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Comparison of a new gold-immunochromatographic assay for the detection of antibodies against avian influenza virus with hemagglutination inhibition and agar gel immunodiffusion assays
Veterinary Immunology and Immunopathology 117 (2007) 17–25 www.elsevier.com/locate/vetimm
Comparison of a new gold-immunochromatographic assay for the detection of antibodies against avian influenza virus with hemagglutination inhibition and agar gel immunodiffusion assays DaPeng Peng a,b,1, SiShun Hu a,1, Yan Hua a, YunCai Xiao a, ZiLi Li a, XiLiang Wang a, DingRen Bi a,* b
a College of Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070, China State Reference Laboratory of Veterinary Drug Residues, Huazhong Agricultural University, Wuhan 430070, China
Received 5 April 2006; received in revised form 13 December 2006; accepted 31 January 2007
Abstract A gold-immunochromatographic test-strip kit is used for the detection of IgG antibodies against the nucleocapsid protein of Avian Influenza Virus (AIV). Compared with the ‘‘gold standard’’, i.e. hemagglutination inhibition (HI) and agar gel immunodiffusion (AGID) assays, the gold-immunochromatographic test strip has many advantages, such as high specificity, high sensitivity, convenience, is rapid and has low cost. The gold-immunochromatographic test strip provides a unique tool for the on-site surveillance and diagnosis of Avian Influenza. # 2007 Elsevier B.V. All rights reserved. Keywords: AIV; Antibody; Gold-immunochromatography test strip
1. Introduction Avian influenza (AI) is a highly contagious disease caused by type A influenza viruses that are members of Abbreviations: AGID, agar gel immunodiffusion; AIV, Avian Influenza Virus; AI, Avian influenza; BSA, bovine serum albumin; CGC, colloidal gold conjugate; ELISA, enzyme-linked immunosorbent assay; GICA, gold-immunochromatographic assay; HI, hemagglutination inhibition; HPAIV, highly pathogenic avian influenza viruses; IBD, infectious bursal disease; IB, infectious bronchitis; IgG, immunoglobulin G.; LPAIV, low-pathogenic avian influenza viruses; ND, newcastle disease; rNP, recombinant nucleocapsid protein; SPF, specific-pathogen-free * Corresponding author at: College of Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070, China. Tel.: +86 27 87280208; fax: +86 27 87280408. E-mail address: [email protected] (D. Bi). 1 Both authors have equally contributed to this paper. 0165-2427/$ – see front matter # 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.vetimm.2007.01.022
the family Orthomyxoviridae in the genus Influenzavirus A (OIE, 2005). Infection with low-pathogenic avian influenza viruses (LPAIV) usually is asymptomatic or it induces mild respiratory symptoms, but it can rapidly reduce egg production in poultry. Infection with highly pathogenic avian influenza viruses (HPAIV) is characterized by the sudden onset of fatal systemic disease in susceptible species. Importantly, some strains of LPAIV can serve as progenitors that rapidly mutate into HPAIV, which may infect humans, causing death, as evidenced by the recent outbreaks in Asia (Fisher, 2006; Lahariya et al., 2006). Furthermore, HPAIV infection is easily transmitted between poultry production facilities, leading to severe disease outbreaks or even pandemics. It is difficult to control the spread, so all chickens in the facilities are usually slaughtered. AI causes tremendous economic losses in the poultry
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industry and seriously threatens human health. It is extremely important to develop tools for the epidemiological surveillance of both HPAIV and LPAIV infection in the poultry industry. To prevent and control AI in poultry, vaccination has been employed as a key strategy in many countries since the 1990s. Hemagglutination inhibition (HI) assay and agar gel immunodiffusion (AGID) are the current ‘‘gold standard’’ of serological assays for the surveillance of virus infection and immune responses, and for the diagnosis of AIV by detecting antibodies to AIV (OIE, 2005). Other tests, such as enzyme-linked immunosorbent assay (ELISA), hemagglutination (HA), neuraminidase-inhibition test, microneutralization assay and RTPCR have also been used for the detection of antigens and antibodies to the virus. They are sensitive and specific; however, they are inconvenient and unsuitable for the rapid and on-site characterization of anti-AIV antibodies and AIV infection. These tests either require laboratory operation, skilled technicians, special equipment/facilities and a continual supply of special reagents (such as chicken red blood cells), or they are of lower sensitivity (AGID) and specificity, labor-intensive, have a prolonged time span and the cost is prohibitive (WHO, 2005; Snyder, 1986; Shafer et al., 1998; Zhou et al., 1998; Sala et al., 2003; Jin et al., 2004). Therefore, the development of a rapid and simple test is urgently warranted in routine clinical practice, particularly in rural veterinary clinics, to monitor antibodies to Avian Influenza Virus (AIV). Gold-immunochromatographic assay (GICA) is a new immunochromatographic technique in which a cellulose membrane is used as the carrier and a colloidal goldlabeled antigen or antibody is used as the tracer. This technology has several advantages over traditional immunoassays, such as simple procedure, rapid operation and immediate results, low cost, no requirements for skilled technicians or expensive equipments and is suitable for the on-site detection of antibodies or antigens. The method has been widely used for the diagnosis of many contagious human diseases and the detection of bioactive molecules, hormones, haptens, etc. (Heeschen et al., 1998; Toshio et al., 2001). The technology has also been adapted for the surveillance and diagnosis of poultry infectious diseases, such as newcastle disease (ND) and infectious bursal disease (IBD), but not available for AIV (Yu et al., 2004; Gao et al., 2004). Thus, the development and application of GICA is feasible and significant for the on-site diagnosis and surveillance of AIV and will be of huge economical benefit. Recently, a gold-immunochromatographic assay was developed in our laboratory (patent pending) and this assay could be used for the rapid detection of AIV-
