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A novel liquid-phase piezoelectric immunosensor for detecting Schistosoma japonicum circulating antigen
Parasitology International 60 (2011) 301–306
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Parasitology International j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / p a r i n t
A novel liquid-phase piezoelectric immunosensor for detecting Schistosoma japonicum circulating antigen Zhili Wen a,⁎, Shiping Wang b, Zhaoyang Wu c, Guoli Shen c a b c
Infectious Disease Department, Nanchang NO.1 Hospital ( NO.3 affiliated hospital of Nanchang University), Nanchang 330008, China Pathogen Biology Department, Xiangya medical college, Central South University, Changsha, 410078, China Key Laboratory of Chemometrics & Chemical, Biological Sensing Technologies, Education Ministry of China, Hunan University, Changsha, 410082, China
a r t i c l e
i n f o
Article history: Received 8 December 2010 Received in revised form 5 May 2011 Accepted 7 May 2011 Available online 14 May 2011 Keywords: Piezoelectric immunosensor Schistosomiasis japonica SjCAg Quantitive detection
a b s t r a c t Aims: A new liquid-phase piezoelectric immunosensor (LP-PEIS), which can detect Schistosoma japonicum (Sj) circulating antigens (SjCAg) quantificationally, was developed. Methods: The IgG antibodies were purified from the sera of rabbits which had been infected or immunized by Sj and were immobilized on the surface of piezoelectric quartz crystal in LP-PEIS by staphylococcal protein A (SPA). It was used to detect SjCAg in sera of rabbits which had been infected by Sj in order to acquire some optimum conditions for detecting SjCAg. Finally, the LP-PEIS with optimum conditions was used to detect SjCAg in sera of patients who had been infected by Sj, and was compared with sandwich ELISA. Results: A lot of optimum conditions of LP-PEIS for detecting SjCAg had been acquired. In the detection of patients' sera with acute Schistosomiasis, LP-PEIS has higher positive rate (100%) and lower false positive rate (3.0%) than sandwich ELISA (92.8%, 6.0%). However, there were no significant difference between LP-PEIS and sandwich ELISA. Conclusions: LP-PEIS can quantificationally detect SjCAg in patients' sera as well as sandwich ELISA. © 2011 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Schistosomiasis japonica is currently the most prevalent parasitosis in China [1]. In the recent 5 years, more than 840 thousand people have been infected and 65,000 thousand people are being threatened by Schistosoma japonicum (Sj) in China. There are a lot of serological tests which can diagnose it, such as Enzyme-Linked Immunosorbent Assay (ELISA), indirect hemagglutination test (IHA), circumoval precipitin test (COPT) and Immunogold sliver staining (IGSS) [2–4]. However, these methods can only be used for qualitative analysis, and often are confined to detection of antibody. Piezoelectric immunosensor (PEIS) is a new technique for quantitative analysis. It has some advantages over common serological tests, such as simpler operation, quicker detection and real-time monitoring for immunoreaction in liquid. Its principle is that, when a piezoelectric quartz crystal oscillating in an oscillator circuit adsorbs external substance, the oscillating frequency of crystal will decrease due to the increase of its mass. The decrease of frequency is proportional to the increase of crystal mass, so the concentration of sample can be calculated (Fig. 1). We have successfully used PEIS to diagnose schistosomiasis japonica by detecting Sj antibodies, and therefore exploited a new field for diagnosis of schistosomiasis [5–9]. ⁎ Corresponding author at: Xiangshan Northern Road No.128, Nanchang City, China. Tel.: + 86 13870608072. E-mail address: [email protected] (Z.L. Wen). 1383-5769/$ – see front matter © 2011 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.parint.2011.05.001
As all know, Sj circulating antigens (SjCAg) is more valuable for diagnosis of schistosomiasis japonica than Sj antibodies, because it can be used to diagnose schistosomiasis japonica early and present infection of Sj. However, SjCAg is always very difficult to be detected. In this paper, the availability of detecting SjCAg by liquid-phase PEIS (LP-PEIS) was explored.
2. Materials and methods 2.1. Apparatus and reagents Piezoelectric quartz crystal flake: 9 MHz, AT cut-type, gilt on both sides, 12.5-mm-diameter crystal and 6-mm-diameter electrode (Beijing 707th State-run Factory). Frequency counter: 100 MHz, type of SS3340B (Shijiazhuang 4th Wireless Factory). Electrothermal and isothermal water tank: type of HH.W21.Cr420 (Beijing Scientific Apparatus Factory). Magnetic stirrer: type of 78-1 (Shanghai Nanhui Flash Equipment Factory). High-speed refrigerant centrifuge: type of TGR 16-1.2 (Xinhua Apparatus Factory in Hunan Headquarters of Apparatus and Meter). Time and enumerative collector: type of JSD-2 (Jilin 7th Wireless Factory). Voltage-stabilizing and Current-stabilizing timing electrophoresis apparatus: DYY-4DXING (Beijing Industrial Company of Ironware Tool). SDS-PAGE device and constant-temperature water tank (Sweden LKB Company). Enzyme reader: type of Σ960 (American ERMA Company). TTL oscillator for quartz crystal was made by us. One side of
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Computer
+
-
Reaction pool Magnetic spillikin
Crystal
Oscillator circuit
Cymometer
Stirrer
Fig. 1. Sketch of liquid-phase piezoelectric immunosensor. Liquid-phase Piezoelectric immunosensor is an electronic instrument, which is made of crystal, reaction pool, stirrer, oscillator circuit and cymometer, etc. All of the results from cymometer can be processed in a computer by the numbers. The immunoreaction between the SjCAg in sera sample and the Sj IgG on crystal is speeded by the agitation of a magnetic spillikin.
