Professional Documents
Culture Documents
ii
Abstract
The detector antibody was optimized its conjugation method for the
DNA-streptavidin complex. Two methodologies were tested the effect of the ratio
of DNA to streptavidin and the ratio antibody to DNA-streptavidin complex. The
1:1 ratio of antibody to DNA-streptavidin complex was found to give the best
result and therefore selected for further test.
reagents and assay formats is necessary to reduce the high background problem in
this study.
ii
PCR
DNA DNA
PCR
DNA streptavidin DNA-Streptavidin
DNA-Streptavidin
DNA-Streptavidin 11
DNA 5 0.5
mg/ml human IL6 <0.001 pg/ml
iii
Acknowledgement
iv
Contents
Abstract
................................................................................................................ i
.............................................................................................................. iii
Acknowledgement.................................................................................................. iv
Contents .............................................................................................................. vi
List of Figure........................................................................................................ viii
List of Table ........................................................................................................... ix
Chapter 1. Introduction ........................................................................................... 1
1.1 Antibody-antigen interaction ...................................................................... 1
1.2 Immuno-PCR .............................................................................................. 9
1.3 Lateral flow immunochromatography assay............................................. 13
1.4 Motivation ................................................................................................. 19
1.5 Objective ................................................................................................... 21
Chapter 2. Principle .............................................................................................. 22
2.1 Antibody-DNA conjugation...................................................................... 22
2.2 Lateral flow ............................................................................................... 24
2.3 Membrane-based immuno-PCR................................................................ 26
Chapter 3. Experimental ....................................................................................... 29
3.1 Materials.................................................................................................... 29
3.1.1 Polymerase Chain Reaction ................................................................ 29
3.1.2 Agarose gel electrophoresis ................................................................ 30
3.1.3 Lateral flow immuno-PCR.................................................................. 30
3.1.4 Buffer Solution.................................................................................... 31
3.2 Instruments................................................................................................ 33
3.3 Methods..................................................................................................... 35
3.3.1 Antibody-DNA conjugation................................................................ 35
3.3.2 Preparation of immunochromatographic test strips ............................ 38
3.3.3 Optimization of lateral flow-immuno-PCR assay............................... 40
3.3.4 Determination of the optimal concentration of capture antibody ....... 43
3.3.5 Sensitivity test of lateral flow immuno-PCR for human IL-6 ............ 44
vi
vii
List of Figure
Figure 1-3 Three proposed models described antibody-antigen interaction........... 8
Figure 1-4 The format of immuno-PCR and ELISA.. .......................................... 10
Figure 1-5 The methods employed to conjugate DNA onto antibody.. ................ 12
Figure 1-6 Two formats of the lateral-flow immunochromatography assay. ....... 18
Figure 2-1 Formation of the DNA-conjugated antibody....................................... 22
Figure 2-2 Band distribution of DNA-streptavidin complex on agarose gel
electrophoresis. ..................................................................................... 23
Figure 2-3 Basic principle of the lateral flow immunochromatography assay ..... 26
Figure 2-4 The principle of membrane-based immuno-PCR................................ 28
Figure 3-2 DNA-conjugated antibody formation.................................................. 37
Figure 3-3 Preparation of immunochromatography strips. ................................... 39
Figure 3-4 Optimization of lateral flow immuno-PCR in order to get low
background with two different methods.. ............................................. 42
Figure 4-1 Analysis of DNA-streptavidin complex in 2% agarose gel ................ 48
Figure 4-2 Conjugation of biotinylated antibody with DNA-streptavidin
complex presented in 2% agarose gel electrophoresis. ........................ 49