specific antibodies. Compared with the standard HI and AGID, the new GICA not only retains the advantages of high sensitivity and specificity, but also displayed other attributes, including rapid and simple operation and low cost, which facilitated on-site diagnosis and serologic epidemiological investigations of AI. The application and implications of this new assay are discussed. 2. Materials and methods 2.1. Special reagents and materials The mouse monoclonal antibodies against the Fc fragment of chicken IgG (McAbFc) were prepared as previously described (Hua, 2006). A recombinant nucleocapsid protein (rNP) was generated by constructing a plasmid to express GST-NP fusion proteins and purified using the HiTrap affinity columns (Amersham Biosciences), followed by SDS–PAGE analysis and Western blotting (Hu, 2005). The rabbit anti-mouse IgG was prepared in our laboratory. Hydrogen tetrachloroaurate (III) hydrate (HAuCL4xH2O) was purchased from Sigma–Aldrich Inc. Bovine serum albumin (BSA) was purchased from Roche. Nitrocellulose membranes, glass fibers, sample pads and absorbent pads were purchased from Millipore Corporation (Bedford, MA, USA). 2.2. Serum samples and standard virus antigens Reference positive sera specific for and the antigens of subtypes H5, H7, H9 AIV were obtained from Harbin Veterinary Research Institute. Positive sera for newcastle disease (ND), infectious bursal disease (IBD) and infectious bronchitis (IB) were purchased from the Center for Experimental Animals of Beijing. Negative control sera were obtained from the wings of 4-weekold chickens reared under a specific pathogen-free (SPF) environment. A total of 166 chicken serum samples were collected from chickens of different ages from 17 flocks at three chicken farms in Hubei and Henan Provinces using the protocol approved by the Office of Animal Protection of the University. These chickens were vaccinated with inactivated AIV vaccines 1–5 months prior to sampling. 2.3. Preparation of the Immunochromatography Strip and the Kit 2.3.1. Preparation of colloidal gold-McAbFc Colloidal gold was prepared using a previously reported method with minor modifications (Grabar
D.P. Peng et al. / Veterinary Immunology and Immunopathology 117 (2007) 17–25
et al., 1995). Briefly, an aqueous solution of chloroauric acid (100 ml of 0.01% HAuCl4) was heated to boiling and, with rapid stirring, 2 ml of 1% sodium citrate solution was added. After an additional 10 min of continuous boiling, the colloid gold was gradually cooled with continuous stirring for an additional 15 min. The absorbance of the colloidal gold was measured using an Ultrospec 1100 pro UV/visible spectrophotometer (Amersham Biosciences). The size of colloidal gold particles was analyzed by TEM imaging (Hitachi Instrument Co., Tokyo, Japan). The colloidal gold solution was then stored at 4 8C in a darkcolored glass bottle until use. To prepare colloidal gold–McAbFc, the purified McAbFc (Russo et al., 1983; Perosa et al., 1990) was equilibrated with 2 mM borax buffer (pH 9.0) at 4 8C and then conjugated with the colloidal gold to generate McAbFc–CGC. Briefly, 2 mg of the purified McAbFc in 0.5 ml distilled water was added to 10 ml of gold colloid solution and adjusted to pH 8.0. The mixture was stirred vigorously for 30 min and 1.3 ml of 10% (w/v) bovine serum albumin (BSA) aqueous solution was added to block excess reactivity of the colloidal gold, followed by an additional 30 min stirring. After centrifugation, the resultant McAbFc–CGC pellet was suspended in 2 mM borax buffer (pH 9.0) containing 0.1% (w/v) PEG-20000, washed and then re-suspended in 1 ml of the same buffer. 2.3.2. Preparation of the immunochromatography strip and the kit The GICA test-strip was made of four main elements: the sample pad, the conjugate release pad, the analytical membrane and the absorbent pad, which was backed on a non-porous polyester support and, thus, had a higher tensile strength (Fig. 1A). The sample (absorbent paper) and conjugate pads (glass-fiber membrane) were treated with 20 mM phosphate buffer containing 1% BSA, 0.5% Tween-20 and 0.05% sodium azide, pH 7.4, and dried at 37 8C. Thereafter, the rNP protein (0.2 mg/ml) and the rabbit anti-mouse antibody (1 mg/ml) were loaded on the nitrocellulose membrane at either the test or control line using the BioDot XYZ Platform at a rate of 0.75 ml/cm and then dried at 37 8C. The McAbFc-CGC was loaded on the treated conjugate pad at 6 ml/cm and completely lyophilized. The absorbent pad, nitrocellulose membrane, pretreated conjugate pad and sample pad were assembled to form the strip and attached to the plastic scale-board, which was cut into 65 mm long and 3 mm wide strips. The GICA kit was developed for the detection of antibodies against AIV. The kit consisted of a bag of
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gold-immunochromatographic test strips (10 strips/ bag), a bottle of sample dilution buffer (10 ml/bottle) and a 12-well microtiter plate on a base. 2.3.3. Principle of the GICA The principle of the GICA is based on the following theory. If the tested serum contains the IgG, antibodies against the rNP of AIV, the IgG will be absorbed from the bottom of the strip, which will interact with the McAbFc–CGC on the strip to form an antigen–antibody complex (McAbFc–CGC–chicken IgG). The complex will migrate into the nitrocellulose membrane by capillary action and, subsequently, react with the immobilized rNP of AIV on the testing line, generating a signal band, the density of which will be in proportion to the concentration of the antibody against AIV (McAbFc–CGC–chicken IgG against AIV-rNP). The remaining McAbFc–CGC (blank control) or other antigen–antibody complex from rNP-nonspecific IgG (negative chicken serum control) will run over the test line and then react with the anti-mouse IgG antibody at the control line of the strip to form the second visible red band (chicken IgG–McAbFc–CGC–antimouse IgG) (Fig. 1B). Conceivably, if there is no IgG antibody against AIV in the sample serum, only the control line will be visible (Fig. 1C). Evaluation of the test-strip results can be performed with the naked eye and total assay time is less than 15 min. 2.4. GICA The GICA kit was initially equilibrated to room temperature and then 5 ml of the serum or serially diluted serum was mixed with 95 ml of dilution buffer in a well of the 12-well microtiter plate. The sample pad of an individual testing strip was dipped into the sample solution and left for about 15 min until a red band appeared at the control line. The results were considered positive (if the red band was present at both the test line and the control line), negative (if the red band appeared only at the control line), or invalid (if no red band developed at either lines or only one band appeared at the test line). 2.5. HI assay The HI assays were performed as a routine method (OIE, 2005). Briefly, 0.025 ml of PBS was pipetted into each well of a plastic V-bottomed microtitre plate and 0.025 ml of serum was added into the first well of the plate before a two-fold dilution was made. Four HAU (hemagglutination unit) of virus/antigen in 0.025 ml
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Fig. 1. Schematic diagram of the immunochromatographic strip. (A) Assembly of GICA test strip. (a) Application zone, (b) reaction zone and (c) detection zone. The strip consists of a sample pad, a conjugate pad, a nitrocellulose membrane and an absorption pad. The conjugate pad contains McAbFc-CGC (colloidal gold conjugate). In the detection zone, the nitrocellulose membrane is used as a chromatographic support on which the rNP and rabbit anti-mouse antibody are immobilized. (B) Positive reaction. (C) Negative reaction. The results will be evaluated as negative (if only one band at the control line) or positive (if two bands at the test and control lines).