crystal was sealed. Reaction pool was a plastic cylinder with a capacity of 3.5 ml. SPA (dry congelation of purified product) was from Shanghai Bioticware Institute of Health Ministry). Gel for column chromatography was Sephadex G-200 (Pharmia). (NH4)2SO4, H2SO4, H2O2 and other reagents were all analytically pure. Water was twice distillation. Detergent for crystal (Piranha solution) was a mixture mixed by dense H2SO4 and H2O2 with a proportion of 7:3 (mixed only when needed). 2.2. Methods 2.2.1. Acquisition of infected rabbit's sera (InRS) and immunized rabbit's sera (ImRS) Acquisition of InRS: (1) InRS for pure IgG: Rabbits' blood were collected from the 2.5 kg New Zealand rabbits' carotids when they had been infected by 3000 cercariae of Schistosoma japonicum for 42 days. Rabbits’ sera were separated from the blood and stored in − 20 °C. (2) InRS for detection: Rabbits' sera were collected and stored in − 20 °C when a dozen of 2.5 kg New Zealand rabbits had been infected respectively by 500, 1000, 1500, ……, 5000, 5500, 6000 cercariae of Schistosoma japonicum for 30 days. Acquisition of ImRS: A 2.5 kg New Zealand rabbit was injected with 1 ml complete Freund's adjuvant (CFA, containing 10 mg BCG vaccine) in the skin of its halluces. A week later, 1 mg Schistosoma egg antigen (SEA) and isometric CFA were injected multipoint in the skin of rabbit's back. Then 0.6 mg SEA and isometric CFA were injected in the skin of rabbit's back multipoint once a week. One month later, the titer of antibodies in rabbit's sera was testified to be 1:32 by counter immunoelectrophoresis (CIE test), and rabbit's sera were separated and stored in −20 °C. 2.2.2. Separation and purification of IgG antibodies in InRS and ImRS The method was according to Andrew [10] with a little modification. Firstly, 50% saturated ammonium sulfate (SAS) was used to rid albumin and then 33.3% SAS to rid α globulin and β globulin, leaving only crude IgG (cIgG) in rabbit's sera. Secondly, cIgG was resolved in 2 ml phosphate buffer solution (PBS) and was gel-filtrated in Sephadex G-200. Process of gel filtration: Fully swelling Sephadex G-200 was loaded in 1 cm × 70 cm chromatography column. After gel had been equilibrated in column, 2 ml crude IgG (b1/10 column) was injected in the top of column. The 0.01 M, pH 7.4 PBS (containing 0.14 M NaCl) was used to elute IgG, and the eluted solution was collected timely by automatic collector with 2 ml capacity in every tube. The existences of IgG and SAS were testified respectively by 20% sulfosalicylic acid and 1% BaCl2. When there is no IgG any more in eluted solution, collection was stopped, and elution was kept on until there is no SAS in eluted solution. Thirdly, protein peak was detected according to Smith [11], and activity of antibodies in every tube in protein peak was testified by CIE test. Those antibodies, which were testified to be activated, were mixed together. The mixture was just pure
IgG (pIgG). After the concentration of pIgG was determined by colorimetry on enzyme reader, pIgG was stored in −20 °C. 2.2.3. Confirmation of purity and activity of pure IgG Purity was testified by SDS-PAGE according to traditional methods, while activity was testified by CIE and Immuno-Double-Diffusion (IDD) according to Silva [12]. These tests helped to choose immunoactivated pure IgG for LP-PEIS. 2.2.4. Immobilization of IgG A drop of Piranha's solution was injected onto golden electrode of crystal flake by micrometer syringe, and was ridded by water after 10 min. Then 5 μl of 1 mg/ml SPA was smeared onto the surface of golden electrode, and crystal was put in 37 °C for 30 min. After golden electrode being cleaned and dried, 5 μl of 8 mg/ml pure IgG (from InRS) or 4.5 mg/ml (from ImRS) was laid on it and crystal was put in 37 °C for 1 h. After golden electrode being cleaned and dried again, 1:100-diluted normal rabbit's sera (NRS) was laid on it and crystal was put in 37 °C to block for 30 min. After being cleaned and dried, crystal can be used for experiment. 2.2.5. Methods of detection The 3 ml of 0.01 M, pH 7.0 PBS was added in reaction pool, followed by running of magnetic stirrer under reaction pool. The crystal with immobilized pure IgG was put in PBS. When the oscillating frequency of crystal was stable (Δ f ≤ 1 Hz/min), the frequency at the moment was recorded as the frequency before detection (F1). Then 60ul InRS was added in pool and the changing process of crystal frequency was initially recorded (once a minute) until the frequency became stable again. The last stable frequency was just the frequency after detection (F2). Thus, the change of frequency which resulted from the immunoreactions between sera sample and Sj IgG on crystal, together with that which resulted from non-specific adsorption between proteins, was F1–F2. The solution in pool was drained out and pool was cleaned by water for 5 times. Then 3 ml PBS and another same crystal (with IgG) were added in pool to detect NRS. According to above methods, the frequency before detection (F3) and that after detection (F4) were recorded. Thus the frequency change resulting from non-specific adsorption between proteins was F3-F4. Therefore the change of frequency resulting only from specific antigen-antibody reaction (ΔF) was (F1–F2)–(F3–F4). Meanwhile, in order to discriminate between specific binding of the primary antibody to circulating antigen or an “nonspecific” reaction e.g. involving binding of primary antibodies to anti-idiotype antibodies that emerge during the later phase of an infection, this LP-PEIS was used to detect 60 μl ImRS, which probably contains anti-idiotype antibodies but certainly not any more antigen. 2.2.6. Regeneration of crystal Three common methods were used to clean the crystals that had been used: (1) A drop of Piranha's solution was injected onto crystal and was ridded by water after 10 min. (2) The crystal was put in the pool with 3 ml of 0.01 M NaOH and 0.05 M NaCl and was deterged with stir for 10 min. Then the solution in pool was replaced by 3 ml of 0.01 mol/L HCl, followed by detergency of crystal in it with stir for 10 min. Finally crystal was cleaned by water. (3) The crystal was put in the pool with 3 ml of 0.1 M glycin–HCl buffer solutions and was deterged with stir for 20 min, and then was cleaned by water. 2.2.7. Primary application of LP-PEIS for detecting SjCAg in human sera Sera samples from 180 persons, including 147 patients with schistosomiasis japonica and 33 healthy persons, were randomly collected. The patients were composed of 112 males and 25 females, in which the youngest was 14 and the oldest was 72. The 147 patients were divided into four groups according to their clinical situations, including group A: patients with acute schistosomiasis; group B:
Z.L. Wen et al. / Parasitology International 60 (2011) 301–306
patients with chronic schistosomiasis who had not been treated; group C: patients with chronic schistosomiasis who had been treated with praziquantel for half a year, and group D: patients with chronic schistosomiasis who had been treated with praziquantel for one year. Informed consent was obtained from each patient included in the study and the study protocol conforms to the ethical guidelines of the 1975 Declaration of Helsinki as reflected in a priori approval by the institution's human research committee. Randomization was carried out through random digits table. The 180 sera samples were tested blindly by liquid SjCAg-PEIS and sandwich ELISA using polyclonal antibodies, respectively. The operation of liquid SjCAg-PEIS was according to the optimal conditions which were acquired in former experiments. The operation process of sandwich ELISA was according to Qiu. [13] In liquid SjCAg-PEIS, the frequency change above 300 Hz was judged as positive case. In sandwich ELISA, the ratio of P (OD value from detected sera) to N (OD value from control sera) above 2 was judged as positive case. All of the data were statistically analyzed by SSPS 17.0 statistic software. Measurement data were processed by Student t test, while count data were processed by chi square test.
