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Clinical and Vaccine Immunology, March 2008, p. 569-574, Vol. 15, No. 3
1071-412X/08/$08.00+0     doi:10.1128/CVI.00273-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Development of an Immunochromatographic Strip for Rapid Detection of H9 Subtype Avian Influenza Viruses{triangledown}

Fuhu Peng, Zheng Wang, Shuhui Zhang, Renwei Wu, Sishun Hu, Zili Li, Xiliang Wang, and Dingren Bi*

Laboratory of Animal Microbiology and Immunology, State Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan, People's Republic of China

Received 2 July 2007/ Returned for modification 24 October 2007/ Accepted 9 January 2008


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ABSTRACT
 
An immunochromatographic strip was developed for the detection of the H9 subtype of avian influenza viruses (H9AIVs) in poultry, using two monoclonal antibodies (MAb), 4C4 for H9AIV hemagglutinin (HA) and 4D4 for nucleoprotein. The 4C4 MAb was labeled with colloidal gold as the detection reagent, and the 4D4 MAb was blotted on the test line while a goat anti-mouse antibody was used on the control line of the nitrocellulose membrane. In comparison with the HA and HA inhibition (HI) tests, the strip was specific for the detection of H9AIV, with a sensitivity at 0.25 HA units within 10 min. Storage of the strips at room temperature for 6 months or at 4°C for 12 months did not change their sensitivity and specificity. Evaluation of the strip with experimental tracheal and cloacal swab samples collected from H9N2-infected chickens revealed that the strip detected the H9N2 viruses on day 3 postinoculation, earlier than the appearance of clinical symptoms. Application of the strip for the analysis of 157 tracheal or cloacal samples from potentially infected chickens on five poultry farms showed that four farms had chickens that were infected with H9AIV. Further characterization of 10 positive and 30 negative randomly selected samples showed that no single sample was false positive or negative, as determined by the standard virus isolation and HI assays. Therefore, the immunochromatographic strip for the detection of H9AIVs has high specificity, sensitivity, and stability. This finding, together with the advantages of rapid detection and easy operation and without the requirement for special skills and equipment, makes the strip suitable for onsite detection and the differentiation of H9AIVs from other viruses in poultry.


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INTRODUCTION
 
Influenza viruses (family Orthomyxoviridae) are classified into three types, A, B, and C, based on the antigenicity of viral nucleoproteins and matrix proteins (18). Influenza viruses are further classified into different subtypes according to the antigenicities of the hemagglutinin (HA) and the neuraminidase (NA) surface glycoproteins. Avian influenza virus (AIV) belongs to the type A viruses and has 16 different HA and 9 NA subtypes (5, 31). AIV is categorized into two groups based on its virulence. Infections of poultry with highly pathogenic AIV (HPAIV) usually result in a very high mortality, up to 100%, while infections with low-pathogenicity AIV (LPAIV) can be asymptomatic or often can induce mild respiratory symptoms. Interestingly, infections with LPAIV can rapidly lead to a reduction in egg production in poultry. Importantly, LPAIVs can serve as progenitors to HPAIVs. Therefore, early detection and control of LPAIV infection will be economically and healthfully significant.

Since the H9N2 subtype of AIV was discovered in turkeys in Wisconsin in 1966 (11), the H9N2 AIV-mediated outbreaks have been reported in many countries in the world (1, 7, 16, 20, 24, 25). In mainland China, the H9N2 AIV was first isolated from chickens in Guangdong Province in 1994 (4), and a huge outbreak occurred in 1998, which subsequently spread to several provinces in China. During the outbreak and the spread of the disease, most chickens infected with the H9N2 AIV showed clinical symptoms such as mild respiratory signs, edema around the eyes, and diarrhea, laid soft-shelled eggs, and had a severe drop in egg production and a 5 to 15% mortality rate, leading to severe economic losses in the poultry industries (19). In addition, surveillance in the poultry markets in Hong Kong in 1997 revealed that the H9 subtype AIV (H9AIVs) were cocirculating with the HPAI H5AIVs, raising the concern of genetic recombination between these viruses (37). Importantly, the accumulated evidence has demonstrated that H9N2 AIV can infect mammals, including humans (8, 21). H9AIVs have also been considered to be one of the candidates for the next potential pandemic. Hence, the rapid identification of the virus has important clinical, economic, and epidemiological implications.