D.P. Peng et al. / Veterinary Immunology and Immunopathology 117 (2007) 17–25
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was added to each well and the reaction lasted for at least for 30 min at room temperature. Subsequently, 0.025 ml of 1% (v/v) chicken RBCs was added to each well and gently mixed, allowing the RBCs to settle for about 40 min at room temperature. The HI titre was determined as the highest serum dilution leading to a complete inhibition of the four HAU of virus/antigen. 2.6. AGID AGID assays were performed as previously reported (OIE, 2005). The agarose slides or plates were prepared by pouring 1% (w/v) agarose in 0.1 M PBS buffer onto Petri dishes or glass slides at a thickness of 2–3 mm. Well were then dug, from a template, approximately 5 mm in diameter and 2–5 mm apart. Experimental, control and standard reference sera at different dilutions (50 ul/well) were added and the plates or slides were incubated at room temperature overnight. The titre of positive serum in AGID was determined as the highest serum dilution that formed completely fused immunoprecipited bands with the reference serum. 2.7. Statistical analysis The percentiles of anti-AIV positive sera were statistically analyzed by Chi-square test and a P value of 0.05 was considered significant. 3. Results 3.1. Analysis of prepared colloidal gold Absorbance of the generated colloidal gold particles was measured on a UV–vis spectrometer and plotted against the wavelengths in Fig. 2A. Maximum absorbance occurred at 520 nm. Particle sizes were examined using TEM imaging and average diameters were 18.6 1.2 nm (n = 100) (Fig. 2B). These results indicate that the generated gold particles are suitable for the production of colloidal gold-conjugated antibodies, allowing the generation of signals in ICA. 3.2. Specificity of GICA test strip The specificity of the GICA test strip was determined using the anti-AIV positive reference sera and reference sera against ND, IBD or IB, as well as negative sera from healthy chickens. As shown in Fig. 3, a red band appeared at the control line of the test strip in healthy chickens (negative), which was similar to the blank control, suggesting no AIV-specific IgG was present in the tested
Fig. 2. Characterization of colloidal gold. (A) The absorbance of colloidal gold as determined by UV–vis spectra. Data represent 1 of 20 experiments. (B) The size of colloidal gold particles was characterized by TEM imaging.
serum. Similarly, all the tested sera against ND, IBD or IB showed no positive result at the test line, indicating that the anti-ND, IBD or IB failed to cross-react with the specific rNP antigen of AIV. In contrast, when the reference positive sera were assayed by the GICA test strips, two red bands clearly appeared at both the test and control lines, regardless of the AIV subtypes. Obviously, the GICA test strip has high specificity in detecting the anti-AIV-specific IgG without cross-reactivity to the tested reference sera against other viruses that mediate chicken respiratory diseases. 3.3. Sensitivity of GICA test strip To determine the sensitivity of the GICA test strip, the positive reference sera against H5, H7 or H9 subtype
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D.P. Peng et al. / Veterinary Immunology and Immunopathology 117 (2007) 17–25 Table 1 Comparison of the anti-AIV titers of the reference positive sera Sera
AIV subtype H5 AIV subtype H7 AIV subtype H9
Titer GICA titer
HI titer
AGID titer
1:512 1:1024 1:1024
1:1024 1:1024 1:1024
1:4 1:8 1:8
of AIV were serially diluted and their titers of anti-AIV IgG determined (Fig. 4). Clearly, two red bands developed at the test and control lines, and the highest titers of sera against H7 or H9 AIV reached 1:1024. Significantly, the same serum was replicated 20 times and showed similar results, indicating the high stability and reproducibility of the GICA test strip for analysis of anti-AIV (data not shown). The GICA test strip has high sensitivity and stability in detecting small amount of AIV-specific IgG antibodies reproducibly. 3.4. Analysis of consistency among GICA, HI, and AGID Fig. 3. Specificity of the GICA test strips. Reference positive sera against H5, H7 or H9 subtypes of AIV, ND, IBD or IB, and sera from healthy chickens were simultaneously characterized by the GICA test strips. Similar result patterns were reproduced in repeat experiments (data not shown).
Compared with the gold standard assays, i.e. HI and AGID, three anti-AIV positive reference sera at different dilutions were analyzed by the GICA test strip, HI and AGID assays simultaneously; the results are shown in Table 1. The sensitivity of the GICA test strip was almost the same as HI, except for the anti-H5 serum which was slightly lower than for HI. Both experimental systems could detect anti-AIV IgG in 1:1024 dilutions of anti-H7 or anti-H9 AIV reference sera, which was significantly higher than that determined by AGID. Thus, the GICA test strip has a high sensitivity, which was similar to HI but significantly higher than AGID. The high sensitivity of the GICA test strip was also evidenced from the analysis of 166 clinical serum samples (Table 2). Among the 166 serum samples, 93 samples (56.02%) were positively determined by GICA. The percentage of positive sera was comparable to the rate of 58.43% analyzed by the highly sensitive Table 2 Comparison of the percentages of anti-AIV positive sera Result
Fig. 4. Sensitivity of GICA test strips. Reference positive sera against H5, H7 or H9 subtypes of AIV at different dilutions were used to analyze AIV-specific antibodies by the GICA test strips, HI and AGID assays. Data are presented as the mean dilution of each serum at a single assay.
Positive (case) Negative (case) Ratio of positive (%)
Method GICA
HI
AGID
93 73 56.02
97 69 58.43
50 116 30.12
Note: A total of 166 chicken serum samples were simultaneously analyzed by the GICA test strips, HI and AGID assays. The percentiles of anti-AIV positive sera were analyzed by the Chi-square test.