3. Results 3.1. Comparison among various concentration of SPA for coating of crystal SPA and gold (Au) can form very stable SPA-Au compound with a high affinity constant (10 8 L/mol), so SPA is able to be adsorbed onto golden electrode intensely. Meanwhile, SPA can combine specifically with Fc fraction of IgG, leaving Fab fraction of IgG exposed to the outer layer of decorating membrane and extending to fluxion phase. Fab fraction is just the activity center of IgG antibody which links with determinant of antigen. Thus, SPA will not affect the activity of reaction between antigen and antibody. Therefore, SPA is a good immobilizing reagent for antibody [7]. In this experiment, the relation between concentration of SPA and quantity of adsorbed SPA on crystal was studied. Results showed that change of frequency increased with the increase of concentration of SPA until the concentration was beyond 1 mg/ml (Fig. 2). It was thought as that at the moment the surface of golden electrode became saturated.
303
3.2. Comparison of antibodies between pre-purification and post-purification The unpurified IgG (InRS), crude IgG (cIgG) and purified IgG (pIgG) were immobilized on crystal, respectively. Their immobilization efficiency and detection effect for SjCAg were compared, respectively (Table 1). Results showed that both immobilization efficiency and detection effect of pIgG were best among three kinds of antibody. The reason that InRS and cIgG had a relative poor effect was probably due to that they contained other proteins which can disturbed the combination between IgG and SPA, and link sites were occupied due to non-specific adsorption, which reduced the effective immobilization amount of antibodies.
3.3. Comparison between InRS and ImRS after purification Results showed that purified InRS had better immobilization efficiency but a worse detection effect than purified ImRS. The reason was possibly that the IgG in purified InRS was against several kinds of Schistosoma antigen and thus had a higher concentration, while the IgG in purified ImRS was only against SEA and thus had a lower concentration. Therefore, the combination quantity of purified InRS with SPA was higher than that of purified ImRS with SPA. However, it was just the variety of IgG in purified InRS that disturbed the combination between IgG and circulating antigen in blood, as well as that increased non-specific reaction with negative sample, which led to a worse detection effect than ImRS. Through detecting the InRS sample and ImRS sample, we found that binding of primary antibodies to anti-idiotype antibodies was some weak and could hardly affect the results of detection for SjCAg (Table 2).
3.4. Dilution of sera samples Since sera samples were added in the pool with a constant capacity of 3 ml PBS, dilutions of samples were nearly related to the quantity of samples in pool. The 300 μl, 100 μl, 60 μl, 30 μl, 15 μl, 10 μl and 5 μl of InRS and NRS were added in pool and were diluted by 3 ml PBS to 1:10, 1:30, 1:50, 1:100, 1:200, 1:300 and 1:600, respectively. These samples were respectively detected by LP-PEIS with purified IgG from ImRS (4.5 mg/ml) immobilized on crystal, in order to find optimum dilution of sample. Results showed that frequency change of positive samples (ΔF +), frequency change of negative samples (ΔF −) and difference between ΔF + and ΔF − (ΔF + − ΔF −) all decreased with the increase of sample dilution (Fig. 3). ΔF + − ΔF − came up to maximum (310 Hz) when sample quantity was 60 μl and the dilution was 1:50 (Fig. 4). Thus it can be thought as that combination between SjCAg in sample and immobilized IgG became saturated at the moment. If sample quantity increased right along, ΔF + and ΔF − only increased due to the increase of viscosity of reaction solution but ΔF + − ΔF − remained unchanging. Therefore, 1:50 can be thought as optimum dilution for sample.
Table 1 Characterizations of three kind of immobilized antibody.a
Fig. 2. Relation between concentration of SPA and immobilization effect of Sj IgG. The change of frequency increased with the increase of concentration of SPA until the concentration was beyond 1 mg/ml. It was thought as that at the moment the surface of golden electrode became saturated.
Immobilized antibody
ΔF1(Hz)
ΔF2(Hz)
ΔF3(Hz)
ΔF4(Hz)
Original antibody (InRS) Crude antibody (cIgG) Purified antibody (pIgG)
190 ± 29 325 ± 21 356 ± 18
171 ± 25 234 ± 17 271 ± 14
63 ± 8 52 ± 6 48 ± 5
53 ± 6 65 ± 7 72 ± 7
a ΔF1 represents the frequency changes produced by the immobilization of antibody onto surface of immunosensor by SPA, ΔF2, ΔF3 and ΔF4 represent the frequency changes produced by positive sample (InRS), negative sample (NRS) and control sample (ImRS), respectively. Each value (ΔF) represents the average of four experiments ± the mean values of their arithmetic errors.
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Table 2 Characterizations of two kind of immobilized antibody.a Immobilized antibody
ΔF1(Hz)
ΔF2(Hz)
ΔF3(Hz)
ΔF4(Hz)
Purified InRS Purified ImRS
356 ± 18 254 ± 10
271 ± 14 357 ± 8
48 ± 5 40 ± 3
62 ± 8 57 ± 5
a ΔF1 represents the frequency changes produced by the immobilization of antibody onto surface of immunosensor by SPA, ΔF2, ΔF3 and ΔF4 represent the frequency changes produced by positive sample (InRS), negative sample (NRS) and control sample (ImRS), respectively. Each value (ΔF) represents the average of four experiments ± the mean values of their arithmetic errors.
3.5. Effect of various infection degree of sample on frequency change A serial of positive rabbit's sera infected with various amount of cercariae of Schistosoma japonicum (all be collected 30 days after infection) were respectively detected with the same dilution of 1:50, in order to study the relation between frequency change and infection degree. Results showed that frequency change increased with the increase of infection degree until amount of cercariae was beyond 4500 (Fig. 5). It may be concluded that at the moment combination sites of immobilized IgG had been saturated, and quantity of SjCAg adsorbed by golden electrode had attained to kinetic balance and would not increase with the increase of its own concentration in sample. 3.6. Optimum time for detection The 60ul InRS that was collected after rabbit had been infected with 4500 cercariae for 30 days, and the 60ul NRS were added respectively in 3 ml PBS in a pool with stir. They were detected by a crystal, onto which pure IgG from ImRS was immobilized through 1 mg/ml SPA. The changing process of frequency was recorded (once every 1–5 min) for 70 min while InRS or ImRS was reacting with immobilized IgG, in order to monitor the whole process of immunoreactions in pool. Results showed that frequency of positive sample decreased quickly in first 30 min but slowly in second 30 min, and did not change any more after 60 min. Meanwhile, frequency of negative sample only change a little in first 2 min and tend to be stable after 5 min with a little fluctuation of 1–3 Hz due to stir (Fig. 6). Therefore, the soonest detection time for detecting SjCAg should be 5 min, while the optimum detection time for monitoring the kinetic procedure of
Fig. 3. Relation between dilution of sample and response. The frequency change of positive samples (ΔF+), frequency change of negative samples (ΔF−) and difference between ΔF+ and ΔF− (ΔF+ − ΔF−) all decreased with the increase of sample dilution.