Figure 4-3 Agarose gel electrophoresis of optimization of lateral flow
immuno-PCR. ....................................................................................... 52
Figure 4-4 Optimization of sample pre-treatment................................................. 52
Figure 4-5 Optimization of waiting time. ............................................................. 54
Figure 4-6 Optimization of test waiting time........................................................ 54
Figure 4-7 Optimization of spraying concentration of capture antibody.............. 56
Figure 4-8 Optimization of spraying concentration of the capture antibody ........ 56
Figure 4-9 The sensitivity test of lateral-flow immuno-PCR for detect human
IL-6. ...................................................................................................... 59
Figure 4-10 The sensitivity test of the lateral-flow immuno-PCR on human
IL-6. ...................................................................................................... 59
viii
List of Table
Table 1-1 Overview of applications of lateral-flow immunochromatography
assay...................................................................................................... 14
Table 3-1 Design of the concentration ratio of biotinylated DNA to
streptavidin.. ......................................................................................... 35
Table Appendix 1 The Result of ImageJ measurement on the electrophoresis
band of antibody-antigen interaction. ................................................... 66
Table Appendix 2 The Result of ImageJ measurement on the electrophoresis
band of waiting time optimization........................................................ 66
Table Appendix 3The Result of ImageJ measurement on the electrophoresis
band of the spraying concentration of capture antibody....................... 67
Table Appendix 4 The Result of ImageJ measurement on the electrophoresis
band of the sensitivity test of the lateral-flow immuno-PCR ............... 67
ix
Chapter 1. Introduction
1.1
Antibody-antigen interaction
determinant or epitope. Nucleic acids and lipids are antigenic when these two
molecules conjugated with protein or polysaccharide.
H2
N
NH2
5
3
H2N
NH2
1
6
S
S
S
S
S
HOOC
COOH
HOOC
COOH
similar in the principle that the protein must have precise orientation to fit with the
substrate except that the fitting only occurs after the changes induced by the
substrate itself. Study performed by Betts and Sternberg [5] on the conformational
changes for a set of 39 complexes and predominantly enzyme inhibitors, leading
to the conclusion that proteinprotein recognition occurs by the mechanism of
induced fit. However, Bosshard [6] noted that induced fit is possible only if the
match between the interacting sites is strong enough to provide the initial complex
strength and longevity so that induced fit takes place within a reasonable time. He
also pointed that there is an alternative mechanism to induced fit, called the
preexisting equilibrium/conformational selection model [6]. According to this
model, the antigen binding site on antibody doesnt need to make some change on
conformational to fit with the antigen. The antibody just simply selects the antigen
that has epitope with precise conformation with its binding site or in reciprocal
term, the antigen selects the antibody that has complementary conformation [7].
Studies conducted by Berger [8] and Foote & Milstein [9] validate this model.
However, Bosshard [6] stated that even the conformational selection model was
valuable and alternative for induced fit, it didnt mean that induced fit didnt occur
in protein-protein interaction. Actually, combination of these two models seems to
be the best to describe the interaction between molecules that apparently do not
actually fit to begin with.
antigen
(C)
proposed
by
Bosshard,
is
called
the
preexisting
1.2
Immuno-PCR
The development of immunoassay has grown rapidly since its first
Substrate
Enzyme
DNA
2nd antibody
2nd antibody
PCR
Product
1st antibody
Immuno-PCR
1st antibody
ELISA
10
11
protein A chimera
DNA
DNA
DNA
chemical crosslinker
Figure 1-5 The methods employed to conjugate DNA onto antibody. (A)
ProteinA-strevtavidin chimera is used to connect biotinylated DNA and the Fc
region of IgG. (B) Streptavidin conjugate biotinylated DNA and biotinylated
antibody. (C). DNA covalenty linked to antibody.
12
1.3
Lateral flow immunochromatography is the most widely used membranebased immunoassay application. Compare to other methods, lateral flow gives an
easy of use and short assay time. The application of membrane on immunoassay
was started in 1979 by Towbin et al. in demonstrating that protein can be
transferred to microporous membrane nitrocellulose and detected using antibodies
[25].
proliferated. The first test of lateral flow was made for detection of human
chorionic gonadotropin (GDP). Nowadays, the lateral flow has been used for
monitoring ovulation, detecting infectious disease organisms, analyzing drugs of
abuse, and measuring other analytes important to human physiology. Table 1-1
provides an overview on the variety of biomedical applications realized so far.
