on the well surface. Generally, the more concentrated analyte in the well, the color would get darker. Quench with 100 µL of sulfuric acid, the color would change again and become yellow. At meantime this yellow solution were be measured at 450 nm by using an ELISA reader called Thermo Scientific Multiskan EX microreader.
3.3 References
(1) Hsu, S. M.; Raine, L; Fanger, H. J. Histochem. Cytochem. 1981, 29, 577-580.
(2) Avrameas, S.; Uriel, J. C R Acad. Sci. Hebd. Seances. Acad. Sci. D 1966, 262, 2543-2545.
Chapter 4
Protein Conglomeration Based Amplification Method
4.1 Principles of PCBAM
The development of protein conglomeration based amplification method (PCBAM) may improve the current detection technology. The concept of this method is based on the procedure of ELISA. As noted above, biotinylated antibodies and streptavidin (SA) are the core of this method.1 According to the affinity constant (Ka) between streptavidin and biotin is about 1015 M-1, it is like covalently attach to each other in the solution. Once they bind together, the interaction would be an irreversible reaction essentially. Hence, this reaction regards as a high affinity and specific interaction. In addition, the streptavidin-biotin complex is very stable in a wide range of pH value and temperature. Therefore, the streptavidin-biotin system can be widely used in immunocytochemistry such as cell sorting or immunoprecipitation.2
We can take advantage of the feature of streptavidin-biotin system to synthesize the streptavidin-detection antibody complex (SA-DA). Based on SA has four biotin binding sites, utilize one binding site to attach the biotinylated antibody which was on the surface of wells. While other three sites are bind to another types of detection antibody (DA) (see Figure 2, step 3). Then the secondary antigen was added to attach the SA-DA and the secondary antibody was added again at the following step
to seize antigen. Subsequent go back to add the SA-DA again. These operation steps were repeated layer by layer, and the large protein polymers would form to achieve signal amplification.
4.2 Research of ELISA
4.2.1 The calibration curves of IL-7 and IL-4
Since PCBAM is based on the procedure of the sandwich ELISA, realization of the principle of the ELISA is warranted. We exercised the human interleukin-7 (IL-7) ELISA by following to the procedure of the protocol. Steps are described in Figure 15. The first step was supplying 100 μL of various concentration of IL-7 antigen and 50 μL of IL-7 detection antibody (DA7) in the capture antibody pre-coated micro-well plate and incubated at room temperature (rt) for 120 min. Washed three times by washing buffer afterwards. The second step is adding 100 μL of S-HRP to incubate for 20 min. Then washed three times again and supply the 100 μL of TMB in the dark environment (e.g., warp with aluminum foil or put it in the drawer) to develop for 15 min at third step. Finally, 100 μL of 1 M sulfuric acid is use for quench the reaction. Measured the micro-plate with ELISA reader at wavelength of 450 nm. The data was shown in Table 1 and the calibration curve of IL-7 as shown in Figure 16A.
TMB
Antigen Biotinylated
antibody Primary
antibody
Streptavidin-Horseradish
Peroxidase (S-HRP) 3,3',5,5'
-tetramethylbenzidine TMB =
Figure 15. The procedure of ELISA. Primary capture antibody was coated on the microarray plate. Specific antigen and secondary biotinylated antibody was added to form a sandwich complex. Supplied the S-HRP to seize the biotin which on the biotinylated antibody via streptavidin-biotin system. TMB substrate was added to react with HRP enzyme and produce blue solution at the final step.
Table 1 is simply shown the absorbance of concentration of IL-7 antigens ranging from 50.0 pg/mL to 3.1 pg/mL after subtracting the background. The absorbance of IL-7 antigen decreased along with concentration. The differential value of absorbance was relative low at 6.2 pg/mL. It started to close to zero showing ELISA at this concentration is near to the detection limit.
Table 1. Absorbance in detecting IL-7 antigen with ELISA
IL-7 antigen (pg/mL) Absorbance
50.0 0.160
25.0 0.082 12.5 0.041 06.2 0.017 03.1 0.009
The procedure of human interleukin-4 (IL-4) ELISA is same as IL-7, whereas the antibodies and antigens are using IL-4 detection antibody (DA4) and IL-4 antigen instead. During operate the IL-4, it must be noted that the concentration of IL-4 antigen is different to IL-7. The recommended concentration in the IL-4 protocol is 35.0 pg/mL. As shown in Table 2, the data shows the absorbance which is deducted by background. The concentration ranged from 35.0 pg/mL to 0.1 pg/mL.
Observe in Table 2, when the concentration at about 2.2 pg/mL, the absorbance is beginning not clearly to distinguish the difference.
