Chapter 2. Literature Reviews
2.8 Dendritic amplification
2.8.2 Laminar flow-assisted dendritic amplification
In order to reduce the incubation steps, the Laminar flow-assisted dendritic amplification (LFDA) was devised. LFDA was design by treating two reagents, F-SA and B-anti-SA simultaneously and continuously and
supplied from two laminar streams formed by a Y-shaped microchannel (Figure 14B). As author describe, laminar flow means fluid flow without transverse velocity, when two different solutions (F-SA and B-anti-SA) are pumped into a microchannel, they are mixed only by diffusion through the interface between the two streams. At the contact line among the two streams and the channel floor, the components in the two streams are simultaneously and continuously supplied onto the solid surface. During the flow, the unbind molecule was taken away by the stream, hence needs no washing step and shorten the operation time. The assay can be performed through minimum incubation steps with the LFDA, which exploits the advantage inherent to the microchip format and provides a new opportunity for ultrasensitive point-of-care testing.
From this reference, we find that LFDA is extremely similar to our idea, PCBAM. The PCBAM is using a streptavidin-biotinylate antibody complex and antigen to generate the signal, while LFDA using FITC-labeled streptavidin (F-SA) and biotinylated anti-streptavidin (B-anti-SA) to make amplification. The interaction between B-anti-SA and F-SA is not only use streptavidin-biotin system but also use antibody-antigen sytem (SA and anti-SA). LFDA encourages us that PCBAM is a promising proposal.
2.9 References
(1) Schmitt, O.; Preuße, S.; Haas, S. J. P. Brain. Res. Protoc. 2004, 12, 157-171.
(2) Engvall, E.; Perlmann, P. Immunochem. 1971, 8, 871-874.
(3) Gosling, J. P. Clin. Chem. 1990, 36, 1408-1427.
(4) Sternberger L. A.; Hardy P. H.; Cuculis J. J.; Meyer H. G. J.
Histochem. Cytochem. 1970, 18, 315-333.
(5) Guesdon, J. L.; Ternynck, T.; Avrameas, S. J. Histochem. Cytochem.
1979, 27, 1131-1139.
(6) Hsu, S. M.; Raine, L; Fanger, H. J. Histochem. Cytochem. 1981, 29, 577-580.
(7) Avrameas, S.; Uriel, J. C R Acad. Sci. Hebd. Seances. Acad. Sci. D 1966, 262, 2543-2545.
(8) Sikes, H. D.; Hansen, R. R.; Johnson, L. M.; Jenison, R.; Birks, J. W.;
Rowlen, K. L.; Bowman, C. N. Nat. Mater. 2008, 7, 52-56.
(9) Sikes, H. D.; Jenison, R.; Bowman C. N. Lab Chip 2009, 9, 653-656.
(10) Hosokawa, K.; Omata, M.; Maeda, M. Anal. Chem. 2007, 79, 6000-6004.
(11) Hosokawa, K.; Omata, M.; Sato, K.; Maeda, M. Lab Chip 2006, 6, 236-241.
Chapter 3
Materials and Methods
3.1 Materials 3.1.1 ELISA kit
Human IL-7 and IL-4 kit which contain IL-7 capture antibody B-N18 (CA7) and detection biotinylated antibody B-S16 (DA7), IL-7 and IL-4 antigen standard, IL-4 capture antibody B-R14 (CA4) and IL-4 detection biotinylated antibody B-G28 (DA4), streptavidin-horseradish peroxidase conjugate (S-HRP), 3,3',5,5'-tetramethylbenzidine (TMB) were purchased from Abcam and Gene-probe. All ELISA reagents were used without further purification. The spectrophotometer measurements were acted by Thermo Scientific Multiskan EX microreader.
3.1.2 Buffer
0.010 M phosphate-buffered saline (PBS) (Sigma-Aldrich) consisted of 0.137 M NaCl, and 0.270 mM KCl (pH = 7.4) which is known as coating buffer. Another recipe of PBS buffer is 0.100 M Na2HPO4 and 0.100 M NaH2PO4 mixed together to reach pH 7.4. Blocking buffer is consisted of a PBS with 5% (w/v) bovine serum albumin (BSA) (Sigma-Aldrich). Washing buffer is consisted of a PBS solution with 0.05% (v/v) Tween 20 (pH = 7.4). Standard diluent buffer is PBS
contained 1% BSA. PBS-BSA binding buffer is consisted of 1.00 g BSA and 0.88 g NaCl in 100 mL PBS buffer. HRP-streptavidin diluent buffer is contained PBS with 0.1% tween 20 and 1% BSA (w/v). Tris-buffered saline (TBS) is consisted of 0.025 M Tris (Bio-Rad) and 0.150 M NaCl.
The pH of TBS is adjusted to 7.2 with 12 N HCl (Sigma-Aldrich).
3.1.3 Reagents
Streptavidin (SA) was purchased from Jackson Immuno Research.
Biotin quantitation HABA kits, biotinylated horseradish peroxidase (B-HRP), Poly-HRP, NHS-PEG4-biotin, and NHS-PEG12-biotin were purchase from Thermo Scientific.