13
Antigen
Year
Bacteria
Vibrio harveyi
2007
Virus
Canine distemper
2008
Hormone
19-Nortestosterone
2007
class
Bacteria
Cryptosporidium
parvum.
2000
Remarks
Ref.
Non-
Sithigorngul P
competitive
et al. [26]
Noncompetitive
Competitive
Non-
Kozwich et al.
competitive
[29]
Cardiac
marker
acid-binding protein
Toxin
Aflatoxin B1
2005
Competitive
Hormone
Clenbuterol
2006
Competitive
Antibiotic
Sulfonamides
2007
Competitive
Bacteria
Toxin
Insecticide
Nucleic
acid
Enzyme
Food
additive
Cells
protein
Legionella
pneumophila
Microcystins
Carbaryl and
endosulfan
Single stranded DNA
Canine Trypsin-like
immunoreactivity
2003
2006
Noncompetitive
Noncompetitive
Chan et al.[30]
Delmulle et al.
[31]
2003
Competitive
2006
Competitive
Nucleic acid
Corstjens et al.
lateral flow
[37]
Non-
Waritani et al.
competitive
[39]
2003
2007
Glycyrrhizin
2005
Frataxin
2008
Noncompetitive
Noncompetitive
Putalun et al.[38]
14
15
In the competitive format, the target analytes bind either to the reporter
particles or to the immobilized ligands. In the first case, the target analytes bind to
the ligands and block the ligands from binding to the reporters. This format was
used by Ho and Wauchope [43] to detect aflatoxin B1 (AFB1). The target analytes
was conjugated with liposome and the AFB1 antibody is immobilized at the
capture zone, where the competition occurs between AFB1-conjugated liposome
and AFB1. In the second format of competitive assay, the target analytes was
bound to the reporters and block these reporters from binding to the immobilized
16
17
18
1.4
Motivation
approximately two
hours.
The
application
of
PCR
technique
in
immunoanalytical field has been proved increasing the detection limit of the assay.
On the other hand, immuno-PCR, immunoassay based PCR as the signalgenerated method, has been reported amplifying signal with 100-10000 folds
greater than that by ELISA.
19
20
1.5
Objective
The objective of this study was to develop a membrane-based immuno-
21
Chapter 2. Principle
2.1
Antibody-DNA conjugation
22
23
2.2
Lateral flow
24
25
2.3
Membrane-based immuno-PCR
The principle of membrane-based immuno-PCR is described in Figure 24. The format of membrane-based immuno-PCR is similar with the indirect assay
of immuno-PCR. There are two antibodies used to capture the antigen. The first
antibody is immobilized on membrane nitrocellulose by spraying it onto the
surface of membrane. The membrane binds the antibody with several forces,
mainly hydrophobic interaction and electrostatic forces. The second antibody is
premixed first with the antigen. Premixing can reduce the stoichiometry
26
For the amplification of DNA, the membrane piece, where the antibody
is immobilized, is cut off and put into a PCR tube to mix with PCR cocktail
containing primer, Taq DNA polymerase, dNTP, and water. The mixture is heated
to release the DNA from the membrane so that the amplification process will be
effective. The final product of PCR is then analyzed on gel electrophoresis.
.
27
28
Chapter 3. Experimental
3.1
Materials
3.1.1
Forward primer 20 mer (TAG CAC GGT CAT ATA TGA TG) was
purchased from Purigo Biotech, Inc (Taiwan). The primer was designed to
anneal to the template strand of DNA and allow the Taq Polymerase to
synthesize a strand complementary from the template strand.