Especially between the 1.1 pg/mL and 0.1 pg/mL is extremely closed.
Implies that the detection limit of IL-4 is 2.2 pg/mL, approximately.
Table 2. Absorbance in detecting IL-4 antigen with ELISA
IL-4 antigen (pg/mL) Absorbance
35.0 0.387
The absorbance of IL-7 antigen decreases along with concentration and has regular calibration curve in Figure 16A. The r squared value is 0.9992, which is indicating that the test has a high accuracy. IL-4 also shows linear calibration curve in ELISA method which shows in Figure 16B. The calibration curve in IL-4 has r squared value at 0.9976.
Figure 16. The calibration curves of (A) IL-7 and (B) IL-4. IL-7 antigen has regular calibration curve from 50.0 pg/mL to 3.1 pg/mL. The r square value is 0.9992. IL-4 antigen also shows a linear calibration curve from 35.0 pg/mL to 0.1 pg/mL in ELISA method and r squared value is 0.9976.
4.2.2 The detection limit of IL-7
After understood the operation of ELISA procedure, we further examine the detection limit of IL-7 in ELISA. For the purpose of finding the detection limit of ELISA, we detect various concentration of IL-7 antigen with ELISA. The results present in Table 3 and Figure 17, the concentration range is from 1.0×102 pg/mL to 6.2×10-10 pg/mL and the absorbance are deducted by background which shows in following Table 3.
The absorbance would less than 0.040 after subtracting the background when the concentration lower than 6.2 pg/mL. Additionally, these absorbance which below 6.2 pg/mL are near or less than 0.100 before subtracting the background. Furthermore, the concentration which is lower than 6.2 pg/mL would almost have no difference among these various concentration. It means the limit of detection has been reached in ELISA at about 6.2 pg/mL.
Table 3. The detection limit test in low concentration of IL-7
IL-7 antigen (pg/mL) Absorbance
1.0×10-2 0.815
Take log to each concentration and obtained the log values which can versus to the absorbance to gain the Figure 17. We can obviously find the relationship between concentration and absorbance. After 6.2 pg/mL, the concentration of IL-7 antigen from 6.2×10-2 pg/mL to 6.2×10-10 pg/mL showed a horizontal line. These results indicated the detection limit of IL-7 antigen with ELISA has been reached and cannot measure the properly absorbance of concentration.
Log [IL-7 antigen]
Figure 17. The detection limit test in low concentration of IL-7.
Normalize the curve on log-concentration and versus the absorbance. The range of IL-7 antigen concentration is from 1.0×102 pg/mL to 6.2×10-10 pg/mL. A flat line indicated the ELISA has reached the detection limit.
The raw absorbance which less than 0.100 shows a lack of S-HRP to support the analyte. Hence, the result shows less significance. It is because
S-HRP is the role that responsible for reactive with TMB substrate. If the S-HRP is less on the well, TMB cannot function with enough enzyme, thus did not has color change obviously. Therefore, the absorbance would near to 0.100 or less.
4.2.3 The specific test of IL-4 and IL-7
On the basis of the theory that antibody is specific binds to antigen, this experiment demonstrate the specificity of antigen and antibody. IL-4 and IL-7 antigen was introduced into the IL-4 capture antibody coated well, respectively. In the meantime, treated with DA4 or DA7 to seize different antigen and incubated for 120 min at rt. After wash the well by using wash buffer 3 times, then move on S-HRP step. The result is presented in Table 4.
From Table 4, the result exactly describes the specificity of IL-4 and IL-7.
The absorbance of IL-7 is less than 0.100 indicates both of them would not bind with each other.
Table 4. The specific test of IL-4 and IL-7 IL-7 antigen
(pg/mL) Absorbance IL-4 antigen
(pg/mL) Absorbance
100.0 0.095 35.0 0.584
050.0 0.077 17.5 0.309
025.0 0.095 08.8 0.164
012.5 0.085 04.4 0.099
Microtiter plate was coated with IL-4 capture antibody for ready to use, wash the well with wash buffer 3 times before change into next step.
On account of IL-4 capture antibody (CA4) is not specific to IL-7 antigen so IL-7 antigen just binds to DA7 forming the mixture and do not attach to the surface via binding to CA4. After washing step, the unattached mixture would be washed away. S-HRP would also be washed away due to it cannot find the DA7 in the well. Thus, TMB substrate cannot react with S-HRP cause the low absorbance. In contrast, treat with the IL-4 antigen and DA4 in the IL-4 capture antibody coated well has totally different results. Table 4 shows the absorbance of IL-4 all bigger than IL-7 antigen.
4.3 Studies on PCBAM
4.3.1 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
7with 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
(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