Indirect Biospecific Interactions Of Viral Particles With Latex

Stable latex particle reagent can be used to diagnose a variety of infectious diseases, such as rotavirus, adenovirus, and canine parvovirus (CPV) infections. Indeed, polystyrene beads (latex particles) are agglutinated by antigen when coated with antibodies. The principle of the latex agglutination (LA) test is shown in Fig. 1. Briefly, latex microspheres coated with specific antibodies react in the presence of the antigen. A positive reaction is characterized by macroscopically visible agglutination of the latex particles.

During winter, 50% of children younger than 2 years suffer from severe diarrhea caused by rotavirus infection. Among the several methods for detecting rotavirus (an enveloped virus), electron microscopy, RNA-polyacrylamide gel electrophoresis, ELISA, and EIA are the most popular. The inconvenience of all these techniques is the required time (several days) to obtain results. Thus it is necessary to get rapid, reproducible, sensitive and simple diagnostic test for rotavirus infection. Agglutination is a rapid method for the detection of rotavirus antigen in stool specimens. Amongst all the detection methods available, LA is the simplest one. Indeed, as large amounts of antigens are excreted during rota-virus gastroenteritis, it should be possible to diagnose this disease by latex agglutination reaction. The latex agglutination method for the rotavirus detection was first described by Sanekata et al. in 1981 [8]. Then several LA tests were developed with various latex sizes and different antibody preparations. The most important part of them will be presented and discussed in the following.

Hughes et al. have sensitized commercially latex beads (0.797 pm in diameter, Sigma Chemical Co.) with antirotavirus antibodies from an antiserum preparation or from IgG preparation obtained from the antiserum [9]. The LA test was performed by incubating antibody-coated latex beads with clarified stool suspensions for 15 min at 36°C. Results showed that latex beads were more sensitive for rotavirus detection when coated with IgG than the latex beads coated with whole immune serum. Indeed, the antirotavirus antibody concentration was greater in the IgG preparation than in the whole immune serum. Moreover, in addition to IgG, the whole immune serum contains several proteins that can adsorb onto the latex beads and thus decreased the number of available sites for IgG adsorption. The latex reaction was found to be two- to eightfold less sensitive than the EIA technique (Rotazyme, Abott).

Commercially available LA kits (Slidex Rotakit from bioMerieux, Rotalex from Orion) have been compared with classical techniques [electron micros-

Agglutination Tection Viral
FIG. 1 Latex agglutination test for the detection of viral particles.

copy, Rotazyme (EIA) from Abott] used to detect rotavirus [10] or with their home-made LA test [11]. Results were obtained faster with LA kit (within 25 min) than with classical methods and gave 100% specificity. However, the percent agreement of LA test with classical methods dramatically decreased as the virion count decreased, indicating a lower sensitivity for the LA kit at low viral titer [10]. Treatment of latex beads with 1% bovine serum albumin (BSA) before the coating with antirotavirus IgG fraction led to sensitivity and specificity equal to those of EIA test kit or electron microscopy analysis [11]. Indeed, BSA is a nonspecific protein that adsorbed nonspecifically onto most polymer sur faces. So it was used as precoating of material in order to decrease nonspecific adsorption of proteins [12].

Canine parvovirus (CPV), a nonenveloped virus of 22 nm diameter, causes hemorrhagic enteritis and myocarditis in young dogs and puppies. After symptom onset virus is excreted in the feces for several days. The virus can be infectious for a very long time because of extreme resistance of parvoviruses to environmental inactivation. Thus, there is a real need for a rapid and simple test that can be used by veterinarians to diagnose CPV infections.

Bodeus et al. have compared the sensitivity of the following detection methods: classical slide test with particle counting, and LA test involving polystyrene beads (0.8 pm in diameter) sensitized with purified monoclonal antibodies against CPV antigen [13]. Results were rapidly obtained in 5 min (slide test) to 30 min (particle counting), indicating that the antigen concentration detected by slide test is about 100 ng/mL whereas the particle counter led to 4 ng/mL of detected viral antigen, which is the same sensitivity as RIA technique.

In other respects, Esfandiari et al. have described one-step immunochromato-graphic test (Rapid Parvovirus Test) for the detection of CPV in canine feces samples [14]. In this test, latex particles have 0.3 pm diameter, are dyed blue, and are commercially available as polystyrene beads. The binding of parvovirus particles present in the fecal sample to CPV-mAbj-conjugated latex suspension led to a positive blue band in the T zone within 3-5 min (Fig. 2). This test showed about the same sensitivity and specificity as available ELISA tests.

Nakamura et al. have perfected a latex agglutination test for the detection of infectious bursal disease virus (IBDV). The test is commonly performed using either an agar gel precipitin (AGP) test or ELISA [15]. The minimal titer of virus that agglutinated anti-IBDV monoclonal antibody-bound latex micro-spheres (0.01 mL of 3.98 x 104 PFU strain IQ IBDV-sensitive) was 10-40 times higher than the observed titer value in the AGP test. Therefore, the sensitivity of this LA test is sufficient to detect IBDV antigen in infected bursal tissue within 10 min instead of 48 h.

To conclude on the various latex agglutination tests for the detection of virus particles in a sample, the results presented above are reported in Table 1. It appears that all LA tests described herein have similar sensitivity (about 106 viral particles, or 100 ng/mL) except the LA test using flow cytometry, which displays a higher sensitivity. The simplicity, high specificity, and speed of LA kits make them useful for rapid screening of symptomatic patients. Moreover, the LA test is very simple to perform because it does not require expensive equipment and is sensitive enough for routine diagnostic requirements. Several authors [9,14,15] also indicated the good conservation of biological activity of the antibody-coated latex particles with time. However, even if monoclonal antibodies were used to coat latex microspheres, sensitivity of the LA technique is not optimal. Indeed, antibody can adsorb onto the latex particle surface through

Specific Ige Screening Test

CPV-mAb, conjugated CPV-mAb2 Rabbit-anti-mouse to (atex

Specific Ige Screening Test

-► Positive T-band Control C-band

Migration (blue) (blue)

FIG. 2 Positive result in immunochromatographic test for the detection of canine parvovirus (CPV) in fecal samples. After migration, bound CPV to CPV-mAb1 conjugated to latex was detected in the T zone by reaction with the CPV-mAb2 leading to a blue band.

TABLE 1 Comparison of Latex Agglutination Tests

Ref. 13

Ref. 13

LA test

Ref. 9

Ref. 10

(flow)

(slide)

Ref. 14

Ref. 15

Latex

62.5 |ag

500 |ag

2.5 mg

2 mg

833 |lg

250 |lg

Antibody

0.2 mg

3 mg

0.2 mg

0.2 mg

0.3 mg

dil. 1:400 (ascite)

Sample volume

50 |aL

100 |a,L

25 |a,L

20 |a,L

170 |a,L

10 |xL à 50 %

Incubation time

15 min, 360°C

3 min, R.T.

30 min, R.T.

5 min, R.T.

3-5 min, R.T.

10 min, R.T.

Sensitivity

9.0 105 viruses

3.107 virions/mL

4 ng/mL

100 ng/mL

dil. 1:500

4.104 PFU

R.T., room temperature.

R.T., room temperature.

its Fc fragment and/or via its Fab fragment (Fig. 3), leading to a decrease in the efficiency of the binding with the antigen. This kind of coating, which involves nonspecific interactions between antibodies and polymer surface, is the same as the ELISA one. Therefore, it is not surprising that the sensitivity of both techniques, i.e., LA test and ELISA, is in the same range.

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