Reverse primer 20 mer (GAA GGA AAC AGT TAC ATT TC) was
purchased from Purigo Biotech, Inc (Taiwan). The primer was designed to
anneal to the template strand of DNA and allow the Taq Polymerase to
synthesize a strand complementary from the template strand.
29
PCR standard buffer from New England Biolab (England), to promote Taq
usage and maintain the pH of solution during PCR
3.1.2
Agarose was from Biobasic (Canada) to make agarose gel to analyze DNA.
Ethidium bromide was from BioBasic (Canada) and was used to stain the
agarose gel, so that the gel bands can show on the Gel Imaging System.
3.1.3
30
Absorbent pad CS6 was from Whatman to keep the capillary force in the
lateral flow immuno-PCR assay.
Plastic backing pad was from Adhesive Research, Inc as a backing for
lateral flow materials.
Sample pad 33 Glass was from S&S, used to hold the sample of lateral
flow immuno-PCR.
3.1.4
Buffer Solution
Sodium chloride was from Bio Basic Inc, as material for making PBS
solution and protein lateral flow buffer
Potassium chloride was from Bio Basic Inc, as material for making PBS
solution
31
Tween 20 was from Bio Basic Inc, as material for making protein lateral
flow buffer.
Bovine serum albumin was from Bio Basic Inc, as material for making
protein lateral flow buffer.
HEPES was from Calbiochem, as material for making protein lateral flow
buffer.
Tris (base) was from Bio Basic Inc, as material for making protein lateral
flow wash buffer.
Magnesium chloride was from Bio Basic Inc, as material for making
protein lateral flow wash buffer.
32
3.2
Instruments
Microcentrifuge
(Mikro
120
Hettich
Zentrifugen,
Germany)
for
Biohazard safety hood was from High Ten Scientific Corp, for preparing
PCR and protein dilution.
33
Globals
Enter
Plate
Parameters
Enter
App. Position Y
Track
Assignment
Enter
Track No 1
Track Volume ( 1l)
Sample ID
Save Method
Enter
Default method
Method No. 3
Figure 3-1 Structure and the example of parameter input dialog of Linomat 5
TLC machine for a typical spraying procedure. Following the procedure shown
above to key in appropriate parameters, the TLC machine sprayed the desired
strips for the lateral flow test.
34
3.3
3.3.1
Methods
Antibody-DNA conjugation
The conjugation between antibody and DNA principally follow the
method of Niemeyer et al. [46]. The antibody was conjugated with DNA using
streptavidin (SA) as a bridge, utilizing the high affinity nature between biotin and
streptavidin (Figure 3-2). First, DNA was conjugated with streptavidin to formed
DNA-streptavidin complex. Conjugates of streptavidin and biotinylated dsDNA
fragment were typically prepared by adding DNA (3 M in ddH2O) to PBS and
subsequently added streptavidin (100 g/ml in PBS) until the total volume was 15
l (Table 3-1).
5:1
4:1
3:1
2:1
1:1
1:2
1:3
1:4
1:5
DNA 3 M
(l)
1.5
1.5
1.5
1.5
1.5
1.5
1.5
1.5
1.5
0.5
0.65
0.9
1.35
2.7
5.4
8.1
10.8
13.5
13
12.85
12.6
12.15
10.8
8.1
5.4
2.7
Streptavidin
100 g/ml
(l)
PBS buffer
(l)
35
36
+
Biotinylated DNA
Streptavidin
DNA-Streptavidin Complex
Biotinylated Antibody
Antibody-DNA conjugate
37
3.3.2
38
39
3.3.3
The second parameter was the test waiting time for reducing background
after the immunochromatography strip was applied with a sample. The waiting
40
time was set at 2 min, 5 min, and 7 min. After applying the wash buffer, the
membrane around the test line was cut into about a 2mm piece. This membrane
piece was mixed with the PCR cocktail containing 10 l of 1 M of each primer,
10 l of 1 mM of dNTPs, 5 l of 10x standard Taq buffer and 0.4 l of Taq DNA
polymerase. The mixture was then subjected to PCR with the thermal cycling
conditions 95 C for 5 min as the initial denaturation, then 42 cycles of 95 C for
20 s, 53 C for 30 s and 72 C for 20 s. In the last cycle, 72 C was extended to 5
min and cooled to 4 C for 30 min. The PCR products were finally loaded in a 2%
agarose gel electrophoresis and the intensity of each band was measured using
ImageJ software.