D.P. Peng et al. / Veterinary Immunology and Immunopathology 117 (2007) 17–25 Table 3 Comparison of consistency ratios between GICA and HI
Positive of HI Negative of HI GICA total GICA (%)
Positive of GICA
Negative of GICA
HI total
HI (%)
82 11 93 56.02
15 58 73 43.98
97 69 166
68.67 31.37
Note: The ratio of the positive number of GICA to the positive number of HI is 88.17% and that of the negative number of GICA to the negative number of HI is 79.45%. Table 4 Comparison of consistency ratios between GICA and AGID
Positive of AGID Negative of AGID GICA total GICA (%)
Positive of GICA
Negative of GICA
AGID total
AGID (%)
45 48 93 56.2
5 68 73 43.98
50 116 166
30.12 69.88
Note: The ratio of the positive number of GICA to the positive number of AGID is 48.39% and that of the negative number of GICA to the negative number of AGID is 93.15%.
HI assays (P 0.05), and was notably higher than the 30.12% characterized by AGID (P 0.05). Further analyses revealed that 82 of 93 positive sera samples determined by GICA were also positively analyzed by HI, while 58 of 73 negative sera were negatively confirmed by HI. The ratio of positive and negative consistancy for the two methods was 88.17 and 79.45%, respectively (Table 3), with no significant difference in terms of sensitivity between the methods. Compared with AGID, 45 of 93 positive sera determined by GICA were positively characterized by AGID and 68 of 73 negative sera analyzed by GICA failed to show positive in AGID assays (Table 4). Notably, while very few AGID-positive sera were overlooked by GICA, many GICA-positive sera, which were confirmed by HI, were missed by AGID. This suggests that the sensitivity of the GICA test strip was consistent with HI and much higher than AGID. Importantly, the detection of antiAIV IgG using the GICA test strip took about 15 min; the same serum required a couple of hours with the HI assay and more than 10 h with AGID. In conclusion, the GICA test strip was highly specific and sensitive for the rapid detection of anti-AIV antibodies and is a powerful tool for the on-site surveillance and diagnosis of AI. 4. Discussion AI is a highly infectious disease that currently poses a serious threat to the poultry industry and human
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health. Unfortunately, there is no effective and specific treatment for HPAI in poultry. The precise diagnosis of AIV and effective vaccination, which has been shown to induce immune responses, can help control the spread of the disease. Hence, the on-site and rapid detection of AIVand surveillance of AI in flocks is significant for the economics of poultry production and human health (Beck et al., 2003). There are several methods for the detection of antiAIV antibodies. The AGID assay, one of the gold standard tests, usually detects antibodies against typespecific M and NP antigens of type A influenza virus. This test is very specific, but it has lower sensitivity and commonly takes about 48–72 h to obtain results (WHO, 2005). The HI assay is a labor-intensive and timeconsuming assay, requiring several controls for standardization, which makes it unsuitable for the rapid and on-site characterization of anti-AIV antibodies and AIV infection (Thomas et al., 1999). Similarly, ELISA can detect sub-type antigen with a high sensitivity (Julkunen et al., 1985); however, due to the high sensitivity, application of ELISA to detect antibodies occasionally leads to a high number of false positives. Furthermore, ELISA usually requires laboratory operation, skilled technicians, special equipment/facilities and takes days to get results, making it difficult for use in the rapid and on-site detection of anti-virus antibodies (Bishai and Galli, 1978; Julkunen et al., 1985; Shafer et al., 1998; Zhou et al., 1998; Thomas et al., 1999; Prince and Leber, 2003; Sala et al., 2003; Jin et al., 2004; Adam et al., 2006). Other assays, such as complement fixation, neuraminidase-inhibition test or microneutralization assay, are either less sensitive or require special equipments and complex procedures (WHO, 2005). Therefore, the development of this new and powerful assay system for the rapid detection of AIV-specific antibodies is significant. We have successfully developed a GICA-based test strip kit, which could rapidly detect chicken IgG antibodies against the rNP of AIV, both qualitatively and quantitatively, if using serially diluted chicken serum, without cross-reaction with antibodies against other tested viruses. In comparison with the gold standard assays, the sensitivity of the kit was comparable to the highly sensitive HI assay and significantly higher than the AGID assay. Unlike these commonly used assays, the GICA test-strip kit for the detection of AIV-specific antibodies does not require any equipment or skilled technicians and can be conveniently performed on the poultry farm by a poultry farmer. Importantly, the detection of AIV-specific antibodies by the kit only takes about 15 min, which is much faster
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than the time required for the HI and AGID assays, and the results can be read directly by the naked eye. Therefore, the GICA test strip kit is a highly specific and sensitive assay for the rapid and reproducible detection of AIV specific antibodies, which is easy to operate and low in cost. It could be adapted for on-site surveillance and diagnosis of AI (Paek et al., 2000). The NP of AIV is a type-specific protein and very conservative antigen. It can induce strong immune responses, even with low expression in AIV, and has been widely applied in many serologic assays, such as ELISA and AGID. The GICA test strips, developed with the rNP and McAbFc-CGC, can be used to qualitatively and quantitatively, if using serially diluted serum, detect IgG antibodies against AIV without differentiation of the specific subtype of AIV. This kit could be combined with other antigen detection assays for the early diagnosis of AI, particularly for HPAIV-mediated AI, as the development of AIV-specific IgG antibodies usually appears as early as 3–4 days after onset of AI. Finally, as it is difficult for this kit to distinguish between the anti-AIV IgG induced by an occult infection, recent infection or vaccination, further modifications may make it more effective for the early diagnose of AI. Recent outbreaks of AI throughout the world have resulted in huge economic losses in the poultry industry. Effective vaccination to induce immune responses to AIV is expected to control the spread of AI. Therefore, the epidemiological surveillance of AI and vaccine-induced immune responses require a sensitive and specific assay that can be conveniently operated to rapidly detect antibodies against AIV. The GICA test strip kit has been shown to rapidly detect antibodies to AIV. Its application may economically benefit poultry farmer by monitoring the antibody levels of vaccinated flocks, investigating the epidemiology of AIV in unvaccinated flocks and quarantining live poultry products in market and at customs. Given that HPAIV has potential to spread to humans, which may lead to an outbreak of human influenza or even pandemics, our findings may help in the design and the development of convenient assays for the surveillance of human influenza. In summary, we have successfully developed a new GICA-based test kit, which has been shown to be highly sensitive and specific for the rapid detection of antibodies against AIV. Acknowledgement This project was supported by the key program of Hubei province (2002P0805).