Fig. 4. Relation between dilution of sample and immunoreaction. The ΔF+ − ΔF− came up to maximum (310 Hz) when sample quantity was 60 μl and the dilution was 1:50. It can be thought as that combination between SjCAg in sample and immobilized IgG became saturated at the moment.
immunoreactions between antigen and antibody should be 60 min. (ΔF + − ΔF − was 120 Hz in 5 min and 315 Hz in 60 min). 3.7. Regeneration of crystal Since regeneration of crystal is a key element of developing practical PEIS, three common detergents for used crystal were compared to find an ideal method for regeneration of crystal. Results showed that deterging effect of Piranha's solution was the best among three detergents. It not only ridded proteins on crystal as entirely as possible, but also had a good reappearance. Strong acid and alkali together had a preferable deterging effect but a poor reappearance. Both of deterging effect and reappearance of 0.1 M glycin–HCl buffer solutions (pH 2.0) were the worst among them, perhaps because the detergent was only a dissociation reagent for
Fig. 5. Relation between degree of infection and response. The frequency change increased with the increase of infection degree until amount of cercariae was beyond 4500. It may be believed that at the moment combination sites of immobilized IgG had been saturated, and quantity of SjCAg adsorbed by golden electrode had attained to kinetic balance and would not increase with the increase of its own concentration in sample.
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Table 3 Detection for SjCAg in human sera by LP-PEIS and sandwich ELISA. Kinds of sera
Patients with acute Sj* Patients with chronic Sj* Without treatment Be treated for half a year Be treated for one year Healthy human
Total cases
Measured value (x ± SD)
Positive rate (%)Δ
LP-PEIS (ΔF, Hz)
ELISA (OD value)
LP-PEIS
ELISA
P
42
388 ± 27
0.244 ± 0.061
100.0
92.9
0.239
36
349 ± 60
a
0.231 ± 0.073
d
83.3
75.0
0.561
30
176 ± 56
b
0.136 ± 0.035
e
20.0
30.0
0.551
39
150 ± 63
c
0.119 ± 0.034
f
6.7
15.4
0.478
33
173 ± 98
3.0
6.0
1.000
0.102 ± 0.043
*Sj: Schistosomiasis japonica. Δ Standard for positive cases: LP-PEIS: ΔF ≥ 300 Hz; ELISA: OD P/N ≥ 2. There is significant difference between “a” and “b” (P = 0.0003), “d” and “e” (P = 0.026), while there is no significant difference between “b” and “c” (P = 0.274), “e” and “f” (P = 0.624).
Fig. 6. Relation between reaction time and response. The frequency of positive sample decreased quickly in first 30 min but slowly in second 30 min, and did not change any more after 60 min. Meanwhile, frequency of negative sample only change a little in first 2 min and tend to be stable after 5 min with a little fluctuation of 1–3 Hz due to stir.
antigen and antibody and could not rid SPA on golden electrode. Though it could be used again and again without new immobilization of IgG, its detection sensitivity would reduce and disappear after three times. In contrast, Piranha's solution denatured and ridded proteins on crystal quickly by overlapped reaction of two strong oxidants, while NaOH + HCl did so by cooperation of strong oxidant and deoxidizer which increased the damage on crystal and led to shorter service period of crystal. Therefore, in comprehensive consideration, Piranha's solution is the most ideal one among three common detergents for regeneration of crystal. 3.8. Primary application of LP-PEIS for detecting SjCAg in human sera In the detection of patients’ sera with acute Schistosomiasis, the positive rate of PEIS (100%) was higher than that of sandwich ELISA (92.8%). Meanwhile, the false positive rate of PEIS (3.0%) was lower than that of sandwich ELISA (6.0%). However, there were no significant difference between PEIS and sandwich ELISA (P = 0.239, P = 1.000). In the detection of patients' sera with chronic Schistosomiasis, the positive rates of those who had not been treated, or had been treated with praziquantel for half a year or one year were 83.3%, 20.0% and 6.7% respectively in PEIS, while were 75.0%, 30.0% and 15.4% respectively in sandwich ELISA. The measurement data from both of LP-PEIS and sandwich ELISA showed that there were significant difference between the patients without treatment and those who had been treated with praziquantel for half a year (P= 0.0003, P = 0.026). The difference in LPPEIS seem more obvious than that in sandwich ELISA, which apparently suggested that LP-PEIS has better evaluation effect than sandwich ELISA. However, there were no significant difference between PEIS and sandwich ELISA (P = 0.561, P = 0.551, P = 0.478) (Table 3).
In this study, piezoelectric immunosensor was used to detect SjCAg. Purified IgG was immobilized on surface of piezoelectric crystal by SPA to establish a novel LP-PEIS for detecting SjCAg in sera. The whole process of reaction between antigen and antibody can be monitored dynamically by this LP-PEIS. A lot of important data about optimum condition for detection was acquired. Sera samples of 147 patients with schistosomiasis japonica and 33 healthy persons were tested blindly by the liquid SjCAg-PEIS with optimum condition and sandwich ELISA with polyclonal antibodies, respectively. The results showed that both of LP-PEIS and sandwich ELISA can detect SjCAg in human sera and evaluate the curative effect of praziquantel very well. In the detection of patients’ sera with acute schistosomiasis, LP-PEIS has higher positive rate (100%) and lower false positive rate (3.0%) than sandwich ELISA (92.8%, 6.0%, respectively). However, there were no significant difference between LP-PEIS and sandwich ELISA. Therefore, this novel LP-PEIS was believed to be a good method for quantificationally detecting SjCAg in sera. It has more advantages over common serological tests, such as simple operation, quick reaction, quantitive detection and real-time monitoring for antigen-antibody reaction in liquid. This study lays a foundation for early diagnosis of schistosomiasis japanica and evaluation of curative effect by LP-PEIS. Moreover, based on the study, an automatic electronic analysator which can diagnose schistosomiasis quickly will be invented in the future. A lot of samples can be detected at the same time through installing many small reaction pools, in each of which a crystal is put and connected to an independent cymometer. If it becomes true, diagnosis of schistosomiasis will probably be in a great revolution and a new epoch for the diagnosis of schistosomiasis will come.
Acknowledgements The study was provided financial assistance by National Natural Science Foundation of China and Ministry of Health and Hunan Province's Scientific Committee of China.
4. Discussion References For a long time, early diagnosis of schistosomiasis japanica is always a difficult problem to all of the parasitologists. Detection for SjCAg is a good idea, but there are few methods that can detect it very well until now. At the present time, the most common method for detecting SjCAg is sandwich ELISA [13–15]. However, sandwich ELISA still has some defects, such as perplexing operation process, slow detection speed and nonspecific detection result.
