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Determination of Nerve Growth Factor in Rat Spinal Cord by Capillary
Electrophoresis-Based Immunoassay with a Laser Induced Fluorescence Detector
Xiaoli Zou Yuanqian Li Tinghua Wang Chunyan Zhou Hongyan Zeng
Department of Sanitary Technology, West China school of public health, Sichuan University,
610041, P.R.China.
Abstract In order to study the relationship between spinal cord injury and the change of nerve
growth factor (NGF), an analytical method for NGF by capillary electrophoresis–based
immunoassay (CEIA) with laser induced fluorescence (LIF) was developed. Having been
dissolved in phosphate buffer solution (PBS) and concentrated with vacuum freeze-drying, NGF
in spinal cord of rat was allowed to react with NGF monoclonal antibody labeled with
fluorescence isothiocyanate (FITC). Then the immuno-complex, FITC-labeled anti-NGF and
FITC were separated and determined by LIF-CEIA using Kiton red as the internal standard. The
linear range of the method was 2 ng mL-1 ~30 ng mL-1and the limit of detection was 0.35 ng mL-1.
The relative standard derivations (RSDs) for relative migration time and relative fluorescence
intensity ratio were 7.98 % and 6.52 % respectively. The contents of NGF from spinal cord of rat
were determined by both the proposed method and western blotting. The results with the two
methods agreed well. The spiked recoveries of the samples were 88.5 %~116.3 %. The proposed
method was rapid, precise and inexpensive.
Key words: Laser induced fluorescence (LIF), Capillary electrophoresis-based immunoassay
(CEIA), Nerve growth factor, Spinal cord of rat
Introduction
There is more and more evidence that nerve growth factor (NGF) may function as a
biologically active molecule in spinal cord plasticity [1]. Recently, NGF showed to play some
roles in neuroprotection such as stimulation of sprouting and synaptic recorganization. It was
applied in clinic treatment to protect injured neurons and improve their function [2~4]. But it is
difficult to know the time window and dosage of NGF in clinic medication because the content of
NGF and its change in organisms can not be determined accurately with a credible method.
Some traditional immunoassay methods, such as western blotting, immuno-histochemistry
and enzyme-lined immunosorbent assay were usually applied to study the change of NGF content
[5~8]. Immunoassay, indeed, has high selectivity due to the specific reaction between antibody
and antigen. However, it is tedious, high solvent and sample consumption. Furthermore,
traditional immuno-assay suffers from poor reproducibility owing to difficulties in attaching the
reagent to the support surface and strong background. At present, the capillary
electrophoresis-based immunoassay (CEIA) has been used as a viable alternative to conventional
immunoassay. Because of combining high selectivity of immuno-reactivity with high separation
efficiency of CE, CEIA has the advantages of low sample and solvent consumption, rapid
immuno- reaction and separation, automation and the ability to simultaneously determine multiple
analytes [9,10]. Moreover, its high separation efficiency can provide the way to solve the
problems of the cross reaction and the background noise interference.
Schultz and Kennedy first applied CEIA-LIF to analyze insulin. Labeled insulin and the Fab
fragment of monoclonal anti-insulin were used as the tracer and the immuno-reagent respectively
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in this study. The separation was completed within 3 min and the detection limit of insulin was
about 3 nmol L-1 or 420 zmol for competitive immunoassay [11]. Up to now, CEIA-LIF has been
employed to a wide range of compounds including protein [12,13], pesticide[14], hormone[15,16]
and so on.
There are two types of immunoassay used in CEIA-LIF: One is noncompetitive and the other
is competitive. In their paper, Kalish et al, have analyzed the content of neurotropins in serum by
combining the competitive assay and online immuno-affinity capillary electrophoresis [17]. The
anti-neurotrophin antibodies were digested into Fab fragments and coated on the inside wall of
fused silica capillary. After being labeled with AlexaFluor 633, neurotrophins in serum were
captured by the immobilized antibodies in capillary and determined by capillary electrophoresis.
In order to immobilize the antibodies on the capillary internal surface, it took about near twenty
hours to modify the inside wall of capillary including silylation, rinsing and incubation. To
simplify the analytical procedure and propose a method for quantitative determination of NGF in
spinal cord, in our work, CEIA-LIF was used for rapid noncompetitive immunoassay of NGF
from spinal cord of rat. In noncompetitive assay, the excess fluorescently labeled antibody is
added to a sample. The principle can be expressed as follows.