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Comparison of a new gold-immunochromatographic assay for the detection of antibodies against avian influenza virus with hemagglutination inhibition and agar gel immunodiffusion assays DaPeng Peng a,b,1, SiShun Hu a,1, Yan Hua a, YunCai Xiao a, ZiLi Li a, XiLiang Wang a, DingRen Bi a,* b
a College of Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070, China State Reference Laboratory of Veterinary Drug Residues, Huazhong Agricultural University, Wuhan 430070, China
Received 5 April 2006; received in revised form 13 December 2006; accepted 31 January 2007
Abstract A gold-immunochromatographic test-strip kit is used for the detection of IgG antibodies against the nucleocapsid protein of Avian Influenza Virus (AIV). Compared with the ‘‘gold standard’’, i.e. hemagglutination inhibition (HI) and agar gel immunodiffusion (AGID) assays, the gold-immunochromatographic test strip has many advantages, such as high specificity, high sensitivity, convenience, is rapid and has low cost. The gold-immunochromatographic test strip provides a unique tool for the on-site surveillance and diagnosis of Avian Influenza. # 2007 Elsevier B.V. All rights reserved. Keywords: AIV; Antibody; Gold-immunochromatography test strip
1. Introduction Avian influenza (AI) is a highly contagious disease caused by type A influenza viruses that are members of Abbreviations: AGID, agar gel immunodiffusion; AIV, Avian Influenza Virus; AI, Avian influenza; BSA, bovine serum albumin; CGC, colloidal gold conjugate; ELISA, enzyme-linked immunosorbent assay; GICA, gold-immunochromatographic assay; HI, hemagglutination inhibition; HPAIV, highly pathogenic avian influenza viruses; IBD, infectious bursal disease; IB, infectious bronchitis; IgG, immunoglobulin G.; LPAIV, low-pathogenic avian influenza viruses; ND, newcastle disease; rNP, recombinant nucleocapsid protein; SPF, specific-pathogen-free * Corresponding author at: College of Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070, China. Tel.: +86 27 87280208; fax: +86 27 87280408. E-mail address: [email protected] (D. Bi). 1 Both authors have equally contributed to this paper. 0165-2427/$ – see front matter # 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.vetimm.2007.01.022
the family Orthomyxoviridae in the genus Influenzavirus A (OIE, 2005). Infection with low-pathogenic avian influenza viruses (LPAIV) usually is asymptomatic or it induces mild respiratory symptoms, but it can rapidly reduce egg production in poultry. Infection with highly pathogenic avian influenza viruses (HPAIV) is characterized by the sudden onset of fatal systemic disease in susceptible species. Importantly, some strains of LPAIV can serve as progenitors that rapidly mutate into HPAIV, which may infect humans, causing death, as evidenced by the recent outbreaks in Asia (Fisher, 2006; Lahariya et al., 2006). Furthermore, HPAIV infection is easily transmitted between poultry production facilities, leading to severe disease outbreaks or even pandemics. It is difficult to control the spread, so all chickens in the facilities are usually slaughtered. AI causes tremendous economic losses in the poultry
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industry and seriously threatens human health. It is extremely important to develop tools for the epidemiological surveillance of both HPAIV and LPAIV infection in the poultry industry. To prevent and control AI in poultry, vaccination has been employed as a key strategy in many countries since the 1990s. Hemagglutination inhibition (HI) assay and agar gel immunodiffusion (AGID) are the current ‘‘gold standard’’ of serological assays for the surveillance of virus infection and immune responses, and for the diagnosis of AIV by detecting antibodies to AIV (OIE, 2005). Other tests, such as enzyme-linked immunosorbent assay (ELISA), hemagglutination (HA), neuraminidase-inhibition test, microneutralization assay and RTPCR have also been used for the detection of antigens and antibodies to the virus. They are sensitive and specific; however, they are inconvenient and unsuitable for the rapid and on-site characterization of anti-AIV antibodies and AIV infection. These tests either require laboratory operation, skilled technicians, special equipment/facilities and a continual supply of special reagents (such as chicken red blood cells), or they are of lower sensitivity (AGID) and specificity, labor-intensive, have a prolonged time span and the cost is prohibitive (WHO, 2005; Snyder, 1986; Shafer et al., 1998; Zhou et al., 1998; Sala et al., 2003; Jin et al., 2004). Therefore, the development of a rapid and simple test is urgently warranted in routine clinical practice, particularly in rural veterinary clinics, to monitor antibodies to Avian Influenza Virus (AIV). Gold-immunochromatographic assay (GICA) is a new immunochromatographic technique in which a cellulose membrane is used as the carrier and a colloidal goldlabeled antigen or antibody is used as the tracer. This technology has several advantages over traditional immunoassays, such as simple procedure, rapid operation and immediate results, low cost, no requirements for skilled technicians or expensive equipments and is suitable for the on-site detection of antibodies or antigens. The method has been widely used for the diagnosis of many contagious human diseases and the detection of bioactive molecules, hormones, haptens, etc. (Heeschen et al., 1998; Toshio et al., 2001). The technology has also been adapted for the surveillance and diagnosis of poultry infectious diseases, such as newcastle disease (ND) and infectious bursal disease (IBD), but not available for AIV (Yu et al., 2004; Gao et al., 2004). Thus, the development and application of GICA is feasible and significant for the on-site diagnosis and surveillance of AIV and will be of huge economical benefit. Recently, a gold-immunochromatographic assay was developed in our laboratory (patent pending) and this assay could be used for the rapid detection of AIV-
