Received Date: March 14, 2015; Accepted Date: March 30, 2015; Published Date: April 04, 2015
Citation: Liu X,Qin B, He T,Chen J,Cao D, et al.(2015) Development of a Specific Latex Agglutination Test to Detect Antibodies of Enterovirus 71. Metabolomics 5:142. doi: 10.4172/2153-0769.1000142
Copyright: © 2015 Qin B, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
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A latex agglutination test (LAT) was developed for the rapid detection of antibodies against the VP1 or VP1 proteins of Enterovirus 71 (EV71). The proteins of interest, including prokaryotically expressed VP1 and two strains of anti-VP1 Monoclonal antibody (McAb) against EV71 were covalently linked to carboxylated latex using Ethyl-dimethyl-aminopropyl carbodiimide (EDC) to prepare sensitized latex beads. The LAT was evaluated by an enzyme-linked immunosorbent assay (ELISA) as a reference test. The VP1-LAT showed a sensitivity of 87.0%, a specificity of 88.9%, and an agreement ratio of 90.0% in detecting VP1 in 100 serum samples from experimentally infected mice, while these values were 86.8%, 96.7%, and 93.3%, respectively, for 608 clinical human serum samples. The VP1-LAT has advantages over other assays in terms of low cost, rapidity, chemical stability, high sensitivity, repeatability, and specificity. The LAT established in the present study is a rapid and simple test suitable for field monitoring of antibodies against VP1-EV71.
Enterovirus 71; Hand-foot-and-mouth disease; Latex agglutination test; Homogeneity; Ethyl-dimethyl-amino-propyl carbodiimide
Hand, foot and mouth disease (HFMD) is a common childhood disease that is characterized by fever, a rash on the hands and feet, and mouth ulcers [1,2]. Most children, usually under five years old, with HFMD carry Coxsackievirus A and/or Enterovirus 71 (EV71) . It is usually a mild and self-limiting febrile disease in children, however, EV71 infection has been associated with various neurological diseases, such as aseptic meningitis, polio-like paralysis, and acute encephalitis with neurological pulmonary edema, mainly in young children and infants [1,4,5].
The neurovirulence of EV71 first drew public concern in 1975 in Bulgaria when 44 people died of a polio-like disease . EV71 was correlated with epidemics of neuroinvasive diseases in New York, Malaysia, Japan, Australia, Taiwan, China, Hungary, and Hong Kong ; thus, it is regarded as an emerging neurotropic enterovirus, as is poliovirus [6,8].
EV71 is a non-enveloped virus, belonging to the genus Enterovirus of the Picornaviridae family, with a positive, single-stranded positivepolarity RNA of about 7.5 kb in size [9,10], with single Open reading frame (ORF) encodes a huge polyprotein of 2,194 amino acids and is flanked by a 5’-untranslated region (UTR) and 3’-UTR, which can be divided into three regions: P1 consisting of the four structural proteins VP1 to VP4, and P2 and P3 .
For seroepidemiological surveys and evaluations of vaccine effects, EV71 IgG from serum samples needs to be detected. At present, the serologic test for EV71 is the Enzyme-linked immunosorbent assay (ELISA), which takes a minimum of several hours and requires trained personnel and special equipment [12,13]. In our study, we isolated epidemic strains of EV71 from clinical specimens. After being identified by reverse transcribed PCR (RT-PCR) [14,15], the VP1 was amplify and the protein was expressed . For the detection of IgG against EV71 VP1, we used prokaryotically expressed VP1 as bait to establish the latex agglutination method to detect antibodies against EV71. After optimizing the experimental parameters, the latex agglutination method could specifically detect the relevant target. The anti-VP1 IgG detection method was not dependent upon a secondary antibody, such as in an ELISA or immunofluorescence, and it can detect IgG from all kinds of serum samples.
Latex agglutination, among all antigen detection assays, is the most rapid and simplest to perform [17,18]. In this study, we describe a simple and specific Latex agglutination test (LAT) that is based on the VP1 protein for the rapid detection of VP1-specific antibodies in sera. The specificity and sensitivity of the assay was compared with a commercial ELISA kit. LAT is a specific, sensitive, accurate, fast and simple IgG-detection method which has potential value for field applications.