Ag + Ab*
AgAb*
A capillary electrophoresis separation of the mixture is expected to show two peaks with
fluorescence detection: one for the complex (AgAb*) and the other for the free labeled antibody
(Ab*). If antigen (Ag) can quantitatively form an AgAb* complex and the complex is stable
during the period of subsequent CE separation, the fluorescence intensity of the complex would be
proportionate to the content of antigen. Therefore, it is possible to determine the amount of antigen
in the samples according to the fluorescence intensity of the complex [18]. In our experiment,
NGF monoclonal antibody was labeled with FITC and set to react with NGF. The
immuno-complex was separated and determined with CE-LIF. The proposed method combined
the specificity of immuno-reaction with the high separation efficiency of CE, as well as high
sensitivity of LIF to form the analytical system for NGF from spinal cord to study the relationship
between the spinal cord hurt and NGF. The electrophoresis can be carried out directly after the
solution of spinal cord samples was incubated with the antibody for 15 min. There is no need for
the modification of the capillary inside wall.
Experimental
Chemicals
NGF, monoclonal anti-NGF-β (Sigma, produced from mouse clone 25623.1), FITC (Beijing
Yinjing Science and Technology Ltd., China), 25 mmol L-1 phosphate buffer solution (PBS, pH 9.4)
as the derivation buffer, 20 mmol L-1 PBS (pH 8.0) as the running buffer, 10 mmol L-1 PBS (pH 7.2),
100 μg mL-1 NGF in PBS (pH 7.2), 10 mg mL-1 monoclonal anti-NGF in PBS (pH 7.2), 1 mg mL-1
FITC in PBS (pH 9.4), Kiton red (Sigma), NaOH, Sodium dodecyl sulfate (SDS).
Deionized water obtained from a Milli-Q water purification system was used throughout the
experiment (Millipore, synergy185, USA). Other reagents were of analytical grade (A.R.). The
running buffer was filtered through 0.45 μm nylon membrane filters and sonicated for 10 min prior
to use.
Apparatus
The CEIA-LIF detection system used for this work has been described elsewhere [19]. Briefly,
the electrophoresis was driven by a high voltage power supply (CZE1000R, Spell Misman,
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Plainview, NY, USA), and carried out in an uncoated fused-silica capillary of 60 cm (effective length
of 52 cm), 100 µm I.D.×375 µm O.D. (Hebei Optical Fiber Factory, China) with an electric field of
166 V.cm-1. LIF detection was done using an argon ion laser (2 mW, Edmund Scientific, Barrington,
NJ) with excitation at 488 nm and emission at 520 nm. Fluorescence signals were recorded using a
personal computer with a program written in Visual C++ for windows.
Experimental procedure
Preparation of FITC-labeled anti-NGF
3 µL of 10 mg mL-1 antibody and 10 µL of 1 mg mL-1 FITC solution were mixed in a 0.5 mL
micro-centrifuge tube and incubated at 4 ℃ overnight in 25 mmol L-1 PBS (pH 9.4). The labeled
anti-NGF was used directly throughout the experiments for immuno-reaction and its working
concentration was determined by direct immuno-fluorescence assay (DFA).
Immuno-reaction between NGF and anti-NGF
1μL labeled anti-NGF was diluted to 0.5 mL with 10 mmol L-1 PBS（pH7.2）. Then 1 μL
diluted anti-NGF solution, a certain amount of NGF and 1 μL of 2×10-8 mol L-1 Kiton red
solution as the internal standard were mixed in a 0.5 mL micro-centrifuge tube. The mixture was
incubated in 10 mmol L-1 PBS（pH7.2）at room temperature for 15 min and then analyzed by CE.
The concentration range of NGF standard solutions was 2 ng mL-1~30 ng mL-1 for this work.
Capillary electrophoresis
Electrophoresis was performed in an uncoated fused-silica capillary of 60 cm (effective
length of 52 cm), 100 µm I.D.×375 µm O.D. using 20 mmol L-1 PBS（pH 8.0）as the running
buffer at 10 kV. The sample solution was injected at 15 kV for 10 s in anode. To obtain
reproducible results, the capillary was rinsed with water, 0.01 mol L-1 NaOH and the running
buffer for 2 min sequentially between runs.