specific antibodies. Compared with the standard HI and AGID, the new GICA not only retains the advantages of high sensitivity and specificity, but also displayed other attributes, including rapid and simple operation and low cost, which facilitated on-site diagnosis and serologic epidemiological investigations of AI. The application and implications of this new assay are discussed. 2. Materials and methods 2.1. Special reagents and materials The mouse monoclonal antibodies against the Fc fragment of chicken IgG (McAbFc) were prepared as previously described (Hua, 2006). A recombinant nucleocapsid protein (rNP) was generated by constructing a plasmid to express GST-NP fusion proteins and purified using the HiTrap affinity columns (Amersham Biosciences), followed by SDS–PAGE analysis and Western blotting (Hu, 2005). The rabbit anti-mouse IgG was prepared in our laboratory. Hydrogen tetrachloroaurate (III) hydrate (HAuCL4xH2O) was purchased from Sigma–Aldrich Inc. Bovine serum albumin (BSA) was purchased from Roche. Nitrocellulose membranes, glass fibers, sample pads and absorbent pads were purchased from Millipore Corporation (Bedford, MA, USA). 2.2. Serum samples and standard virus antigens Reference positive sera specific for and the antigens of subtypes H5, H7, H9 AIV were obtained from Harbin Veterinary Research Institute. Positive sera for newcastle disease (ND), infectious bursal disease (IBD) and infectious bronchitis (IB) were purchased from the Center for Experimental Animals of Beijing. Negative control sera were obtained from the wings of 4-weekold chickens reared under a specific pathogen-free (SPF) environment. A total of 166 chicken serum samples were collected from chickens of different ages from 17 flocks at three chicken farms in Hubei and Henan Provinces using the protocol approved by the Office of Animal Protection of the University. These chickens were vaccinated with inactivated AIV vaccines 1–5 months prior to sampling. 2.3. Preparation of the Immunochromatography Strip and the Kit 2.3.1. Preparation of colloidal gold-McAbFc Colloidal gold was prepared using a previously reported method with minor modifications (Grabar
D.P. Peng et al. / Veterinary Immunology and Immunopathology 117 (2007) 17–25
et al., 1995). Briefly, an aqueous solution of chloroauric acid (100 ml of 0.01% HAuCl4) was heated to boiling and, with rapid stirring, 2 ml of 1% sodium citrate solution was added. After an additional 10 min of continuous boiling, the colloid gold was gradually cooled with continuous stirring for an additional 15 min. The absorbance of the colloidal gold was measured using an Ultrospec 1100 pro UV/visible spectrophotometer (Amersham Biosciences). The size of colloidal gold particles was analyzed by TEM imaging (Hitachi Instrument Co., Tokyo, Japan). The colloidal gold solution was then stored at 4 8C in a darkcolored glass bottle until use. To prepare colloidal gold–McAbFc, the purified McAbFc (Russo et al., 1983; Perosa et al., 1990) was equilibrated with 2 mM borax buffer (pH 9.0) at 4 8C and then conjugated with the colloidal gold to generate McAbFc–CGC. Briefly, 2 mg of the purified McAbFc in 0.5 ml distilled water was added to 10 ml of gold colloid solution and adjusted to pH 8.0. The mixture was stirred vigorously for 30 min and 1.3 ml of 10% (w/v) bovine serum albumin (BSA) aqueous solution was added to block excess reactivity of the colloidal gold, followed by an additional 30 min stirring. After centrifugation, the resultant McAbFc–CGC pellet was suspended in 2 mM borax buffer (pH 9.0) containing 0.1% (w/v) PEG-20000, washed and then re-suspended in 1 ml of the same buffer. 2.3.2. Preparation of the immunochromatography strip and the kit The GICA test-strip was made of four main elements: the sample pad, the conjugate release pad, the analytical membrane and the absorbent pad, which was backed on a non-porous polyester support and, thus, had a higher tensile strength (Fig. 1A). The sample (absorbent paper) and conjugate pads (glass-fiber membrane) were treated with 20 mM phosphate buffer containing 1% BSA, 0.5% Tween-20 and 0.05% sodium azide, pH 7.4, and dried at 37 8C. Thereafter, the rNP protein (0.2 mg/ml) and the rabbit anti-mouse antibody (1 mg/ml) were loaded on the nitrocellulose membrane at either the test or control line using the BioDot XYZ Platform at a rate of 0.75 ml/cm and then dried at 37 8C. The McAbFc-CGC was loaded on the treated conjugate pad at 6 ml/cm and completely lyophilized. The absorbent pad, nitrocellulose membrane, pretreated conjugate pad and sample pad were assembled to form the strip and attached to the plastic scale-board, which was cut into 65 mm long and 3 mm wide strips. The GICA kit was developed for the detection of antibodies against AIV. The kit consisted of a bag of
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gold-immunochromatographic test strips (10 strips/ bag), a bottle of sample dilution buffer (10 ml/bottle) and a 12-well microtiter plate on a base. 2.3.3. Principle of the GICA The principle of the GICA is based on the following theory. If the tested serum contains the IgG, antibodies against the rNP of AIV, the IgG will be absorbed from the bottom of the strip, which will interact with the McAbFc–CGC on the strip to form an antigen–antibody complex (McAbFc–CGC–chicken IgG). The complex will migrate into the nitrocellulose membrane by capillary action and, subsequently, react with the immobilized rNP of AIV on the testing line, generating a signal band, the density of which will be in proportion to the concentration of the antibody against AIV (McAbFc–CGC–chicken IgG against AIV-rNP). The remaining McAbFc–CGC (blank control) or other antigen–antibody complex from rNP-nonspecific IgG (negative chicken serum control) will run over the test line and then react with the anti-mouse IgG antibody at the control line of the strip to form the second visible red band (chicken IgG–McAbFc–CGC–antimouse IgG) (Fig. 1B). Conceivably, if there is no IgG antibody against AIV in the sample serum, only the control line will be visible (Fig. 1C). Evaluation of the test-strip results can be performed with the naked eye and total assay time is less than 15 min. 2.4. GICA The GICA kit was initially equilibrated to room temperature and then 5 ml of the serum or serially diluted serum was mixed with 95 ml of dilution buffer in a well of the 12-well microtiter plate. The sample pad of an individual testing strip was dipped into the sample solution and left for about 15 min until a red band appeared at the control line. The results were considered positive (if the red band was present at both the test line and the control line), negative (if the red band appeared only at the control line), or invalid (if no red band developed at either lines or only one band appeared at the test line). 2.5. HI assay The HI assays were performed as a routine method (OIE, 2005). Briefly, 0.025 ml of PBS was pipetted into each well of a plastic V-bottomed microtitre plate and 0.025 ml of serum was added into the first well of the plate before a two-fold dilution was made. Four HAU (hemagglutination unit) of virus/antigen in 0.025 ml
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Fig. 1. Schematic diagram of the immunochromatographic strip. (A) Assembly of GICA test strip. (a) Application zone, (b) reaction zone and (c) detection zone. The strip consists of a sample pad, a conjugate pad, a nitrocellulose membrane and an absorption pad. The conjugate pad contains McAbFc-CGC (colloidal gold conjugate). In the detection zone, the nitrocellulose membrane is used as a chromatographic support on which the rNP and rabbit anti-mouse antibody are immobilized. (B) Positive reaction. (C) Negative reaction. The results will be evaluated as negative (if only one band at the control line) or positive (if two bands at the test and control lines).