41
42
3.3.4
43
3.3.5
3.3.6
ImageJ software was used to analyze the intensity of agarose gel image.
The software can be obtained freely from NIH website (http://rsb.info.nih.gov/ij/).
The tutorial for 1D gel image analysis using Image J can be loaded from internet.
In briefly, the band was selected using the rectangular selection tool by outlining
the first lane. In the ImageJ toolbar, selected Analyze>Gels>Select First Lane (or
44
press "1") and "Lane 1 selected" will be displayed in the status bar (Figure 3-5.A).
Analyze>Gels>Plot Lanes (or press "3") was selected to generate the lane profile
plots. The base lane was drawn in each peak using the straight line selection tool
(Figure 3-5.B) . The size for each peak was measured by clicking inside with the
wand tool (Figure 3-5.C). The data was saved and analyze further using Microsoft
Excel.
45
A)
B)
C)
D)
Figure 3-5 Analysis of gel image using ImageJ software. (A) Outlining the band using the rectangular selection tools. (B) Plotting
the line. (C) Measuring the area of each peak. (D) Saving the data.
46
47
Lane
DNA ratio
SA ratio
Free
DNA
1
5
1
2
4
1
3
3
1
4
2
1
5
1
1
6
1
2
7
1
3
8
1
4
9
1
5
48
Lane
DNA-SA
ratio
Antibody
ratio
1
M
Free
10
1-5
DNA
49
4.2
To get a high signal to noise ratio, the lateral flow immuno-PCR was
optimized. Two approaches were conducted for this optimization. In the first
approach, the reactions between antigen and antibody-DNA conjugate was
50
premixed at a tube; whereas in the second approach, they were step by step added
on membrane. Their immuno-PCR products were run on a 2% gel electrophoresis
as shown in Figure 4-3. In this Figure 4-3, the premixing approach gave a lower
background and higher test intensity than the step-by-step approach. In order to
more clearly indicate the optimization result, the intensity of each band was
further measured by ImageJ software and presented in signal to noise ratio as
shown in Figure 4-4. In the figure, premixing method gave about twice higher
ratio compared with the approach of step by step addition.
51
1a
1b
2a
2b
2.5
signal to noise ratio
2
1.5
1
0.5
0
sequential
Pre-mixed
52
53
1a
1b
2a
2b
3a
3b
Figure 4-5 Optimization of waiting time. The immuno-PCR products were run
on a 2% gel electrophoresis. The waiting time set at (1) 7 min, (2) 5 min and (3) 2
min. Signals from (a) sample and (b) background were compared with each other.
2.5
2
1.5
1
0.5
0
2 min
o
5 min
Waiting time
7 min
54
The graphic of signal to noise ratio showed that 5 min waiting time was
the best choice for the next experiment. It should give enough time for antibody
and antigen to reach the stoichiometry for reaction. In other case, 2 minute waiting
time was not long enough for antigen and antibody to reach the stoichiometry for
reaction, such that when a wash was applied, the capture antibody was not strong
enough to bind with the antigen and therefore washed away. Two minutes were
also possibly not long enough to allow all samples to flow through the test line
and thus the residual samples were carried by the wash buffer to cause a
background. For the case of 7-minute waiting time, its sample signal was lower
than the 5-minute one, but its background signal was also higher. The higher
background was possibly caused by unwashed antibody-DNA conjugate on
nitrocellulose membrane. The antibody-DNA conjugate already bound strongly to
the membrane for a long waiting time, such that it couldnt be removed when the
wash was applied.