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Parasitology International j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / p a r i n t
A novel liquid-phase piezoelectric immunosensor for detecting Schistosoma japonicum circulating antigen Zhili Wen a,⁎, Shiping Wang b, Zhaoyang Wu c, Guoli Shen c a b c
Infectious Disease Department, Nanchang NO.1 Hospital ( NO.3 affiliated hospital of Nanchang University), Nanchang 330008, China Pathogen Biology Department, Xiangya medical college, Central South University, Changsha, 410078, China Key Laboratory of Chemometrics & Chemical, Biological Sensing Technologies, Education Ministry of China, Hunan University, Changsha, 410082, China
a r t i c l e
i n f o
Article history: Received 8 December 2010 Received in revised form 5 May 2011 Accepted 7 May 2011 Available online 14 May 2011 Keywords: Piezoelectric immunosensor Schistosomiasis japonica SjCAg Quantitive detection
a b s t r a c t Aims: A new liquid-phase piezoelectric immunosensor (LP-PEIS), which can detect Schistosoma japonicum (Sj) circulating antigens (SjCAg) quantificationally, was developed. Methods: The IgG antibodies were purified from the sera of rabbits which had been infected or immunized by Sj and were immobilized on the surface of piezoelectric quartz crystal in LP-PEIS by staphylococcal protein A (SPA). It was used to detect SjCAg in sera of rabbits which had been infected by Sj in order to acquire some optimum conditions for detecting SjCAg. Finally, the LP-PEIS with optimum conditions was used to detect SjCAg in sera of patients who had been infected by Sj, and was compared with sandwich ELISA. Results: A lot of optimum conditions of LP-PEIS for detecting SjCAg had been acquired. In the detection of patients' sera with acute Schistosomiasis, LP-PEIS has higher positive rate (100%) and lower false positive rate (3.0%) than sandwich ELISA (92.8%, 6.0%). However, there were no significant difference between LP-PEIS and sandwich ELISA. Conclusions: LP-PEIS can quantificationally detect SjCAg in patients' sera as well as sandwich ELISA. © 2011 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Schistosomiasis japonica is currently the most prevalent parasitosis in China [1]. In the recent 5 years, more than 840 thousand people have been infected and 65,000 thousand people are being threatened by Schistosoma japonicum (Sj) in China. There are a lot of serological tests which can diagnose it, such as Enzyme-Linked Immunosorbent Assay (ELISA), indirect hemagglutination test (IHA), circumoval precipitin test (COPT) and Immunogold sliver staining (IGSS) [2–4]. However, these methods can only be used for qualitative analysis, and often are confined to detection of antibody. Piezoelectric immunosensor (PEIS) is a new technique for quantitative analysis. It has some advantages over common serological tests, such as simpler operation, quicker detection and real-time monitoring for immunoreaction in liquid. Its principle is that, when a piezoelectric quartz crystal oscillating in an oscillator circuit adsorbs external substance, the oscillating frequency of crystal will decrease due to the increase of its mass. The decrease of frequency is proportional to the increase of crystal mass, so the concentration of sample can be calculated (Fig. 1). We have successfully used PEIS to diagnose schistosomiasis japonica by detecting Sj antibodies, and therefore exploited a new field for diagnosis of schistosomiasis [5–9]. ⁎ Corresponding author at: Xiangshan Northern Road No.128, Nanchang City, China. Tel.: + 86 13870608072. E-mail address: [email protected] (Z.L. Wen). 1383-5769/$ – see front matter © 2011 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.parint.2011.05.001
As all know, Sj circulating antigens (SjCAg) is more valuable for diagnosis of schistosomiasis japonica than Sj antibodies, because it can be used to diagnose schistosomiasis japonica early and present infection of Sj. However, SjCAg is always very difficult to be detected. In this paper, the availability of detecting SjCAg by liquid-phase PEIS (LP-PEIS) was explored.
2. Materials and methods 2.1. Apparatus and reagents Piezoelectric quartz crystal flake: 9 MHz, AT cut-type, gilt on both sides, 12.5-mm-diameter crystal and 6-mm-diameter electrode (Beijing 707th State-run Factory). Frequency counter: 100 MHz, type of SS3340B (Shijiazhuang 4th Wireless Factory). Electrothermal and isothermal water tank: type of HH.W21.Cr420 (Beijing Scientific Apparatus Factory). Magnetic stirrer: type of 78-1 (Shanghai Nanhui Flash Equipment Factory). High-speed refrigerant centrifuge: type of TGR 16-1.2 (Xinhua Apparatus Factory in Hunan Headquarters of Apparatus and Meter). Time and enumerative collector: type of JSD-2 (Jilin 7th Wireless Factory). Voltage-stabilizing and Current-stabilizing timing electrophoresis apparatus: DYY-4DXING (Beijing Industrial Company of Ironware Tool). SDS-PAGE device and constant-temperature water tank (Sweden LKB Company). Enzyme reader: type of Σ960 (American ERMA Company). TTL oscillator for quartz crystal was made by us. One side of
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Computer
+
-
Reaction pool Magnetic spillikin
Crystal
Oscillator circuit
Cymometer
Stirrer
Fig. 1. Sketch of liquid-phase piezoelectric immunosensor. Liquid-phase Piezoelectric immunosensor is an electronic instrument, which is made of crystal, reaction pool, stirrer, oscillator circuit and cymometer, etc. All of the results from cymometer can be processed in a computer by the numbers. The immunoreaction between the SjCAg in sera sample and the Sj IgG on crystal is speeded by the agitation of a magnetic spillikin.