Various laboratory methods are currently available for the detection and surveillance of H9AIVs, and they include virus isolation, the HA assay, the HA inhibition (HI) test (14), and reverse transcription (RT)-PCR (3). However, these assays are laborious, time-consuming, and difficult to incorporate into an automated procedure, and they require laboratory operations, skilled technicians, and special equipment/facilities. Therefore, the development of a sensitive, specific, and easily performed assay is crucial for the rapid detection and surveillance of H9AIV infection and spread.

The immunochromatographic assay is a new immunochromatographic technique in which a cellulose membrane is used as the carrier and a colloidal gold-labeled antigen or antibody is used as the tracer. The method has been widely used for the diagnosis of many contagious human diseases and for the detection of bioactive molecules, hormones, haptens, and others (9, 30). Recently, it has been efficiently applied to the detection of bovine virus diarrhea and white spot syndrome viruses (15, 27). We have successfully developed immunochromatographic strips for detecting antibodies against AIV (22) and detecting sulfadiazine residue in eggs and chickens (28, 29). To extend these studies, we have recently developed an immunochromatographic strip which can specifically, sensitively, and rapidly detect H9AIV. Here, we report the development and validation of this assay system and discuss its implication in the surveillance of H9AIV.


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MATERIALS AND METHODS
 
Equipment. Transmission electron microscopy images were recorded with a Hitachi H600 transmission electron microscope (Hitachi Instrument Co., Tokyo, Japan). A model ZX1000 dispensing platform and a model CM4000 guillotine cutter (BioDot, Irvine, CA) were used for the preparation of test strips.

Chemicals and special reagents. Gold chloride (HAuCl4·3H2O), sodium citrate (C6H5Na3O7·2H2O), bovine serum albumin (BSA), polyvinylpolypyrrolidone K30, and Tween 20 were purchased from Sigma (St. Louis, MO). The recombinant nucleocapsid protein was generated and purified in our laboratory, as previously described (12). In brief, the cDNA for the H9AIV nucleoprotein (NP) gene (1,497 bp) was obtained from H9N2 virus by RT-PCR and cloned into an expression vector, pGEX-KG, in Escherichia coli to generate the recombinant plasmid pKG-NP. Following transformation, the expression of the fusion protein glutathione S-transferase (GST)-NP was induced using 1 mmol/liter isopropyl-β-D-thiogalactopyranoside (IPTG) in E. coli. Subsequently, the recombinant GST-NP was purified with a GSTrap high-performance column (Amersham Biosciences), according to the manufacturer's instructions. The eluted NP were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The goat anti-mouse antibody was obtained from Sino-American Biotechnology Co. (Luoyang, China). Nitrocellulose membranes, glass fibers, and absorbent paper were purchased from Millipore Corporation (Bedford, MA).

Viruses and antigens. Influenza A virus subtype H9N2 (influenza A/chicken/China/HSS/1999) was isolated from an AIV-infected chicken in Hubei Province in 1998 (19). Reference antigens for each subtype of H5, H7, or H9 AIV and specific sera against each subtype of AIV were obtained from Harbin Veterinary Research Institute (Harbin, China). Other reference viruses, including Newcastle disease virus (NDV), infectious bronchitis virus (IBV), infectious bursal disease virus (IBDV), infectious laryngotracheitis virus (ILTV), and fowl adenovirus (FAV), and reference sera specific for NDV were obtained from the China Institute of Veterinary Drug Control (Beijing, China).

Chickens, infection, and sample preparations. Influenza A H9N2 virus was propagated in the allantoic cavity of 10-day-old specific-pathogen-free (SPF) embryonated chicken eggs (Spafas Poultry Co., Jinan, China). Following incubation of the allantoic fluids at 37°C for 48 h, the fluids were harvested (26), and their viral content was determined by using an HA assay, mixing 25 µl allantoic fluids with an equal volume of 0.5% (vol/vol) chicken red blood cells in phosphate-buffered saline (PBS) in V-bottom microtiter plates (10).