55
a
b
1 mg/ml
b
a b
b
a
a
0.5 mg/ml 100 g/ml 10 g/ml
2.5
signal to noise ratio
2
1.5
1
0.5
0
0.01 mg/ml
0.1 mg/ml
0.5 mg/ml
1 mg/ml
56
57
4.3
sensitivity. The conditions previously optimized were applied in this test. A serial
of ten fold dilution from 10 pg/ml to 0.001 pg/ml of human IL-6 was detected by
the membrane-based immuno-PCR methodologies. The result of the sensitivity
test was presented in Figure 4-9 and Figure 4-10. From the result of gel
electrophoresis analysis, membrane-based immuno-PCR was showed to be able to
detect <0.001 pg/ml of human IL-6. The signal of membrane-based immuno-PCR
in this research was not linear. The critical point of this technique was on how to
cut the membrane correctly. The dimension of the membrane from each antigen
concentration should be cut in the uniform dimension. High variability in the
dimension will result in non linear signal.
58
Figure 4-9 The sensitivity test of lateral-flow immuno-PCR for detect human
IL-6. The lateral-flow immuno-PCR was able to detect 0.001 pg/ml of humanIL6. M = marker, 1-5) Human IL-6 with ten fold serial dilution from 10 pg/ml to
0.001 pg/ml, 6) = negative control,
3000
2500
2000
1500
1000
500
0
0
0.001
0.01
0.1
10
59
Chapter 5. Conclusion
A membrane-based immuno-PCR was developed in this study to increase
the sensitivity of the lateral flow immunochromatography assay. The method was
similar with the conventional lateral flow immunochromatography assay. But
instead the gold nanoparticles-conjugated antibody, this study employed a DNAconjugated antibody as the signal reporter. By the traditional PCR procedure, the
reporter DNA on membrane was amplified to improve the detection sensitivity.
The optimal condition for sample preparation was premixing antigen and
DNA-conjugated antibody and waiting for five minutes after sample application.
Premixing ensured the reaction between antigen and antibody and also prevented
the reaction from creating variable stoichiometry. Five minutes gave enough time
for antibody to react with antigen but did not trap the DNA-antibody in the pore
of the membrane. The concentration of receptor antibody was optimized as 0.5
mg/ml to achieve the highest signal to noise ratio. In addition, the membranebased immuno-PCR can detect <0.001 pg/ml of human IL-6. Further development
on the reagent and method is necessary to reduce the background and to increase
the linearity of the signal.
60
Reference
[1]
Ravetch J and Bolland S (2001). IgG Fc receptors. Annu Rev Immunol 19:
275290.
[2]
[5]
12: 271283.
[6]
Bosshard HR. 2001. Molecular Recognition by Induced Fit: How Fit is the
Concept? News Physiol. Sci. 16: 171173.
[7]
HR.
Demonstration
1995.
of
Spectroscopic,
Conformational
Calorimetric,
Adaptation
in
and
Kinetic
Peptide-Antibody
61
Receptor, and Interleukin-6 on Individual Mouse Embryos by ImmunoPolymerase Chain Reaction. [Thesis]. University of Maine.
[14] Ruzicka V, Marz W, Russ A and Gross W. 1993. Immuno-PCR with a
commercially available avidin system, Science 260: 260261.
[15] Joerger RD, Truby TM, Hendrickson ER, Young RM, and Ebersole RC.
1995. Analyte Detection with DNA-Labeled Antibodies and Polymerase
Chain Reaction. Clin. Chem. 41(9): 1371-1377.
[16] Sims PW, Vasser M, Wong WL, William PW and Meng YG. 2000.
Immunopolymerase chain reaction using real-time polymerase chain
reaction for detection, Anal. Biochem. 281: 230232.