EV71 isolation from clinical samples
Throat swabs anal swab samples of patients diagnosed with HFMD were collected, and were sorted into EV71 or coxsackievirus A16 (Cox A16) by real-time PCR. Swab samples identified as EV71-positive were filtered using a 0.22 μm filter (Millipore, Billerica, MA, USA) and added to the wells of Vero cells cultured in Dulbecco’s modified Eagle’s medium (DMEM; Invitrogen, Carlsbad, CA, USA) at 37°C in a 5% CO2 atmosphere supplemented with 10% fetal bovine serum (Gibco, Grand Island, NY, USA), 2 mM of glutamine, 100 IU/ml of penicillin, and 100 IU/ml of streptomycin. The supernatant was collected and stored at -80°C until the cytopathic effect (CPE) took place, regardless of whether it was the first or second passage. Over time, the growth of the vero cells gradually declined. CPE appeared as early as 72 hours, and as late as the second passage. Two samples resulted with typical CPE are shown in Figures 1A and 1B.
To clarify the sequence and genotype of the local epidemic EV71 strain, 10 EV71 strains were isolated from clinical samples of HFMD children . Positive samples identified by RT-PCR were added to the wells seeded with Vero cells.
Genotype analysis of isolated EV71
The cDNA of EV71 was acquired by reverse transcription using the extracted RNA from the culture supernatant as template and an oligo (dT) primer. Then, the sequence of VP1 was amplified by PCR using pEV71-F and -R primers (Table 1) and then subjected to sequencing. Both the nucleotide and amino acid sequences of VP1 of isolated EV71 strains were compared with that of A, B, and C genotype reference EV7l strains .
Primer-EV-forward coupled with primer-EV-reverse were designed as universal primers of enterovirus, and used to amplify a 154-bp segment shared by enteroviruses, including EV71, EV70, coxsackie virus A, and poliovirus. Primer-EV71-forward and -reverse were designed to detect EV71, while pCA16-forward and -reverse were for CoxA16 detection. Primer EV71-VP1-forward and -reverse were used to construct pET28a-VP1.
Table 1: Primer sequences for pET28a-VP1 construction and qRT-PCR.
Production and purification of recombinant VP1 protein
The production of recombinant VP1 was conducted as follows. After EV71 RNA was extracted, cDNA was acquired by reverse transcription and used as template to amplify the VP1 with the pEV71-F and -R primers (Table 1), which contained the unique restriction sites BamHI and HindIII at the 5’ and 3’ ends, respectively. A 1,015-bp PCR product was detected by electrophoresis and then recovered, digested with BamHI and HindIII, and ligated into the same sites in pET28(a)+ using T4 DNA ligase. The resulting vector, pET-28a-VP1, in which VP1 is fused at the C-terminal end of the Glutathione-S-transferase (GST) protein, was transformed into E. coli strain BL21, and GSTVP1 expression was induced by adding isopropyl-b-D-thiogalactoside (IPTG). Proteins were purified on a GST-affinity column, and dialyzed in Phosphate-buffered saline (PBS) for three days in the next step . The purity of the proteins was analyzed by Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and confirmed by Western blot analysis with mouse sera against the VP1 or EV71 .
Serum samples in our study include mouse serum, rabbit serum, and human serum (adult and infant). Mice and rabbits were challenged with live/inactive isolated EV71 viruses or expressed VP1 protein to acquire different positive sera. 400 serum samples from 3 to 5-yearold children were collected, some of whom were previously infected with HFMD. 100 serum samples from adults were also collected for detection.
Preparation and optimization of sensitized latex beads
Sensitized latex beads were prepared as follows. In brief, 0.7 μm radius 10% latex beads (Ke Xin Company, Shanghai, China) were bridged with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and then conjugated with different concentrations of VP1 protein. The beads were washed gently three times with 0.1M carbonate buffer (pH 9.6) and then three times with 0.02M phosphate buffer (pH 4.5), then suspended in 2% EDC solution and 2-hours-incubated on a shaker at 37°C. After washing three times with boric acid buffer (pH 8.4), VP1 was added and rocked gently for 6 h. Finally, the bead pellet was suspended in latex storage buffer (1% BSA, 5% glycerol, 0.1% NaN3 in PBS, pH 7.4) and stored at 4°C prior to use [17,18,22]. For the best sensitivity and specificity of the LAT, a series of experiments were conducted to identify the optimal concentrations for protein and latex beads, as well as the length of the conjugation reaction. 100μl of VP1 (500 ng/μl) (serial two-fold dilutions from 1:1 to 1:16) was used to sensitize 200 μl of latex beads (1%) [18,23]. The primed beads were mixed with PBS or positive serum and observed for autoagglutination or agglutination. The optimum concentration was defined as the most sensitive and economical concentration that did not result in autoagglutination. Determination of the optimal reaction time for the sensitization and concentration of the latex beads was performed similarly in a systematic fashion .