A total of 25 White Leghorn bio-clean chickens at 6 weeks of age were housed in animal experimental facilities at Huazhong Agricultural University, China, and experimentally infected intranasally with 0.2 ml of H9N2-infected allantoic fluid (128 HA units of H9N2). A group of White Leghorn bio-clean chickens inoculated with allantoic fluid from unmanipulated eggs was used as the control. Their cloacal and tracheal swab samples were collected 1 day before inoculation and every other day after inoculation, for up to 11 days postinoculation (p.i.). On day 3 p.i., four infected and two control chickens were sacrificed, and their tracheas, lungs, hearts, livers, spleens, kidneys, and muscles were collected. The collected cloacal and tracheal samples were pretreated by dipping the swabs into 1 ml distilled water in 1.5-ml centrifuge tubes with gentle stirring and extrusion. After samples were allowed to settle for a couple of minutes, their supernatants were collected for the strip tests. The individual organ sample, including trachea, lung, heart, liver, spleen, kidney, and muscle samples collected from each chicken, was homogenized in 10 volumes of distilled water. After samples settled for 15 min, their supernatants were collected as samples for the strip tests.

Preparation of anti-AIV MAbs. The monoclonal antibodies (MAbs) against the NP or the H9 subtype hemagglutinin of AIV were produced as previously described (33, 36). Briefly, BALB/c mice at 6 to 8 weeks of age were immunized subcutaneously with 0.1 ml of H9N2 virus (512 units of HA) emulsified in 50% complete Freund's adjuvant. The mice were boosted with the same amount of antigen in 50% incomplete Freund's adjuvant every 15 days, four times, followed by an intraperitoneal injection with 0.2 ml of H9N2 virus (1,024 units of HA). Three days later, their splenic mononuclear cells were isolated and fused with murine myeloma cells (SP2/0), using 50% polyethylene glycol. The hybridomas were generated through the selection of hypoxanthine-aminopterin-thymidine medium. The supernatants of hybridoma cultures from each well were screened using a recombinant nucleocapsid protein-based enzyme-linked immunosorbent assay (ELISA) and H9 subtype hemagglutinin-specific HI assays. The positive hybridoma cells were cloned by a limiting dilution, and the stable hybridoma clones were injected into BALB/c nude mice.

The immunoglobulin G1 MAbs 4C4, which recognizes the H9 subtype hemagglutinin, and 4D4, which recognizes NP, were purified from mouse ascitic fluids, using sequential precipitation with caprylic acid and ammonium sulfate (23) and dialyzed against the phosphate buffer (pH 7.4) at 4°C. Their purities were characterized by SDS-PAGE, and their specificities and affinities were demonstrated by ELISA (13).

Preparation of colloidal gold and colloidal gold-MAb conjugate. Colloidal gold was prepared as previously reported (6), with minor modifications. Briefly, 200 ml of 0.01% (wt/vol) HAuCl4 in doubly distilled water in a 500-ml round-bottom flask was heated with rapid stirring up to boiling, and then 3.6 ml of 1% trisodium citrate was added to the solution. After the colloidal gold solution was boiled for an additional 15 min, it was stirred continually and allowed to cool down gradually. Its pH was adjusted to 8.2 by using 1% potassium carbonate (wt/vol), followed by storage at 4°C in a dark-colored glass bottle.

MAb 4C4 (300 µl; 1 mg/ml) was mixed with 20 ml of colloidal gold solution. The mixture was stirred vigorously for 30 min and added to 2.5 ml of 10% (wt/vol) BSA to block excess reactivity of the gold colloid, followed by stirring the mixture for an additional 30 min. After the mixture was centrifuged at 6,000 x g at 4°C for 45 min, the resulting conjugate pellet was resuspended and washed twice with 2 mM borax buffer (pH 9.0) containing 0.1% (wt/vol) polyethylene glycol (molecular weight, 20,000), followed by resuspension in 1 ml of the same buffer. The size and shape of the unconjugated colloidal gold and those of colloidal gold conjugated to antibodies were characterized by using transmission electron microscopy measurements according to a standard procedure (32).