crystal was sealed. Reaction pool was a plastic cylinder with a capacity of 3.5 ml. SPA (dry congelation of purified product) was from Shanghai Bioticware Institute of Health Ministry). Gel for column chromatography was Sephadex G-200 (Pharmia). (NH4)2SO4, H2SO4, H2O2 and other reagents were all analytically pure. Water was twice distillation. Detergent for crystal (Piranha solution) was a mixture mixed by dense H2SO4 and H2O2 with a proportion of 7:3 (mixed only when needed). 2.2. Methods 2.2.1. Acquisition of infected rabbit's sera (InRS) and immunized rabbit's sera (ImRS) Acquisition of InRS: (1) InRS for pure IgG: Rabbits' blood were collected from the 2.5 kg New Zealand rabbits' carotids when they had been infected by 3000 cercariae of Schistosoma japonicum for 42 days. Rabbits’ sera were separated from the blood and stored in − 20 °C. (2) InRS for detection: Rabbits' sera were collected and stored in − 20 °C when a dozen of 2.5 kg New Zealand rabbits had been infected respectively by 500, 1000, 1500, ……, 5000, 5500, 6000 cercariae of Schistosoma japonicum for 30 days. Acquisition of ImRS: A 2.5 kg New Zealand rabbit was injected with 1 ml complete Freund's adjuvant (CFA, containing 10 mg BCG vaccine) in the skin of its halluces. A week later, 1 mg Schistosoma egg antigen (SEA) and isometric CFA were injected multipoint in the skin of rabbit's back. Then 0.6 mg SEA and isometric CFA were injected in the skin of rabbit's back multipoint once a week. One month later, the titer of antibodies in rabbit's sera was testified to be 1:32 by counter immunoelectrophoresis (CIE test), and rabbit's sera were separated and stored in −20 °C. 2.2.2. Separation and purification of IgG antibodies in InRS and ImRS The method was according to Andrew [10] with a little modification. Firstly, 50% saturated ammonium sulfate (SAS) was used to rid albumin and then 33.3% SAS to rid α globulin and β globulin, leaving only crude IgG (cIgG) in rabbit's sera. Secondly, cIgG was resolved in 2 ml phosphate buffer solution (PBS) and was gel-filtrated in Sephadex G-200. Process of gel filtration: Fully swelling Sephadex G-200 was loaded in 1 cm × 70 cm chromatography column. After gel had been equilibrated in column, 2 ml crude IgG (b1/10 column) was injected in the top of column. The 0.01 M, pH 7.4 PBS (containing 0.14 M NaCl) was used to elute IgG, and the eluted solution was collected timely by automatic collector with 2 ml capacity in every tube. The existences of IgG and SAS were testified respectively by 20% sulfosalicylic acid and 1% BaCl2. When there is no IgG any more in eluted solution, collection was stopped, and elution was kept on until there is no SAS in eluted solution. Thirdly, protein peak was detected according to Smith [11], and activity of antibodies in every tube in protein peak was testified by CIE test. Those antibodies, which were testified to be activated, were mixed together. The mixture was just pure
IgG (pIgG). After the concentration of pIgG was determined by colorimetry on enzyme reader, pIgG was stored in −20 °C. 2.2.3. Confirmation of purity and activity of pure IgG Purity was testified by SDS-PAGE according to traditional methods, while activity was testified by CIE and Immuno-Double-Diffusion (IDD) according to Silva [12]. These tests helped to choose immunoactivated pure IgG for LP-PEIS. 2.2.4. Immobilization of IgG A drop of Piranha's solution was injected onto golden electrode of crystal flake by micrometer syringe, and was ridded by water after 10 min. Then 5 μl of 1 mg/ml SPA was smeared onto the surface of golden electrode, and crystal was put in 37 °C for 30 min. After golden electrode being cleaned and dried, 5 μl of 8 mg/ml pure IgG (from InRS) or 4.5 mg/ml (from ImRS) was laid on it and crystal was put in 37 °C for 1 h. After golden electrode being cleaned and dried again, 1:100-diluted normal rabbit's sera (NRS) was laid on it and crystal was put in 37 °C to block for 30 min. After being cleaned and dried, crystal can be used for experiment. 2.2.5. Methods of detection The 3 ml of 0.01 M, pH 7.0 PBS was added in reaction pool, followed by running of magnetic stirrer under reaction pool. The crystal with immobilized pure IgG was put in PBS. When the oscillating frequency of crystal was stable (Δ f ≤ 1 Hz/min), the frequency at the moment was recorded as the frequency before detection (F1). Then 60ul InRS was added in pool and the changing process of crystal frequency was initially recorded (once a minute) until the frequency became stable again. The last stable frequency was just the frequency after detection (F2). Thus, the change of frequency which resulted from the immunoreactions between sera sample and Sj IgG on crystal, together with that which resulted from non-specific adsorption between proteins, was F1–F2. The solution in pool was drained out and pool was cleaned by water for 5 times. Then 3 ml PBS and another same crystal (with IgG) were added in pool to detect NRS. According to above methods, the frequency before detection (F3) and that after detection (F4) were recorded. Thus the frequency change resulting from non-specific adsorption between proteins was F3-F4. Therefore the change of frequency resulting only from specific antigen-antibody reaction (ΔF) was (F1–F2)–(F3–F4). Meanwhile, in order to discriminate between specific binding of the primary antibody to circulating antigen or an “nonspecific” reaction e.g. involving binding of primary antibodies to anti-idiotype antibodies that emerge during the later phase of an infection, this LP-PEIS was used to detect 60 μl ImRS, which probably contains anti-idiotype antibodies but certainly not any more antigen. 2.2.6. Regeneration of crystal Three common methods were used to clean the crystals that had been used: (1) A drop of Piranha's solution was injected onto crystal and was ridded by water after 10 min. (2) The crystal was put in the pool with 3 ml of 0.01 M NaOH and 0.05 M NaCl and was deterged with stir for 10 min. Then the solution in pool was replaced by 3 ml of 0.01 mol/L HCl, followed by detergency of crystal in it with stir for 10 min. Finally crystal was cleaned by water. (3) The crystal was put in the pool with 3 ml of 0.1 M glycin–HCl buffer solutions and was deterged with stir for 20 min, and then was cleaned by water. 2.2.7. Primary application of LP-PEIS for detecting SjCAg in human sera Sera samples from 180 persons, including 147 patients with schistosomiasis japonica and 33 healthy persons, were randomly collected. The patients were composed of 112 males and 25 females, in which the youngest was 14 and the oldest was 72. The 147 patients were divided into four groups according to their clinical situations, including group A: patients with acute schistosomiasis; group B:
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patients with chronic schistosomiasis who had not been treated; group C: patients with chronic schistosomiasis who had been treated with praziquantel for half a year, and group D: patients with chronic schistosomiasis who had been treated with praziquantel for one year. Informed consent was obtained from each patient included in the study and the study protocol conforms to the ethical guidelines of the 1975 Declaration of Helsinki as reflected in a priori approval by the institution's human research committee. Randomization was carried out through random digits table. The 180 sera samples were tested blindly by liquid SjCAg-PEIS and sandwich ELISA using polyclonal antibodies, respectively. The operation of liquid SjCAg-PEIS was according to the optimal conditions which were acquired in former experiments. The operation process of sandwich ELISA was according to Qiu. [13] In liquid SjCAg-PEIS, the frequency change above 300 Hz was judged as positive case. In sandwich ELISA, the ratio of P (OD value from detected sera) to N (OD value from control sera) above 2 was judged as positive case. All of the data were statistically analyzed by SSPS 17.0 statistic software. Measurement data were processed by Student t test, while count data were processed by chi square test.