Latex agglutination test
The LAT was performed by mixing 15 μl samples with 15 μl prepared beads on a microscope slide with a plastic tip or toothpick, the slide was rocked manually for about 1 to 2 minutes which depend on temperature, rocking amplitude and IgG titer . Test results were scored as follows: “++++”, rapid agglutination of 100% of the sensitized latex beads; “+++”, 75% sensitized latex beads agglutinated; “++”, agglutination of 50% of the sensitized latex beads; “+”, fine particle agglutinated, about 25% of the sensitized latex beads with a questionable interpretation; “–”, no visible agglutination greater than negative control. Agglutination reactions “+” to “++++” were considered to be positive results.
Construction of pET-28(a)-VP1 and expression of VP1
As described above, VP1 was PCR-amplified, and then pET-28a- VP1 was constructed successfully. Then VP1 protein was expressed in E. coli strain BL21 and purified on a GST-affinity column and then dialyzed in PBS for three days. The purity of the protein was analyzed by SDS-PAGE [13,18]. As shown in Figure 2A, VP1, which has a molecular weight of 37 kDa, was successfully expressed. Purified VP1 was identified by Western blot analysis with sera from mice challenged with EV71 (Figure 2B, lane 1), sera from mice immunized with VP1 (Figure 2B, lane 2), and a commercially available VP1 polyclonal antibody (Figure 2B, lane 3). It turned out that expressed VP1 protein in our study keeps approving immunogenicity and reactogenicity.
Figure 2: Prokaryotic expression, purification and identification of VP1. The vector pET-28a-VP1 was transformed into E. coli strain BL21, and GST-VP1 was expressed and purified on a GST-affinity column, and then analyzed by western blot. A. SDS-PAGE detection of unpurified (lane 1) and purified (lane 2) VP1 protein, 37 kDa in size. B. Western blot analysis of VP1 using sera from mice challenged with EV71 (lane 1), mice immunized with expressed VP1 (lane 2), and a commercialized VP1 polyclonal antibody (lane 3).
Optimization of latex bead sensitization
Different antigen concentrations were used to sensitize 10% latex beads with an overnight reaction time (as the starting condition, as recommended by the latex bead manufacturer). A standard positive serum sample was serially diluted to react with the sensitized latex beads; the final serum dilution with positive agglutination was recorded as the serum titer .
Samples containing the VP1 protein were prepared at an initial concentration of 500 ng/μl, and then serially diluted two-fold, from 1:1 to 1:64. Next, 200 μl of latex beads were washed as previously described . Serially diluted 100 μl samples containing the VP1 protein were added to the EDC-processed latex beads solution. After 6 hours of gentle rocking, sensitized latex beads were prepared. As shown in Table 2, the most appropriate dilution that did not exhibit autoagglutination was economical, and which had the highest titer for positive sera were the 1:8 dilution for the VP1 protein. That is to say that 6,250 ng of VP1 protein per 200 μl of latex beads stored in 750 μl of buffer could be used to screen 500 samples.
Different antigen concentrations were used to sensitize 1% latex beads with an overnight reaction time (as the starting condition recommended by the manufacturer). Standard positive serum was serially diluted for reaction with the sensitized latex beads.