Preparation of the immunochromatographic strip. The immunochromatographic strip was composed of four components, a sample pad, a conjugate pad, a nitrocellulose membrane, and an absorbent pad, as illustrated in Fig. 1. The sample pads (cellulose fiber; catalog no. CFSP223000; Millipore) and the conjugate pads (glass-fiber membrane, catalog no. GFCP203000; Millipore) were treated with 20 mM phosphate buffer containing 2% BSA, 2.5% sucrose, 1% Tween 20, 0.3% polyvinylpyrrolidone K30, and 0.02% sodium azide (pH 7.4) and dried at 37°C. The MAb 4D4 (1 mg/ml) or the goat anti-mouse antibody (1 mg/ml) in PBS was dispensed at the test or the control line on the nitrocellulose membrane (catalog no. SHF01200225; Millipore), using a BioDot XYZ platform at a rate of 0.9 µl/cm and a speed of 4 cm/s and then dried at 37°C. The MAb 4C4-colloidal gold conjugate was applied to the treated conjugate pad at a rate of 10 µl/cm (about 1.5 µg/cm) and then lyophilized completely. The absorption pad, nitrocellulose membrane, pretreated conjugate pad, and sample pad were assembled as a strip and attached to a plastic scale board with a 1- to 2-mm overlap, sequentially. The assembled plate was cut into 3-mm-wide pieces, using a CM 4000 cutter (Bio-Dot). The generated strip products were packaged in a plastic bag with desiccant and stored at 4°C or under the indicated condition.


Figure 1
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  		FIG. 1. Schematic diagram of the immunochromatographic strip.

The principle of immunochromatographic assay and procedure for the test. During the assay process, the liquid sample is applied to the sample pad, and it rapidly diffuses into the conjugate pad. If the sample contains H9AIV antigen, the sample will react with the colloidal gold-4C4 conjugate to form an antigen-colloidal gold-4C4 complex. The complex will move along on the nitrocellulose membrane chromatographically, due to capillary action. Eventually, the complex will react with immobilized anti-NP MAb 4D4 on the test line to form a colored band. The excess conjugate, or free conjugate if the sample does not contain H9AIV antigen, will migrate along the membrane to the control line, where it will interact with immobilized goat anti-mouse antibody to form a colored band. Therefore, a positive sample will display two bands, one at the test line and one at the control line, while a negative sample will show only one band at the control line within 10 min. The composition of the immunochromatographic strip test is illustrated in Fig. 2.


Figure 2
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  		FIG. 2. Sample results of the immunochromatographic strip test.

Specificity, sensitivity, and stability of the immunochromatographic strip. The allantoic fluid harvested from H9N2-infected eggs at 256 HA units, the allantoic fluid from unmanipulated eggs, the standard H5, H7, and H9 subtypes of AIV antigens (5 mg/ml), and other viruses commonly infecting poultry, including NDV (256 HA units/ml), IBV (105.0 50% egg infective dose [EID50]/ml), IBDV (105.0 50% egg lethal dose [ELD50]/ml), ILTV (107.2 EID50/ml), and FAV (egg drop syndrome 76 virus, 1,024 HA units/ml), were simultaneously tested by using the immunochromatographic strips. Allantoic fluids with different units of HA were used to evaluate the sensitivity of the immunochromatographic strip. To determine the stability, immunochromatographic strips were stored at room temperature or at 4°C and used for testing positive (16 HA units) and negative samples every 15 or 30 days.

Diagnosis of H9AIV infection in the field. The immunochromatographic strips were applied in diagnosing H9AIV infection. A total of 157 tracheal or cloacal swabs were collected from chickens with respiratory symptoms consistent with AIV infection on five chicken farms in Hubei Province. Potential H9AIV infection was assessed in duplicate by using the immunochromatographic strips. Ten positive and thirty negative samples tested by the immunochromatographic strips were randomly selected and retested using the standard virus isolation and HI assays (10, 34). Briefly, sample supernatants were pretreated with an antibiotic-antimycotic solution. Approximately 200-µl supernatants of individual samples were inoculated into 9- to 10-day-old SPF embryonated chicken eggs. On day 5 p.i., the allantoic fluids were harvested and tested for the presence of H9AIV, using the HA and HI tests and the H9N2-specific sera (Harbin Veterinary Research Institute, Harbin, China).