D.P. Peng et al. / Veterinary Immunology and Immunopathology 117 (2007) 17–25
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was added to each well and the reaction lasted for at least for 30 min at room temperature. Subsequently, 0.025 ml of 1% (v/v) chicken RBCs was added to each well and gently mixed, allowing the RBCs to settle for about 40 min at room temperature. The HI titre was determined as the highest serum dilution leading to a complete inhibition of the four HAU of virus/antigen. 2.6. AGID AGID assays were performed as previously reported (OIE, 2005). The agarose slides or plates were prepared by pouring 1% (w/v) agarose in 0.1 M PBS buffer onto Petri dishes or glass slides at a thickness of 2–3 mm. Well were then dug, from a template, approximately 5 mm in diameter and 2–5 mm apart. Experimental, control and standard reference sera at different dilutions (50 ul/well) were added and the plates or slides were incubated at room temperature overnight. The titre of positive serum in AGID was determined as the highest serum dilution that formed completely fused immunoprecipited bands with the reference serum. 2.7. Statistical analysis The percentiles of anti-AIV positive sera were statistically analyzed by Chi-square test and a P value of 0.05 was considered significant. 3. Results 3.1. Analysis of prepared colloidal gold Absorbance of the generated colloidal gold particles was measured on a UV–vis spectrometer and plotted against the wavelengths in Fig. 2A. Maximum absorbance occurred at 520 nm. Particle sizes were examined using TEM imaging and average diameters were 18.6 1.2 nm (n = 100) (Fig. 2B). These results indicate that the generated gold particles are suitable for the production of colloidal gold-conjugated antibodies, allowing the generation of signals in ICA. 3.2. Specificity of GICA test strip The specificity of the GICA test strip was determined using the anti-AIV positive reference sera and reference sera against ND, IBD or IB, as well as negative sera from healthy chickens. As shown in Fig. 3, a red band appeared at the control line of the test strip in healthy chickens (negative), which was similar to the blank control, suggesting no AIV-specific IgG was present in the tested
Fig. 2. Characterization of colloidal gold. (A) The absorbance of colloidal gold as determined by UV–vis spectra. Data represent 1 of 20 experiments. (B) The size of colloidal gold particles was characterized by TEM imaging.
serum. Similarly, all the tested sera against ND, IBD or IB showed no positive result at the test line, indicating that the anti-ND, IBD or IB failed to cross-react with the specific rNP antigen of AIV. In contrast, when the reference positive sera were assayed by the GICA test strips, two red bands clearly appeared at both the test and control lines, regardless of the AIV subtypes. Obviously, the GICA test strip has high specificity in detecting the anti-AIV-specific IgG without cross-reactivity to the tested reference sera against other viruses that mediate chicken respiratory diseases. 3.3. Sensitivity of GICA test strip To determine the sensitivity of the GICA test strip, the positive reference sera against H5, H7 or H9 subtype
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D.P. Peng et al. / Veterinary Immunology and Immunopathology 117 (2007) 17–25 Table 1 Comparison of the anti-AIV titers of the reference positive sera Sera
AIV subtype H5 AIV subtype H7 AIV subtype H9
Titer GICA titer
HI titer
AGID titer
1:512 1:1024 1:1024
1:1024 1:1024 1:1024
1:4 1:8 1:8
of AIV were serially diluted and their titers of anti-AIV IgG determined (Fig. 4). Clearly, two red bands developed at the test and control lines, and the highest titers of sera against H7 or H9 AIV reached 1:1024. Significantly, the same serum was replicated 20 times and showed similar results, indicating the high stability and reproducibility of the GICA test strip for analysis of anti-AIV (data not shown). The GICA test strip has high sensitivity and stability in detecting small amount of AIV-specific IgG antibodies reproducibly. 3.4. Analysis of consistency among GICA, HI, and AGID Fig. 3. Specificity of the GICA test strips. Reference positive sera against H5, H7 or H9 subtypes of AIV, ND, IBD or IB, and sera from healthy chickens were simultaneously characterized by the GICA test strips. Similar result patterns were reproduced in repeat experiments (data not shown).
Compared with the gold standard assays, i.e. HI and AGID, three anti-AIV positive reference sera at different dilutions were analyzed by the GICA test strip, HI and AGID assays simultaneously; the results are shown in Table 1. The sensitivity of the GICA test strip was almost the same as HI, except for the anti-H5 serum which was slightly lower than for HI. Both experimental systems could detect anti-AIV IgG in 1:1024 dilutions of anti-H7 or anti-H9 AIV reference sera, which was significantly higher than that determined by AGID. Thus, the GICA test strip has a high sensitivity, which was similar to HI but significantly higher than AGID. The high sensitivity of the GICA test strip was also evidenced from the analysis of 166 clinical serum samples (Table 2). Among the 166 serum samples, 93 samples (56.02%) were positively determined by GICA. The percentage of positive sera was comparable to the rate of 58.43% analyzed by the highly sensitive Table 2 Comparison of the percentages of anti-AIV positive sera Result
Fig. 4. Sensitivity of GICA test strips. Reference positive sera against H5, H7 or H9 subtypes of AIV at different dilutions were used to analyze AIV-specific antibodies by the GICA test strips, HI and AGID assays. Data are presented as the mean dilution of each serum at a single assay.
Positive (case) Negative (case) Ratio of positive (%)
Method GICA
HI
AGID
93 73 56.02
97 69 58.43
50 116 30.12
Note: A total of 166 chicken serum samples were simultaneously analyzed by the GICA test strips, HI and AGID assays. The percentiles of anti-AIV positive sera were analyzed by the Chi-square test.