Table 2: Optimization of protein concentration for latex bead sensitization
To determine the optimal sensitization time, 100 μl of VP1 in a 1:8 dilution of the original concentration (500 ng/μl) was placed into a solution of EDC-processed latex beads. The optimal time for the conjugation reaction was reported as 5 hours in our previous study [18,23]. However, as shown in Table 3, from 2 to 6 hours, there was a time-dependent increase in sensitivity, while 7 and 8 hours of conjugation did not further increase the sensitivity (Table 3). Thus, 6 hours was adopted in the VP1-LAT due to a slightly higher sensibility, timeliness, and lack of autoagglutination .
|S T||LAT||S T||LAT||S T||LAT||S T||LAT||S T||LAT||S T||LAT||S T||LAT|
VP1 proteins at a 1:8 dilution were used to sensitize 1% latex beads with various reaction times (2, 3, 4, 5, and 6 hours). The best reaction time was determined via a reaction with standard positive serum in serial twofold dilutions (from 1:1 to 1:256).
Table 3: Optimization of reaction time for latex bead sensitization
VP1-conjugated latex beads react well with anti-VP1 in sera. The LAT was performed by mixing 15 μl of positive serum with 15 μl of sensitized latex beads on a microscope slide with a plastic tip or toothpick, and then the slide was rocked manually for 30-60 second [18,23]. According to the results shown in Figure 3, VP1-sensitized latex beads can react with the sera of EV71-infected patients (Figure 3B), rabbits (Figure 3C), and mice (Figure 3D) quickly and thoroughly compared with the negative control (Figure 3A). A positive reaction demonstrated a mixture of clear solution and macroscopic particles, while the negative control exhibited a uniform emulsion. We also selected 8 samples of patients to do LAT, the results shown in Fig.3E-L ranged from negative (–) to positive (+ to ++++).
Figure 3: VP1-conjugated latex beads react well with anti-VP1 in different kinds of sera. 15 μl of sensitized latex beads were mixed and rocked with 15 μl of different kinds of sera: negative control (A), serum from an EV71-infected patient (B), serum from EV71-challenged rabbit (C), serum from an EV71-challenged mice (D), and 8 serum samples of patients (E-L).
Specificity, repeatability, and stability of VP1-LAT
To examine the specificity of the latex beads, standard sera positive for a wide range of enteroviruses (enteric cytopathogenic human orphan virus, coxsackievirus A16, norovirus, poliovirus, EV70, and hepatitis A virus), and these virus strains were collected and verified using a commercial ELISA kit and real-time PCR . Six lots of LAT (both VP1 and anti-VP1) were involved in this assay, and used to detect these standard positive sera. These standard sera did not react with the EV71-VP1-LAT, except for the EV71-positive serum (Table 4), indicating that EV71-VP1-LAT established in the present assay was specific for EV71 only.
For specificity determination, six lots of theVP1-LAT were used to test standard serum positive for a wide range of enteroviruses, such as coxsackievirus A16, norovirus, enteric cytopathic human orphan (ECHO)-virus, EV70, HAV, in addition to EV71 as a positive control.
Table 4: Specificity, repeatability, and stability of the VP1-LAT
These six lots of the LAT were also used to test 50 serum samples, all detections were repeated three times with the same batch of beads. The stability of the sensitized latex was judged by performing the VP1- LAT on 50 serum samples three times with the same batch of beads: on the day of preparation, and after 1.5, 3, 4.5, and 6 months of storage at 2-8°C in a refrigerator . Assuringly, all of the repeated tests yielded the same results (data not shown). Thus far, the LAT shows high sensitivity, satisfactory repeatability, and stability.
Comparison of VP1-LAT and ELISA
The sensitivity and specificity of the LAT were evaluated by performing both the LAT and ELISA on 100 serum samples from experimentally infected mice and 608 clinical serum samples. In the 100 serum samples from experimentally infected mice, 87 were positive and 13 were negative by ELISA, whereas 84 were positive and 16 were negative by LAT, and 79 were positive by both assays and 11 were negative in both assays. According to this data, the specificity, sensitivity, and agreement ratio with the ELISA for the VP1-LAT was 90.8%, 84.6%, and 90.0% (Table 5). In the 608 clinical serum samples, including 408 samples from children from one kindergarten in which there was a HFMD outbreak and 200 samples from healthy adults. 212 were positive and 396 were negative by ELISA, whereas 189 were positive and 419 were negative by LAT, while 184 were positive in both assays and 383 were negative in both assays. According to this data, the specificity, sensitivity, and agreement ratio with ELISA for the VP1-LAT was 86.8%, 96.7%, and 93.3% (Table 5). For the total of 708 samples, the specificity, sensitivity, and agreement ratio with ELISA for the VP1-LAT was 88.0%, 96.3%, and 92.8%.
|100 experimental samples||608 clinical serum samples|
Experimental samples Sensitivity: 79/87=90.8%; specificity: 11/13= 84.6%; agreement ratio: (79+11)/100=90.0%.
