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, SichuanUniversity, 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 ngmL-1 ~30 ngmL-1and the limit of detection was 0.35 ngmL-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 in this study. The separation was completed within 3 min and the detection limit of insulin was about 3 nmolL-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 etal, 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 wasincubated with the antibody for15 min.There is no need for the modification ofthe 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 mmolL-1 phosphate buffer solution (PBS, pH 9.4) as the derivation buffer, 20 mmolL-1 PBS (pH 8.0) as the running buffer, 10 mmolL-1 PBS (pH 7.2), 100 μgmL-1 NGF in PBS (pH 7.2), 10 mgmL-1 monoclonal anti-NGF in PBS (pH 7.2), 1 mgmL-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, 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 mgmL-1 antibody and 10 µL of 1 mgmL-1 FITC solution were mixed in a 0.5 mL micro-centrifuge tube and incubated at 4 ℃ overnight in 25 mmolL-1 PBS (pH9.4). 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 mmolL-1 PBS（pH7.2）. Then 1 μL diluted anti-NGF solution, a certain amount of NGF and 1 μL of 2×10-8 molL-1Kiton red solution as the internal standard were mixed in a 0.5 mL micro-centrifuge tube. The mixture was incubated in 10 mmolL-1 PBS（pH7.2）at room temperature for 15 min and then analyzed by CE. The concentration range of NGF standard solutions was 2 ngmL-1~30 ngmL-1 for this work.
Capillary electrophoresis
Electrophoresis was performed in an uncoated fused-silica capillary of 60cm (effective length of 52cm), 100 µm I.D.×375 µm O.D. using 20 mmolL-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 molL-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 mLkg-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 mmolL-1 PBS (pH 7.2). The right side was prepared according to the abovementioned procedure and considered as the control group.
The electrophoregram of NGF standard solution under conditions described above was shown in Fig1.
Results and discussion
Labeling reaction of anti-NGF with FITC
The optimal medium in which the labeling reaction could be conducted efficientlywas 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-complexandanti-NGF. The satisfactory results were obtained in both CBS and PBS. PBS 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, 25mmolL-1PBS 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 1:1200determined 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 separationparameters
To obtain a baseline resolution of free Ab* and immuno-complex, the main analytical parameters of CE were optimized respectively.
20 mmolL-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 mmolL-1 to 25 mmolL-1) on the separation was also investigated and the results were presented in Fig 6. Taking both resolution and peak shape into account, 20 mmolL-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. 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 ngmL-1 to 30 ngmL-1. The linear range was from 2 ngmL-1 to 30 ngmL-1 with a correlation coefficient of 0.993. The limit of detection was 0.35 ngmL-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 blottingwas just asemi-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-LIFwith 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 0.35ngmL-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).
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