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RESULTS
 
Specificity and sensitivity of the immunochromatographic strip. To determine the specificity of the immunochromatographic strip, allantoic fluids harvested from H9N2-infected eggs at 64 units of HA and allantoic fluids from unmanipulated eggs, together with standard antigens of H5, H7, or H9AIV and other viruses, were characterized using the immunochromatographic strips simultaneously (Fig. 3A). Clearly, while all of the samples tested showed one strong band on the control line, only allantoic fluid harvested from H9N2-infected eggs and the standard H9AIV antigen displayed an additional band on the test line on the immunochromatographic strips. This suggests that the immunochromatographic strip can specifically detect H9AIV but not the other tested AIVs, NDV, IBV, IBDV, ILTV, and FAV, common infectious viruses in poultry. This high specificity of the immunochromatographic strip allows discrimination of H9AIV from other common virus-mediated infections in poultry.


Figure 3
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  		FIG. 3. Specificity and sensitivity of the immunochromatographic strip. Negative allantoic fluids (AF) of healthy SPF embryonated chicken eggs, positive allantoic fluids from H9N2 AIV-infected chicken eggs, reference standard antigens of H9, H5, and H7 subtypes of AIV, and other viruses, including NDV, IBV, IBDV, ILTV, and FAV, were simultaneously characterized by the strips (A). Similar patterns of results were observed for experiments repeated 20 times. The 64 HA units of H9N2 AIV was serially diluted and tested with the strips for determining the sensitivity of the strip (B).

The allantoic fluids containing H9N2 AIV at 64 units/ml of HA were serially diluted and evaluated for the sensitivity of the immunochromatographic strip (Fig. 3B). When the allantoic fluids were diluted from 1:2 to 1:256 (from 32 to 0.25 HA units, respectively), two clear bands at the test and control lines were observed. Therefore, the sensitivity of the immunochromatographic strip reached a level of 0.25 units of HA antigens. Notably, the densities of the bands for various concentrations of HA antigens on the test lines gradually declined from the strongest band at 32 units of HA to 0.25 units of HA, suggesting that the efficacy of detecting H9AIVs by this strip is dose dependent. Furthermore, similar results were observed by repeating the test of these samples 10 times (data not shown), indicating the high reproducibility of this experimental system. Therefore, the immunochromatographic strip for the detection of H9AIVs is both highly specific and sensitive and is reproducible.

Stability of the immunochromatographic strip. To determine their stability, the immunochromatographic strips were randomly sampled. They were stored at room temperature or at 4°C for up to 12 months, and their specificity and sensitivity levels for the detection of H9AIVs were tested every 15 to 30 days (Table 1). The immunochromatographic strips stored at 4°C for 12 months showed persistent sensitivity and could detect 0.25 HA units of H9AIVs, the same sensitivity level as that of a strip that was freshly produced. In contrast, although the strips kept at room temperature for 6 months did not alter their sensitivity for detecting H9AIVs, continual storage at room temperature for 9 months reduced their sensitivity by 50%, and extension of the storage time to 12 months further decreased the sensitivity of the strips. Importantly, the specificity of the immunochromatographic strip for the detection of H9AIVs did not change, as evidenced by the fact that no single negative sample became a false positive regardless of the storage conditions tested. Apparently, the immunochromatographic strip can be stored at 4°C for at least 12 months and at room temperature for 6 months without losses in sensitivity and specificity for the detection of H9AIVs.