3.2 Methods for development of PCBAM 3.2.1 Synthesis of biotinylated BSA
Biotinylated BSA (bBSA) is an important reagent in our methods. It combines protein and biotin together and plays a role to bind to streptavidin or S-HRP. The procedure as following: Add 170 µL of DI water into NHS-PEG4-biotin or NHS-PEG12-biotin, the concentration would become 0.020 M. Took 100 µL of this reagent and mixed with 2.0 mg/mL of BSA. The more NHS-PEG-biotin reagent reacts with BSA would obtain more biotin on the BSA surface. Synthesis was at 4 °C for
overnight. One day later, filter the unreacted NHS-PEG4-biotin or NHS-PEG12-biotin with spin column (cutoff 10 kDa). Loading the bBSA in the spin column and centrifuge at 10000 gravity (g) for 20 min. Discard the filtrate and add PBS into spin column then centrifuge at 10000 g for 20 min again. Operate the filter step 3 times and then reverse the spin column to spin down the bBSA. Centrifuge at 1000 g for 30 min. Reconstitute the bBSA with PBS at last.
3.2.2 Quantify the amount of biotin on the bBSA and B-HRP
Equilibrate the HABA/Avidin Premix to room temperature first, and then add 100 μL of DI water to dissolve the powder and mix with pipette tip. Pipette 800 μL of PBS into a 1 mL cuvette. Use this cuvette with PBS to zero the spectrophotometer. Add the 100 μL of the HABA solution from to the cuvette. Mix by inversion, pipettes it up and down. Measure the absorbance of the solution in the cuvette at 500 nm. Add 100 μL of bBSA, to the cuvette and mix well. Use B-HRP as positive control. Measure the absorbance at 500 nm again. The result shows there has 6 or 7 biotins on each bBSA while B-HRP has 2 biotins.
3.2.3 ELISA
Took 50 µL of IL-7 capture antibody (1 mg/mL) into 10 mL PBS and dispense 100 µL in each well and coating at 4 °C for overnight. Wash 3 times with wash buffer and block with 5% BSA for 2 h, each well needs 250 µL. Dump the solution out and tap the plate on paper towel after 2 h and put the plate dry on the bench at rt for 24 h. Then the plate is ready to use. Incubated with 100 µL of IL-7 antigen at various concentrations and 50 µL of IL-7 biotinylated detection antibody (DA, 1.2×10-3 µg/mL) incubated for 2 h to form the putative complexes which shown in Figure 6C. After 3 times wash, add 100 µL of S-HRP (1.5×10-4 mg/mL) into well incubated for 20 min. Treat with 100 µL TMB and put in the dark place for 15 min in final step.
3.2.4 ABC method
The protocol of ABC method in this study is a little different to original protocol which describe in the paper.1-2 The reagents are included streptavidin and B-HRP. Therefore, this method is applied in detecting the biotinylated protein such as bBSA or DA. For detecting the bBSA, it should coating the bBSA on the plate first, each well need 100 µL, and the concentration range is from 1.0×103 pg/mL to 3.0×10 pg/mL. Put the plate at 4 °C overnight. Wash 3 times and then add the 100 µL of streptavidin (1.5×10-4 µg/µL) incubate for 20 min. After 20 min, wash 3 times and then
add 100 µL of B-HRP (1.5×10-4 µg/µL) for 20 min. Repeat adding streptavidin and B-HRP until final step. The final step is wash 3 times and adds 100 µL of TMB in each well develops for 15 min in dark place.
3.2.5 BiHRP method
For detecting the bBSA, dispense 100 µL of bBSA (1.0×102 pg/mL) in each well surface first. Put this 96 wells plate at 4 °C for overnight.
Dump out the solution and wash 3 times with wash buffer. Tap the plate on paper towel and then dispense 100 µL of S-HRP (1.5×10-4 mg/mL) in each well for 20 min. After incubated the S-HRP, wash 3 times and then add 100 µL of B-HRP (1.5×10-4 mg/mL) incubate for 20 min. Repeat adding S-HRP and B-HRP layer by layer until final step. Wash 3 times then adds 100 µL of TMB in each well and put in the dark place develops for 15 min.
3.2.6 SHRPSA method
SHRPSA method is using S-HRP and bBSA as reagents. In detecting bBSA experiment, dispense 100 µL of bBSA (1.0×102 pg/mL) in each well first; while in DA experiment, dispense various concentration of DA (From 2.0×102 pg/mL to 3.0×10 pg/mL) 100 µL in the well. Put the plate at 4 °C refrigerator for overnight coating. Dump out the bBSA and wash 3 times with wash buffer. Then dispense 100 µL of S-HRP (1.5×10-4 µg/µL)
incubate for 20 min. After 20 min, wash 3 times and then add 100 µL of bBSA (1.5×10-4 µg/µL) for 20 min. Repeat adding S-HRP and bBSA about 3 to 5 cycles. 1 cycle means the operation include adding S-HRP and bBSA.
Wash 3 times, treat with 100 µL of TMB and put in the dark place for 15 min in final step.
3.2.7 Poly-SHRPSA method
The significant reagent in this method is use poly-HRP, a polymer conjugate with plentiful S-HRP. In detecting DA experiment, coating the plate as following: Dispense various concentration of DA (From 8.0×102 pg/mL to 1.2×10 pg/mL) 100 µL in the well and put in the 4 °C refrigerator for overnight. Dump out the DA and wash 3 times with wash buffer.
Subsequent dispense 100 µL of poly-HRP (1.0×10-4 µg/µL) incubate for 20 min. Put the plate on the orbital shaker at 160 rpm in every step. After 20 min, add 100 µL of bBSA (1.5×10-4 µg/µL) directly into the well for reacting 20 min without wash, the total volume in the well would become 200 µL. After another 20 min, dump out the reagents and wash 3 times.
Treat with 100 µL of poly-HRP again. Repeat adding poly-HRP and bBSA for 3 cycles. The cycle means the operation includes adding poly-HRP and bBSA. The final step is wash 3 times and adds 100 µL of TMB in each well develops for 15 min in dark place.
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
means of treating reagents continuously layer by layer would amplified the