3. Results 3.1. Comparison among various concentration of SPA for coating of crystal SPA and gold (Au) can form very stable SPA-Au compound with a high affinity constant (10 8 L/mol), so SPA is able to be adsorbed onto golden electrode intensely. Meanwhile, SPA can combine specifically with Fc fraction of IgG, leaving Fab fraction of IgG exposed to the outer layer of decorating membrane and extending to fluxion phase. Fab fraction is just the activity center of IgG antibody which links with determinant of antigen. Thus, SPA will not affect the activity of reaction between antigen and antibody. Therefore, SPA is a good immobilizing reagent for antibody [7]. In this experiment, the relation between concentration of SPA and quantity of adsorbed SPA on crystal was studied. Results showed that change of frequency increased with the increase of concentration of SPA until the concentration was beyond 1 mg/ml (Fig. 2). It was thought as that at the moment the surface of golden electrode became saturated.
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3.2. Comparison of antibodies between pre-purification and post-purification The unpurified IgG (InRS), crude IgG (cIgG) and purified IgG (pIgG) were immobilized on crystal, respectively. Their immobilization efficiency and detection effect for SjCAg were compared, respectively (Table 1). Results showed that both immobilization efficiency and detection effect of pIgG were best among three kinds of antibody. The reason that InRS and cIgG had a relative poor effect was probably due to that they contained other proteins which can disturbed the combination between IgG and SPA, and link sites were occupied due to non-specific adsorption, which reduced the effective immobilization amount of antibodies.
3.3. Comparison between InRS and ImRS after purification Results showed that purified InRS had better immobilization efficiency but a worse detection effect than purified ImRS. The reason was possibly that the IgG in purified InRS was against several kinds of Schistosoma antigen and thus had a higher concentration, while the IgG in purified ImRS was only against SEA and thus had a lower concentration. Therefore, the combination quantity of purified InRS with SPA was higher than that of purified ImRS with SPA. However, it was just the variety of IgG in purified InRS that disturbed the combination between IgG and circulating antigen in blood, as well as that increased non-specific reaction with negative sample, which led to a worse detection effect than ImRS. Through detecting the InRS sample and ImRS sample, we found that binding of primary antibodies to anti-idiotype antibodies was some weak and could hardly affect the results of detection for SjCAg (Table 2).
3.4. Dilution of sera samples Since sera samples were added in the pool with a constant capacity of 3 ml PBS, dilutions of samples were nearly related to the quantity of samples in pool. The 300 μl, 100 μl, 60 μl, 30 μl, 15 μl, 10 μl and 5 μl of InRS and NRS were added in pool and were diluted by 3 ml PBS to 1:10, 1:30, 1:50, 1:100, 1:200, 1:300 and 1:600, respectively. These samples were respectively detected by LP-PEIS with purified IgG from ImRS (4.5 mg/ml) immobilized on crystal, in order to find optimum dilution of sample. Results showed that frequency change of positive samples (ΔF +), frequency change of negative samples (ΔF −) and difference between ΔF + and ΔF − (ΔF + − ΔF −) all decreased with the increase of sample dilution (Fig. 3). ΔF + − ΔF − came up to maximum (310 Hz) when sample quantity was 60 μl and the dilution was 1:50 (Fig. 4). Thus it can be thought as that combination between SjCAg in sample and immobilized IgG became saturated at the moment. If sample quantity increased right along, ΔF + and ΔF − only increased due to the increase of viscosity of reaction solution but ΔF + − ΔF − remained unchanging. Therefore, 1:50 can be thought as optimum dilution for sample.
Table 1 Characterizations of three kind of immobilized antibody.a
Fig. 2. Relation between concentration of SPA and immobilization effect of Sj IgG. The change of frequency increased with the increase of concentration of SPA until the concentration was beyond 1 mg/ml. It was thought as that at the moment the surface of golden electrode became saturated.
Immobilized antibody
ΔF1(Hz)
ΔF2(Hz)
ΔF3(Hz)
ΔF4(Hz)
Original antibody (InRS) Crude antibody (cIgG) Purified antibody (pIgG)
190 ± 29 325 ± 21 356 ± 18
171 ± 25 234 ± 17 271 ± 14
63 ± 8 52 ± 6 48 ± 5
53 ± 6 65 ± 7 72 ± 7
a ΔF1 represents the frequency changes produced by the immobilization of antibody onto surface of immunosensor by SPA, ΔF2, ΔF3 and ΔF4 represent the frequency changes produced by positive sample (InRS), negative sample (NRS) and control sample (ImRS), respectively. Each value (ΔF) represents the average of four experiments ± the mean values of their arithmetic errors.
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Table 2 Characterizations of two kind of immobilized antibody.a Immobilized antibody
ΔF1(Hz)
ΔF2(Hz)
ΔF3(Hz)
ΔF4(Hz)
Purified InRS Purified ImRS
356 ± 18 254 ± 10
271 ± 14 357 ± 8
48 ± 5 40 ± 3
62 ± 8 57 ± 5
a ΔF1 represents the frequency changes produced by the immobilization of antibody onto surface of immunosensor by SPA, ΔF2, ΔF3 and ΔF4 represent the frequency changes produced by positive sample (InRS), negative sample (NRS) and control sample (ImRS), respectively. Each value (ΔF) represents the average of four experiments ± the mean values of their arithmetic errors.
3.5. Effect of various infection degree of sample on frequency change A serial of positive rabbit's sera infected with various amount of cercariae of Schistosoma japonicum (all be collected 30 days after infection) were respectively detected with the same dilution of 1:50, in order to study the relation between frequency change and infection degree. Results showed that frequency change increased with the increase of infection degree until amount of cercariae was beyond 4500 (Fig. 5). It may be concluded that at the moment combination sites of immobilized IgG had been saturated, and quantity of SjCAg adsorbed by golden electrode had attained to kinetic balance and would not increase with the increase of its own concentration in sample. 3.6. Optimum time for detection The 60ul InRS that was collected after rabbit had been infected with 4500 cercariae for 30 days, and the 60ul NRS were added respectively in 3 ml PBS in a pool with stir. They were detected by a crystal, onto which pure IgG from ImRS was immobilized through 1 mg/ml SPA. The changing process of frequency was recorded (once every 1–5 min) for 70 min while InRS or ImRS was reacting with immobilized IgG, in order to monitor the whole process of immunoreactions in pool. Results showed that frequency of positive sample decreased quickly in first 30 min but slowly in second 30 min, and did not change any more after 60 min. Meanwhile, frequency of negative sample only change a little in first 2 min and tend to be stable after 5 min with a little fluctuation of 1–3 Hz due to stir (Fig. 6). Therefore, the soonest detection time for detecting SjCAg should be 5 min, while the optimum detection time for monitoring the kinetic procedure of
Fig. 3. Relation between dilution of sample and response. The frequency change of positive samples (ΔF+), frequency change of negative samples (ΔF−) and difference between ΔF+ and ΔF− (ΔF+ − ΔF−) all decreased with the increase of sample dilution.