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  		TABLE 1. Specificity and sensitivity of the immunochromatographic strip under various storage conditionsa

Evaluation of the immunochromatographic strip for testing samples from experimental infected chickens. To evaluate whether the immunochromatographic strip could detect clinical samples, groups of White Leghorn chickens were inoculated intranasally with allantoic fluids containing H9N2 viruses or healthy allantoic fluids. Following the chickens’ infection, their tracheal and cloacal swab samples were longitudinally collected and tested using the immunochromatographic strips described in Table 2. Control chickens inoculated with healthy allantoic fluid showed no single positive for the H9AIV infection throughout the observation period. In contrast, 25 out of 25 tracheal and 23 out of 25 cloacal swab samples from the H9N2-infected chickens were identified as positive for H9AIV, by using the strips on day 3 p.i., when no clinical sign was observed in all experimental chickens. Analysis of their sera revealed that no anti-H9AIV antibody was detected before infection and the titers of anti-H9AIV antibodies reached about 1:16 in most infected chickens on day 5 p.i., as determined by the HI assays (data not shown). Hence, the immunochromatographic strip can detect early H9AIV infection in chickens. Notably, the positive rates of the detection of H9AIVs in this experimental model gradually declined with time. On day 11 or 9 p.i., there was no detectable H9AIVs in tracheal or cloacal swab samples, respectively, which may reflect specific antibody-mediated viral clearance.


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  		TABLE 2. Evaluation of the immunochromatographic strip's ability to detect H9N2 in tracheal and cloacal swab samples from experimentally infected chickens

Given that all of the chickens experimentally infected showed no clinical sign on day 3 p.i., four infected and two control chickens were randomly selected and sacrificed. Their trachea, lung, heart, liver, spleen, kidney, and muscle samples were analyzed by using the strips. All the tracheal samples from the infected chickens were positive for H9AIV infection; however, the results from other organ samples were negative, except that some organ samples from one infected chicken were questionably positive. These results are consistent with the notion that the H9AIVs spread mainly via a respiratory route. Our data suggest that the tracheal or the cloacal swab sample is the most appropriate for the immunochromatographic strip test.

Clinical application of the immunochromatographic strip. To further validate the application of the immunochromatographic strip at the clinic, a total of 157 cloacal and tracheal swab samples were collected from chickens on five poultry farms where the housed chickens were suspected of having viral infections. Characterization of these samples revealed that 26 out of 157 cloacal and tracheal swab samples were positive for H9AIV infection (Table 3). Notably, 10 positive and 30 negative samples were randomly selected from the pools of strip-positive or strip-negative samples and inoculated into SPF embryonated chicken eggs. Five days later, the eggs’ allantoic fluids were harvested and tested using the HA and HI assays. Allantoic fluids of the 10 positive samples showed various titers (4 to 64 units) of HA and were all positive for H9AIV as determined by the HI assay (Table 4). Interestingly, allantoic fluids from the 30 negative samples tested by the strip tests were still negative for H9AIVs in the HI assay, although 14 of these were positive by HA, and allantoic fluids of 7 had evidence of NDV, by HI assay. Thus, the results from the immunochromatographic strip tests were consistent with the data from the highly sensitive and specific virus isolation test. These findings suggest that the immunochromatographic strip may be used for the detection and differentiation of H9AIV in clinical diagnosis.


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  		TABLE 3. Diagnosis of H9AIV infection in the field


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  		TABLE 4. Comparative analysis of samples randomly selecteda


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DISCUSSION
 
The epidemic of AI has resulted in a tremendous economic loss and has potentially affected human health. Since the 1990s, specific vaccination has been employed in many countries. However, epidemiological studies have indicated that the LPAIV infections, caused especially by the H9 subtype of LPAIVs, have become ubiquitous and endemic in poultry in many countries (2). Furthermore, infection with H9AIVs usually is asymptomatic or induces mild respiratory symptoms but causes a rapid drop in egg production in poultry, which is similar to that of other OIE-listed viruses, such as NDV, IBV, or ILTV, in vaccinated poultry. Therefore, the development of an easily operated assay for the on-site detection of H9AIVs will be significant in the surveillance of H9AIV infection and spreading.