D.P. Peng et al. / Veterinary Immunology and Immunopathology 117 (2007) 17–25 Table 3 Comparison of consistency ratios between GICA and HI
Positive of HI Negative of HI GICA total GICA (%)
Positive of GICA
Negative of GICA
HI total
HI (%)
82 11 93 56.02
15 58 73 43.98
97 69 166
68.67 31.37
Note: The ratio of the positive number of GICA to the positive number of HI is 88.17% and that of the negative number of GICA to the negative number of HI is 79.45%. Table 4 Comparison of consistency ratios between GICA and AGID
Positive of AGID Negative of AGID GICA total GICA (%)
Positive of GICA
Negative of GICA
AGID total
AGID (%)
45 48 93 56.2
5 68 73 43.98
50 116 166
30.12 69.88
Note: The ratio of the positive number of GICA to the positive number of AGID is 48.39% and that of the negative number of GICA to the negative number of AGID is 93.15%.
HI assays (P 0.05), and was notably higher than the 30.12% characterized by AGID (P 0.05). Further analyses revealed that 82 of 93 positive sera samples determined by GICA were also positively analyzed by HI, while 58 of 73 negative sera were negatively confirmed by HI. The ratio of positive and negative consistancy for the two methods was 88.17 and 79.45%, respectively (Table 3), with no significant difference in terms of sensitivity between the methods. Compared with AGID, 45 of 93 positive sera determined by GICA were positively characterized by AGID and 68 of 73 negative sera analyzed by GICA failed to show positive in AGID assays (Table 4). Notably, while very few AGID-positive sera were overlooked by GICA, many GICA-positive sera, which were confirmed by HI, were missed by AGID. This suggests that the sensitivity of the GICA test strip was consistent with HI and much higher than AGID. Importantly, the detection of antiAIV IgG using the GICA test strip took about 15 min; the same serum required a couple of hours with the HI assay and more than 10 h with AGID. In conclusion, the GICA test strip was highly specific and sensitive for the rapid detection of anti-AIV antibodies and is a powerful tool for the on-site surveillance and diagnosis of AI. 4. Discussion AI is a highly infectious disease that currently poses a serious threat to the poultry industry and human
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health. Unfortunately, there is no effective and specific treatment for HPAI in poultry. The precise diagnosis of AIV and effective vaccination, which has been shown to induce immune responses, can help control the spread of the disease. Hence, the on-site and rapid detection of AIVand surveillance of AI in flocks is significant for the economics of poultry production and human health (Beck et al., 2003). There are several methods for the detection of antiAIV antibodies. The AGID assay, one of the gold standard tests, usually detects antibodies against typespecific M and NP antigens of type A influenza virus. This test is very specific, but it has lower sensitivity and commonly takes about 48–72 h to obtain results (WHO, 2005). The HI assay is a labor-intensive and timeconsuming assay, requiring several controls for standardization, which makes it unsuitable for the rapid and on-site characterization of anti-AIV antibodies and AIV infection (Thomas et al., 1999). Similarly, ELISA can detect sub-type antigen with a high sensitivity (Julkunen et al., 1985); however, due to the high sensitivity, application of ELISA to detect antibodies occasionally leads to a high number of false positives. Furthermore, ELISA usually requires laboratory operation, skilled technicians, special equipment/facilities and takes days to get results, making it difficult for use in the rapid and on-site detection of anti-virus antibodies (Bishai and Galli, 1978; Julkunen et al., 1985; Shafer et al., 1998; Zhou et al., 1998; Thomas et al., 1999; Prince and Leber, 2003; Sala et al., 2003; Jin et al., 2004; Adam et al., 2006). Other assays, such as complement fixation, neuraminidase-inhibition test or microneutralization assay, are either less sensitive or require special equipments and complex procedures (WHO, 2005). Therefore, the development of this new and powerful assay system for the rapid detection of AIV-specific antibodies is significant. We have successfully developed a GICA-based test strip kit, which could rapidly detect chicken IgG antibodies against the rNP of AIV, both qualitatively and quantitatively, if using serially diluted chicken serum, without cross-reaction with antibodies against other tested viruses. In comparison with the gold standard assays, the sensitivity of the kit was comparable to the highly sensitive HI assay and significantly higher than the AGID assay. Unlike these commonly used assays, the GICA test-strip kit for the detection of AIV-specific antibodies does not require any equipment or skilled technicians and can be conveniently performed on the poultry farm by a poultry farmer. Importantly, the detection of AIV-specific antibodies by the kit only takes about 15 min, which is much faster
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than the time required for the HI and AGID assays, and the results can be read directly by the naked eye. Therefore, the GICA test strip kit is a highly specific and sensitive assay for the rapid and reproducible detection of AIV specific antibodies, which is easy to operate and low in cost. It could be adapted for on-site surveillance and diagnosis of AI (Paek et al., 2000). The NP of AIV is a type-specific protein and very conservative antigen. It can induce strong immune responses, even with low expression in AIV, and has been widely applied in many serologic assays, such as ELISA and AGID. The GICA test strips, developed with the rNP and McAbFc-CGC, can be used to qualitatively and quantitatively, if using serially diluted serum, detect IgG antibodies against AIV without differentiation of the specific subtype of AIV. This kit could be combined with other antigen detection assays for the early diagnosis of AI, particularly for HPAIV-mediated AI, as the development of AIV-specific IgG antibodies usually appears as early as 3–4 days after onset of AI. Finally, as it is difficult for this kit to distinguish between the anti-AIV IgG induced by an occult infection, recent infection or vaccination, further modifications may make it more effective for the early diagnose of AI. Recent outbreaks of AI throughout the world have resulted in huge economic losses in the poultry industry. Effective vaccination to induce immune responses to AIV is expected to control the spread of AI. Therefore, the epidemiological surveillance of AI and vaccine-induced immune responses require a sensitive and specific assay that can be conveniently operated to rapidly detect antibodies against AIV. The GICA test strip kit has been shown to rapidly detect antibodies to AIV. Its application may economically benefit poultry farmer by monitoring the antibody levels of vaccinated flocks, investigating the epidemiology of AIV in unvaccinated flocks and quarantining live poultry products in market and at customs. Given that HPAIV has potential to spread to humans, which may lead to an outbreak of human influenza or even pandemics, our findings may help in the design and the development of convenient assays for the surveillance of human influenza. In summary, we have successfully developed a new GICA-based test kit, which has been shown to be highly sensitive and specific for the rapid detection of antibodies against AIV. Acknowledgement This project was supported by the key program of Hubei province (2002P0805).
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