Fig. 4. Relation between dilution of sample and immunoreaction. The ΔF+ − ΔF− came up to maximum (310 Hz) when sample quantity was 60 μl and the dilution was 1:50. It can be thought as that combination between SjCAg in sample and immobilized IgG became saturated at the moment.
immunoreactions between antigen and antibody should be 60 min. (ΔF + − ΔF − was 120 Hz in 5 min and 315 Hz in 60 min). 3.7. Regeneration of crystal Since regeneration of crystal is a key element of developing practical PEIS, three common detergents for used crystal were compared to find an ideal method for regeneration of crystal. Results showed that deterging effect of Piranha's solution was the best among three detergents. It not only ridded proteins on crystal as entirely as possible, but also had a good reappearance. Strong acid and alkali together had a preferable deterging effect but a poor reappearance. Both of deterging effect and reappearance of 0.1 M glycin–HCl buffer solutions (pH 2.0) were the worst among them, perhaps because the detergent was only a dissociation reagent for
Fig. 5. Relation between degree of infection and response. The frequency change increased with the increase of infection degree until amount of cercariae was beyond 4500. It may be believed that at the moment combination sites of immobilized IgG had been saturated, and quantity of SjCAg adsorbed by golden electrode had attained to kinetic balance and would not increase with the increase of its own concentration in sample.
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Table 3 Detection for SjCAg in human sera by LP-PEIS and sandwich ELISA. Kinds of sera
Patients with acute Sj* Patients with chronic Sj* Without treatment Be treated for half a year Be treated for one year Healthy human
Total cases
Measured value (x ± SD)
Positive rate (%)Δ
LP-PEIS (ΔF, Hz)
ELISA (OD value)
LP-PEIS
ELISA
P
42
388 ± 27
0.244 ± 0.061
100.0
92.9
0.239
36
349 ± 60
a
0.231 ± 0.073
d
83.3
75.0
0.561
30
176 ± 56
b
0.136 ± 0.035
e
20.0
30.0
0.551
39
150 ± 63
c
0.119 ± 0.034
f
6.7
15.4
0.478
33
173 ± 98
3.0
6.0
1.000
0.102 ± 0.043
*Sj: Schistosomiasis japonica. Δ Standard for positive cases: LP-PEIS: ΔF ≥ 300 Hz; ELISA: OD P/N ≥ 2. There is significant difference between “a” and “b” (P = 0.0003), “d” and “e” (P = 0.026), while there is no significant difference between “b” and “c” (P = 0.274), “e” and “f” (P = 0.624).
Fig. 6. Relation between reaction time and response. The frequency of positive sample decreased quickly in first 30 min but slowly in second 30 min, and did not change any more after 60 min. Meanwhile, frequency of negative sample only change a little in first 2 min and tend to be stable after 5 min with a little fluctuation of 1–3 Hz due to stir.
antigen and antibody and could not rid SPA on golden electrode. Though it could be used again and again without new immobilization of IgG, its detection sensitivity would reduce and disappear after three times. In contrast, Piranha's solution denatured and ridded proteins on crystal quickly by overlapped reaction of two strong oxidants, while NaOH + HCl did so by cooperation of strong oxidant and deoxidizer which increased the damage on crystal and led to shorter service period of crystal. Therefore, in comprehensive consideration, Piranha's solution is the most ideal one among three common detergents for regeneration of crystal. 3.8. Primary application of LP-PEIS for detecting SjCAg in human sera In the detection of patients’ sera with acute Schistosomiasis, the positive rate of PEIS (100%) was higher than that of sandwich ELISA (92.8%). Meanwhile, the false positive rate of PEIS (3.0%) was lower than that of sandwich ELISA (6.0%). However, there were no significant difference between PEIS and sandwich ELISA (P = 0.239, P = 1.000). In the detection of patients' sera with chronic Schistosomiasis, the positive rates of those who had not been treated, or had been treated with praziquantel for half a year or one year were 83.3%, 20.0% and 6.7% respectively in PEIS, while were 75.0%, 30.0% and 15.4% respectively in sandwich ELISA. The measurement data from both of LP-PEIS and sandwich ELISA showed that there were significant difference between the patients without treatment and those who had been treated with praziquantel for half a year (P= 0.0003, P = 0.026). The difference in LPPEIS seem more obvious than that in sandwich ELISA, which apparently suggested that LP-PEIS has better evaluation effect than sandwich ELISA. However, there were no significant difference between PEIS and sandwich ELISA (P = 0.561, P = 0.551, P = 0.478) (Table 3).
In this study, piezoelectric immunosensor was used to detect SjCAg. Purified IgG was immobilized on surface of piezoelectric crystal by SPA to establish a novel LP-PEIS for detecting SjCAg in sera. The whole process of reaction between antigen and antibody can be monitored dynamically by this LP-PEIS. A lot of important data about optimum condition for detection was acquired. Sera samples of 147 patients with schistosomiasis japonica and 33 healthy persons were tested blindly by the liquid SjCAg-PEIS with optimum condition and sandwich ELISA with polyclonal antibodies, respectively. The results showed that both of LP-PEIS and sandwich ELISA can detect SjCAg in human sera and evaluate the curative effect of praziquantel very well. In the detection of patients’ sera with acute schistosomiasis, LP-PEIS has higher positive rate (100%) and lower false positive rate (3.0%) than sandwich ELISA (92.8%, 6.0%, respectively). However, there were no significant difference between LP-PEIS and sandwich ELISA. Therefore, this novel LP-PEIS was believed to be a good method for quantificationally detecting SjCAg in sera. It has more advantages over common serological tests, such as simple operation, quick reaction, quantitive detection and real-time monitoring for antigen-antibody reaction in liquid. This study lays a foundation for early diagnosis of schistosomiasis japanica and evaluation of curative effect by LP-PEIS. Moreover, based on the study, an automatic electronic analysator which can diagnose schistosomiasis quickly will be invented in the future. A lot of samples can be detected at the same time through installing many small reaction pools, in each of which a crystal is put and connected to an independent cymometer. If it becomes true, diagnosis of schistosomiasis will probably be in a great revolution and a new epoch for the diagnosis of schistosomiasis will come.
Acknowledgements The study was provided financial assistance by National Natural Science Foundation of China and Ministry of Health and Hunan Province's Scientific Committee of China.
4. Discussion References For a long time, early diagnosis of schistosomiasis japanica is always a difficult problem to all of the parasitologists. Detection for SjCAg is a good idea, but there are few methods that can detect it very well until now. At the present time, the most common method for detecting SjCAg is sandwich ELISA [13–15]. However, sandwich ELISA still has some defects, such as perplexing operation process, slow detection speed and nonspecific detection result.
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