[17] Kozlov IA, Melnyk PC, Stromsborg KE, Chee MS, Barker DL and Zhao C.
2003. Efficient Strategies for the Conjugation of Oligonucleotides to
Antibodies Enabling Highly Sensitive Protein Detection. Biopolymers. 73:
621630.
[18] Adler M, Wacker R, and Niemeyer CM. 2003. A real-time immuno-PCR
assay for routine ultrasensitive quantification of proteins. Biochem. Biophys.
62
[23] Sims PW, Vasser M, Wong WL, Williams PM and Meng GY. 2000.
Immunopolymerase chain reaction using real-time polymerase chain
reaction for detection, Anal. Biochem. 281: 230232.
[24] Lind K and Kubista M. 2005. Development and evaluation of three real-time
immuno-PCR assemblages for quantification of PSA. Journal of
53: 3364-3368.
63
[32] Zhang GP, Wang XN, Yang JF, Yang YY, Xing GX, Li QM, Zhao D, Chai
SJ and Guo JQ. 2006. Development of an immunochromatographic lateral
flow test strip for detection of -adrenergic agonist Clenbuterol residues.
69(6): 669-671.
64
[40] Willis JH, Isaya G, Gakh O, Capaldi RA and Marusich MF. 2008. Lateralflow immunoassay for the frataxin protein in Friedreichs ataxia patients and
carriers. Molecular Genetics and Metabolism.
[41] Harvey MA, Audette CA, and McDonogh R. 1996. The use of microporous
polymer membranes in immunoassays. IVD Tech.
[42] Jones KD. 1999. Troubleshooting protein binding in nitrocellulose
membranes, Part 1: Principles. IVD Tech.
[43] Ho JAA and Wauchope RD. 2002. A strip liposome immunoassay for
aflatoxin B-1. Anal. Chem. 74: 14931496.
[44] Esch MB, Baeumner AJ and Durst RA. 2001. Detection of Cryptosporidium
parvum using oligonucleotide-tagged liposomes in a competitive assay
format. Anal. Chem. 73: 31623167.
[45] Schweitzer B, Wiltshire S, Lambert J, O'Malley S, Kukanskis K, Zhu Z,
Kingsmore SF, Lizardi PM and Ward DC. 2000. Immunoassays with rolling
circle DNA amplification: A versatile platform for ultrasensitive antigen
detection. Proc Natl Acad Sci USA. 97(18): 1011310119.
[46] Niemeyer CM, Adler M, Pignataro B, Lenhert S, Gao S, Chi L, Fuchs H and
Blohm D. 1999. Self-assembly of DNA-Streptavidin nanostructure and their
use as reagents in immuno-PCR. Nucleic Acid Research. 27:4553-4567.
[47] Rasband WS. ImageJ. US National Institutes of Health, Bethesda, Maryland,
USA, http://rsb.info.nih.gov/ij/, 1997-2007.
65
Appendix
Table Appendix 1 The Result of ImageJ measurement on the electrophoresis
band of antibody-antigen interaction for Figure 4-4.
Premixed
Step-by-step
Sample intensity
Background intensity
12969.66
13692.34
5597.024
2.317242
1.247065
10979.65
Sample intensity
Background intensity
2 min
5521.225
4315.773
5 min
7503.125
3222.468
1.279313
2.328378
7 min
4453.225
2843.276
1.56623
66
Sample intensity
Background intensity
1 mg/ml
3975.912
2447.912
1.624205
0.5 mg/ml
6583.912
3154.397
2.087217
0.1 mg/ml
4789.962
3722.79
1.286659
0.01 mg/ml
5537.175
4015.347
1.379003
antibody
Intensity
10 pg/ml
2441.397
1 pg/ml
1539.548
0.1 pg/ml
1534.598
0.01 pg/ml
2166.426
0.001 pg/ml
1749.669
0 pg/ml
962.012
67