Preparation of spinal cord sample solution
Animals were coelio-anesthetized by 2 % pentobarbital sodium solution (2.0 mL kg-1 body
weight). After the unilateral hemilaminectomy was carried out at L1-L4 and L6 lumbar, all the
dorsal root ganglions (DRGs) with 1 mm ~2 mm segments of the associated dorsal roots on the
left side were removed at the intervertebral foramina, sparing the L5 DRG untouched. Surviving
for seven days after the operation, all the rats were first anesthetized, and then the spinal cords
were removed. The spinal cord was divided along the posterior median sulcus and homogenized in
10 mmol L-1 PBS (pH 7.2) containing 10 μL phenylmethyl sulfonyl fluoride (PMSF). Then the
fluid was extracted with ultrasonic for 10min and centrifuged (1120×g) for 30 min at 4 ℃. The
supernatant was dried in a vacuum refrigeration drier to prepare the sample powder, which was
used for CEIA analysis after being dissolved in 10 mmol L-1 PBS (pH 7.2). The right side was
prepared according to the above mentioned procedure and considered as the control group.
The electrophoregram of NGF standard solution under conditions described above was
shown in Fig 1.
Results and discussion
Labeling reaction of anti-NGF with FITC
The optimal medium in which the labeling reaction could be conducted efficiently was
investigated. Three buffers, i.e., carbonate buffer solution (CBS), borax buffer solution (BBS) and
phosphate buffer (PBS), as the labeling reaction media were tested in the experiments. FITC was
easy to decompose in BBS and the decomposition products could not be separated from the
immnuo-complex and anti-NGF. The satisfactory results were obtained in both CBS and PBS. PBS
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was selected as a medium of the labeling reaction, as it is also the running buffer for CE. The effect
of pH, the concentration of PBS and the concentration ratio of anti-NGF to FITC on labeling
reaction of anti-NGF with FITC were studied. The change of the fluorescence intensity of
FITC-anti-NGF with pH of PBS was shown in Fig 2. The experimental results indicated that the
labeling reaction between anti-NGF and FITC did not proceed in acid medium. When pH of the
buffer was 9.2~9.6, the fluorescence intensity of FITC-anti-NGF was higher and stable. Therefore,
PBS with pH 9.4 was finally selected for the labeling reaction. The experiments also showed that the
concentration of PBS had little effect on the labeling reaction. However, the higher concentration of
the buffer was used, the current was higher at sample injection for CE. Therefore, 25mmol L-1 PBS
was selected in the experiments. Concentration ratio of FITC to anti-NGF was also influenced on
the labeling reaction. The fluorescence intensity of FITC-anti-NGF increased with the ratio up to 1:4.
So, the ratio of 1:3 for FITC to anti-NGF was chosen in the experiments. The working concentration
of labeled anti-NGF obtained in our experiments was 1:1200 determined by direct
immuno-fluorescence assay (DFA).
The immuno-reaction time
In order to investigate the incubation time of immuno-reaction, the mixtures of NGF and
FITC-labeled anti-NGF (Ab*) were incubated for 5 min-30 min at room temperature. The
fluorescence intensity of the complex could be detected as soon as NGF and Ab* were mixed and
it increased rapidly within 10 min and kept stable in 10 min-20 min. Therefore, the incubation
time of 15 min was selected in the experiment. It can be seen in Fig 3 that there was a change of
the fluorescence intensity for the immuno-complex and Ab* against the immuno-reaction time..
Optimization of CE separation parameters
To obtain a baseline resolution of free Ab* and immuno-complex, the main analytical
parameters of CE were optimized respectively.
20 mmol L-1 PBS, BBS and Tris-borax-EDTA (TBE) buffer were investigated as the running
buffers for CE. No electrophoretic peak or poor peak was observed in TBE and BBS buffers,
respectively. The satisfactory results could be obtained in the PBS buffer as shown in Fig 4.
Electroosmotic flow (EOF) and the status of the capillary wall changed with the concentration and
pH of the running buffer. The effect of pH of PBS from 7.0 to 11.0 was investigated in the
experiments (in Fig 5). The experimental results indicated that the better resolution and
symmetrical peaks were obtained with CE at pH 8.0. The complex was decomposed in the running
buffer of pH>9.0. The influence of the concentrations of PBS buffer (from 5 mmol L-1 to 25 mmol
L-1) on the separation was also investigated and the results were presented in Fig 6. Taking both
resolution and peak shape into account, 20 mmol L-1 PBS (pH 8.0) was used as the running buffer
in the experiments.
The effect of the electrophoretic voltage from 8 kV~12 kV on CE separation was studied.
Increasing of electrophoretic voltage had the effect of decreasing analytical time, but high voltage
resulted in widened peaks and the decomposition of the complex with joule heating increasing.