We have successfully developed the immunochromatographic strip for the detection of H9AIVs, using colloidal gold-conjugated 4C4, a MAb specific for the hemagglutinin of H9AIV as the detection antibody, and 4D4, an anti-NP MAb as the precipitation reagent on the membrane. Analysis of the specificity showed that the strip was specific for the detection of H9AIVs and H9AIV antigens and reacted with neither the allantoic fluid from unmanipulated eggs, H5, or H7AIV antigens, nor with other viruses tested, NDV, IBV, IBDV, ILTV, and FAV. Characterization of the sensitivity revealed that the strip could detect H9AIVs at 0.25 HA units within 10 min, which was similar to that of the HI assay. The sensitivity and specificity of the strips did not decrease after strips were stored at room temperature for 6 months or at 4°C for 12 months, a demonstration of their high stability. Furthermore, the evaluation of the strip with experimental tracheal and cloacal swab samples collected from H9N2-infected or control chickens revealed that the strip could be used for detecting H9N2 viruses on day 3 p.i., which was earlier than the appearance of clinical symptoms following H9AIV infection. Conceivably, this strip can be used for the early detection of H9AIV infection. In addition, application of the strip in the analysis of 157 tracheal or cloacal samples from potentially infected chickens on five poultry farms showed that four farms had chickens infected with H9AIV. Further characterization of 10 positive and 30 negative samples randomly selected using the standard virus isolation, the HA, and the HI assays showed that no single sample was a false positive or negative. Therefore, the immunochromatographic strip for the detection of H9AIVs has high specificity, sensitivity, and stability.

Currently, there are several assays available for the detection of H9AIVs. The HA and HI assays, the "gold standard" tests, are labor-intensive and time-consuming and require several controls freshly prepared for standardization, which makes them unsuitable for the rapid and on-site characterization of AIV infection. Similarly, RT-PCR can detect subtypes of virus with high sensitivity (3); however, it usually requires special primers, a laboratory operation, skilled technicians, and specialized equipment, which makes it difficult for the rapid and on-site detection of viruses in the field. In comparison with these assays, the strip we developed can be used for the rapid detection of H9AIVs as the results can be read directly by the naked eye within 10 min. This assay is easily operated and can be performed by farmers. Collectively, the immunochromatographic strip we have developed has many advantages and potentially can be used for the on-site detection of H9AIVs.

The specificity and sensitivity of the immunochromatographic strip are largely dependent on the following factors. First, the quality of the MAbs used in the strip test is crucial for the specificity and sensitivity of the strip. Both of the MAbs we used specifically recognize H9AIVs and have a high affinity for their antigen epitopes. Second, pretreatment of the sample pad and the conjugate pad is important for enhancing the release speed of the conjugate and for reducing nonspecificity. Furthermore, a careful choice of a membrane is critical for high specificity, sensitivity, and rapid detection as the wicking rate and speed of liquid diffusion on the membrane are key characteristics for the suitability of membranes (35). Membranes with a larger pore size give a lower wicking rate and higher diffusion rate, which benefits the rapid detection test. However, this kind of membrane usually has a lower capacity for protein binding, potentially leading to a low sensitivity, as the pore sizes of the membranes are inversely correlated to the protein binding capacity of the membrane. We found that the 120-s/4-cm nitrocellulose membrane was optimal for our experimental system.

In summary, we have successfully developed a highly specific and sensitive immunochromatographic strip for the detection of H9AIVs. Application of this strip at the clinic demonstrated that this strip could be used for the rapid, on-site, and early detection of H9AIVs. The development of this strip provides a screening tool for the differential diagnosis of H9AIV infection from other subtypes of AIV and virus-mediated diseases in poultry. Further extension of this first generation of immunochromatographic strip to a multiple immunochromatographic assay for the detection of other AIVs and viruses may help differentially diagnose common poultry diseases, such as Newcastle disease, avian infectious bronchitis, avian infectious laryngotracheitis, and others. Thus, our findings provide a basis for the design of a new test strip for the surveillance of virus-mediated diseases in poultry.


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FOOTNOTES
 
* Corresponding author. Mailing address: College of Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070, People's Republic of China. Phone: 86-27-87280208. Fax: 86-27-87280408. E-mail: bidingren{at}mail.hzau.edu.cn Back

{triangledown} Published ahead of print on 16 January 2008. Back


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Clinical and Vaccine Immunology, March 2008, p. 569-574, Vol. 15, No. 3
1071-412X/08/$08.00+0     doi:10.1128/CVI.00273-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.




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