Clinical serum samples Sensitivity: 184/212=86.8%; specificity: 383/396=96.7%; agreement ratio: (184+383)/608=93.3%.
Total samples Sensitivity: 79+184/87+212=88.0%; specificity: (11+383)/(13+396)=96.3%; agreement ratio: (79+11+184+383)/(100+608)=92.8%.
Table 5: Comparison of the LAT and ELISA in testing serum samples
HFMD, which generally affects children under the age of five, mainly caused by EV71 and coxsackie virus A16 (CVA16), syndromes caused by them are similar and indistinguishable . However, EV71 infection is more serious than CVA16, and it is frequently associated with serious neurological complications and fatalities.
In China, in 2008, about 490,000 HFMD infections and 126 deaths in infants and young children were reported . To date, neither an effective vaccine nor antiviral treatment is available for EV71 infection . Thus, effective vaccines that could provide protection against EV71-associated HFMD or herpangina are urgently needed [6,9]. To evaluate the level of humoral immunity after vaccination, a fast detection method that is inexpensive and simple and easy to operate is required. The LAT, a proactive response to such demands, was confirmed to detect EV71-IgG from both EV71-infected children and mice and rabbits vaccinated with live or inactivated virus. Because it does not require a secondary antibody, as does ELISA, the LAT could detect anti-VP1 IgG from all kinds of sera from mice, rabbits, people, and so on. EDC is a water-soluble dehydrating agent that could revitalize the carboxyl groups of latex beads and induce the generation of amides or esters. When latex beads were first used in testing, when target proteins were connected to traditional latex beads without EDC, the binding was too weak [17,22]. In our study, EDC plays a key role as a bridge to covalently connect latex beads and target proteins, and this binding was much stronger than with traditional latex beads.
The EV71 particle consists of a naked icosahedral capsid, on which VP1 capsid protein located . VP1 harbors the main neutralizing epitopes of EV71, bears the largest immune selective pressure, is a hot spot in the field of vaccine research, and was reported to carry the primary antigenic determinant of EV71. Anti-VP1 IgG is undoubtedly a logical neutralizing antibody of EV71 [9,27]. Hence, we developed VP1-EV71-LAT using an E. coli-expressed GST-VP1 fusion protein to sensitize latex beads. After a series of conditions were optimized, we used the LAT to detect EV71 in clinical specimens. In comparison with commercial ELISA kits, the specificity, sensitivity, and agreement ratio for a total of 708 serum samples (from both experimentally infected mice and clinical serum samples) were 86.8%, 96.7%, and 93.3%, respectively. The data show that the VP1-LAT exhibited high specificity, sensitivity, and agreement ratio, and could be widely used to detect anti-VP1 IgG from any kind of serum samples, as opposed to ELISA, especially to evaluate the effect of vaccination fast when an inactivated vaccine against EV71 is available.
The LAT established in our study shows satisfactory specificity to selectively differentiate antibodies against EV71 from those of other enteroviruses, and it also exhibits outstanding within-lot and lot-to-lot repeatability, and stability. The VP1-LAT is suitable for the rapid and simple serologic detection of antibodies from humans and a variety of target animals against EV71-VP1, or virons. The VP1-LAT has broad clinical application prospects.
The degree of aggregation, as determined by the naked eye, was slightly subjective. As a matter of fact, latex beads are milk-like before any reaction, and a clear solution and aggregated particles will appear if they react with positive samples. The translucency of the reaction system and particle size was closely correlated to the degree of positive reaction. Furthermore, we will try to improve the method of LAT assessment in a future study by detecting the size and amount of aggregated particles or the translucency of the reaction mixture.
We are grateful to the patients for providing the serum samples. This work was supported by Science Technology Bureau of Shaoxing, Zhejiang province of China (2013B70063 and 2012B70063) and Zhejiang Medical Technology & Education (2013KYA208).