The change of resolution between complex and Ab* with the change of voltage is shown in Fig 7.
It could be seen from Fig 7, electrophoretic voltage of 10 kV provided the best resolution for the
analysis.
Kiton red and Rhodamine B as internal standards were examined following the experimental
procedure, respectively. The experimental results indicated that Kiton red could give a satisfactory
separation with the analytes, but Rhodamine B cannot be separated from the complex and antibody.
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In the optimal separation condition, the resolution of Kiton red and FITC was about 3.72. The
ratio of fluorescence intensity for Kiton red and immuno-complex (relative fluorescence intensity)
was used for quantification of NGF.
noncompetitive immunoassays of NGF based on LIF-CE
A calibration curve of the immuno-complex was constructed by measuring the ratio of peak
height of the complex to that of internal standard at various NGF concentrations from 2 ng mL-1 to
30 ng mL-1. The linear range was from 2 ng mL-1 to 30 ng mL-1 with a correlation coefficient of
0.993. The limit of detection was 0.35 ng mL-1, defined as the concentration of NGF for which the
signal-to-noise was equal to three (S/N=3).
The reproducibility of migration time and peak height was tested for the standard solution.
The relative standard derivations of the migration time and relative fluorescence density were
7.98 % and 6.52 %, respectively. The spiked sample solutions of different concentration were
determined according to the experimental procedure. The average recoveries were
88.5 %~116.3 % as seen in Table1.
Analysis of NGF in Spinal cord
NGF contents of ten spinal cord samples were determined with both the proposed method
and western blot respectively. Western blotting was a traditional method for analyzing the change
of NGF contents after spinal cord injury. Under the optimal conditions of the proposed method,
the mixture was obtained the baseline resolution. NGF contents increased after spinal cord injury
and the ratios of the control group to the operation group were 2.06~3.83 (the average was 3.19)
and 2.8~3.5 (the average was 3.16) determined with the proposed method and western blot
respectively (n=10). There were not significant differences statistically in comparison of the
results between CEIA and western blotting with paired samples t-test (t=0.262, p=0.799) and gave
a good linear correlation (Y=0.9739X, Y-results of Western blotting method, X-results of the
proposed method, γ=0.992, F=657.64, p=0.000). However, Western blotting was just a
semi-quantitative method. It could not obtain the real concentration of NGF just by comparing the
gray scale of the spinal cord slice to get the change for the contents of NGF between the control
group and the operation group. The staining background was influenced on the analysis.
The electrophoregrams of rat spinal cord samples solution are shown in Fig 8.
Conclusion
The proposed method has been developed for analyzing the changes in the content of growth
factor (NGF) during a spinal cord injury in rats. The method is based on CE-LIF with
noncompetitive immunoassay of NGF. CE separation was performed in an uncoated fused-silica
capillary (60 cm×100 µm) with 20 mmol L-1 PBS as the running buffer at pH 8.0. Detection limit
of 0.35ng mL-1 was obtained in the experiment. The proposed method is rapid with 15 min for
incubation, 5 min for separation and the immuno-complex was stable within the period of
determination.
Acknowledgements
This study was supported financially by NATIONAL NATURAL SCIENCE FOUNDATION
OF CHINA (30571566) and THE YOUTH FOUNDATION OF SICHUAN UNIVERSITY
(0040405505016).
Tel: 86-028-85501301.
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Fig 1 The electrophoregram of NGF standard solution
4=Internal standard (Kiton red)
1=Complex, 2=FITC-Ab, 3=FITC,
Fig 2 The change of the relative fluorescence intensity of FITC-anti-NGF with pH of the buffer
(PBS)
Fig 3 The effect of immuno-reaction time of NGF and anti-NGF on determination
Fig 4 The effect of running buffer on the electropheritic separation 1=Complex, 2=FITC-Ab,
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3=FITC
Fig 5 The effect of pH of the running buffer on electrophoretic separation 1=Complex, 2=FITC-Ab,
3=FITC, 4=Internal standard (Kiton red)
Fig 6 The effect of the running buffer concentration on electrophoretic separation 1=Complex,
2=FITC-Ab, 3=FITC, 4=Internal standard (Kiton red)
Fig 7 The effect of electrophoretic voltage on the resolution
Fig 8 The electrophoregram of the spinal cord sample solution
4=Internal standard (Kiton red)
7
1=Complex, 2=FITC-Ab, 3=FITC,