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Laboratory of Animal Virology, College of Veterinary Medicine
Unit of Animal Infectious Diseases, National Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan, Hubei Province, People's Republic of China
ABSTRACT
A latex agglutination test (LAT) based on polystyrene beads sensitized with inactivated avian influenza virus H5N1 particles was developed. Compared with the hemagglutination inhibition test, the sensitivity and specificity of the LAT were 88.8 and 97.6%, respectively, in detecting 830 serum samples from vaccinated chickens. The test has application potential in field practice.
TEXT
Highly pathogenic avian influenza virus (AIV) subtype H5N1 emerged in Hong Kong in 1997 (2) and has since been threatening the poultry industry and human health worldwide. In the spring of 2004, avian influenza due to highly pathogenic subtype H5N1 spread throughout poultry populations in Asia and caused gigantic economic losses.
Vaccination is a key strategy in the prevention and control of avian influenza in poultry. A rapid and simple test is needed in routine field practice to monitor antibodies to AIV H5N1 induced by vaccines in order to validate vaccine efficacy. Currently, the hemagglutination inhibition (HI) assay is the preferred serological assay to detect antibodies to AIV in China (4), but it is laborious to perform, requiring a continuous source of appropriate erythrocytes and pretreatment of serum specimens to eliminate nonspecific inhibitors. Another commonly used method is the enzyme-linked immunosorbent assay (3), which takes several hours at least and needs trained personnel and special equipment.
Here we report the development and evaluation of a latex agglutination test (LAT) based on polystyrene beads sensitized with inactivated AIV H5N1 particles.
To develop the test, the optimal antigen concentration for sensitizing latex beads and the concentration of the blocking agent were first determined. AIV subtype H5N1 strain A/Chicken/HuBei/327/2004(H5N1) was produced by a routine procedure using chicken embryos (4). Allantoic fluids were collected if the HI titer was higher than 2 x 108, pooled, inactivated with formalin, clarified by centrifugation at 8,000 x g, and then ultracentrifuged at 34,000 x g for 2 h. The viral pellet was resuspended in 10 mM phosphate-buffered saline (PBS; pH 7.4) containing 0.05% azide. The concentration of the inactivated virus (antigen) was determined by measuring its optical density at 280 nm (OD280) and OD260 and calculating the content in accordance with the following formula: milligrams of protein per milliliter = (1.45 x OD280) – (0.74 x OD260). The virus suspension was serially diluted twofold with PBS from 1:10 to 1:2,560 and used to sensitize 2% latex beads (polystyrene beads, 0.8-μm diameter; Medical Examination Institute of Shanghai, Shanghai, China) by passive absorption. The latex beads were blocked with bovine serum albumin (BSA; Difco, Detroit, Mich.), and reacted with either PBS (Table 1) or standard anti-H5N1-positive serum (Table 2) to determine spontaneous agglutination and reactivity toward serum samples, respectively. When the virus particle dilution was 1:320 or 1:640 and the blocking BSA concentration was 0 or 0.02, respectively, sensitized latex displayed no spontaneous agglutination with PBS and intense agglutination with positive serum. The two conditions were further compared. The latex beads with 1:640 virus and 0.02% BSA displayed a nonspecific agglutination reaction, while beads with 1:320 virus and no BSA did not after a few days of storage. Thus, the use of latex beads sensitized with virus particles diluted at 1:320 (corresponding to a protein concentration of 0.426 mg/ml) and no BSA in blocking buffer was selected as the optimal condition for the assay.
The specificity of the LAT was first evaluated with standard negative serum samples from 50 specific-pathogen-free chickens and standard serum samples positive for non-AIV pathogens, including the pathogens for Newcastle disease, infectious bursal disease, infectious bronchitis, infectious laryngotracheitis, and Marek's disease, and also serum positive for AIV subtype H9N2. All of the 50 specific-pathogen-free chicken serum samples, the serum samples positive for other non-AIV pathogens, and the AIV H9N2-positive serum samples, were found negative with the LAT, indicating that the method is relatively specific for AIV H5N1.
Tests of the sensitivity and specificity of LAT in detecting AIV H5N1-vaccinated chicken serum samples were carried out with a total of 830 field serum samples with the HI test as the reference. By HI, 748 samples were positive (inhibition index, 2 x 104) and 82 samples were negative (inhibition index, <2 x 104). By the LAT, 666 samples were positive, of which 664 were from the 784 HI-positive samples and 2 were from the 82 HI-negative samples. Compared with that of HI, the sensitivity of the LAT was 88.8% and its specificity was 97.6%. The correlation between the LAT and HI was 89.6% (Table 3).
Furthermore, the LAT showed relatively higher sensitivity (89.1%, 82 positive by the LAT and 92 positive by HI; 91.7%, 400 positive by the LAT and 436 positive by HI) when detecting serum samples collected at 15 days and between 30 and 45 days after vaccination, and relatively lower sensitivity (82.7%, 182 positive by the LAT and 220 positive by HI) when detecting serum samples collected at 60 days after vaccination, all with HI as the reference method for validating the positive samples. This trend is consistent with what was observed by others in the diagnosis of human leptospirosis with the LAT (7). Immunoglobulin M (IgM) is the main subtype of the antibodies detected by the LAT. IgM antibodies appear early after vaccination or infection and subsequently fall at about 1 month after, while IgG antibodies appear later than IgM antibodies but remain much longer than IgM antibodies (8). Moreover, the intensity of the agglutination reaction induced by IgM antibodies is several hundred times stronger than that induced by IgG antibodies (9). Thus, the LAT appears to be suitable for detecting antibodies to AIV H5N1 at an early stage of immunity.
The reproducibility of the LAT was conducted with latex beads sensitized with three lots of virus particle preparations (diluted to the same protein concentration for bead sensitization) to evaluate a total of 100 serum samples. There were 94, 92, and 93 samples tested positive by the three lots, respectively, indicating a high level of reproducibility.
In terms of the stability of the LAT, sensitized latex beads were found to be able to retain similar agglutination sensitivity after storage at 4°C for at least 4 months (data not shown).
The LAT has been proved to be a very useful test for detection of antibodies in clinical microbiology (1, 5, 6). In this study, the LAT with latex beads sensitized with inactivated AIV H5N1 was found to be highly specific, with good sensitivity in detecting antibodies to AIV H5N1 in serum samples from vaccinated chickens. It could also be used to assay serum samples from other poultry species. This LAT is rapid and easy to perform, with no requirement for special equipment or skilled personnel; thus, it has great potential to be used in field practice for monitoring antibodies to AIV H5N1 vaccines.
ACKNOWLEDGMENTS
This work was supported by the Chinese National 10th Five-Year Science and Technology Plan (grant 2004BA519A07).
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