Detection of Foot-and-Mouth Disease Virus Antibodies: II. Use of Fractionated Bovine Antisera for Improving the Specificity of a “Passive” Hemagglutination Test1

Because 7S immunoglobulin (Ig) G antibodies of low type specificity were present in mixtures with highly specific 19S IgM antibodies, many bovine antisera to foot-and-mouth disease virus (FMDV) type A(12), strain 119 cross-reacted with type O of FMDV and to some degree with type C in the passive hemagglutination (HA) test. After 19S IgM antibodies were separated by density gradient centrifugation or precipitated with 4% (w/v) polyethylene glycol, the antigen could be determined with "block" HA tests. Such tests used several antigen concentrations in the titration of each antiserum. Adding 4% (w/v) polyethylene glycol to the serum was especially convenient for rapid precipitation of 19S IgM antibodies for the test. Similar results were obtained with bovine 19S IgM antibodies to FMDV type O, subtype 1, strain Caseros and type C strain Rezende.     

A simple and rapid colloidal gold-based immunochromatogarpic strip test for detection of FMDV serotype A

A sandwich format immunochromatographic assay for detecting foot-and-mouth disease virus (FMDV) serotypes was developed. In this rapid test, affinity purified polyclonal antibodies from Guinea pigs which were immunized with sucking-mouse adapted FMD virus (A/AV88(L) strain) were conjugated to colloidal gold beads and used as the capture antibody, and affinity purified polyclonal antibodies from rabbits which were immunized with cell-culture adapted FMD virus (A/CHA/09 strain) were used as detector antibody. On the nitrocellulose membrane of the immunochromatographic strip, the capture antibody was laid on a sample pad, the detector antibody was printed at the test line(T) and goat anti-guinea pigs IgG antibodies were immobilized to the control line(C). The lower detection limit of the test for a FMDV 146S antigen is 11.7ng/ml as determined in serial tests after the strip device was assembled and the assay condition optimization. No cross reactions were found with FMDV serotype C, Swine vesicular disease (SVD), Vesicular stomatiti svirus (VSV) and vesicular exanthema of swine virus (VES) viral antigens with this rapid test. Clinically, the diagnostic sensitivity of this test for FMDV serotypes A was 88.7% which is as same as an indirect-sandwich ELISA. The specificity of this strip test was 98.2% and is comparable to the 98.7% obtained with indirect-sandwich ELISA. This rapid strip test is simple, easy and fast for clinical testing on field sites; no special instruments and skills are required, and the result can be obtained within 15 min. To our knowledge, this is the first rapid immunochromatogarpic assay for serotype A of FMDV.     

The quantitation of rabies-specific antibodies. II. Factors affecting the sensitivity of the haemagglutination inhibition test

Various factors affecting the HAI test for the quantitation of rabies-specific antibodies have been evaluated with a view to obtaining maximum sensitivity and reproducibility in tests using tissue culture antigens prepared in vero cells and concentrated by dialysis. Goose erythrocytes treated with proteolytic enzyme bromelian at a concentration of 0.025% were much more susceptible to HA than those that were untreated or erythrocytes treated with neuraminidase. In addition, other parameters like the use of a phosphate buffered saline (PBS) as a diluent at pH 6.2, incubation at 0-4 degrees C for 1.5-3 h were found to be most critical for achieving maximum HA activity. To remove non-specific inhibitors, serum samples were treated with aerosil, acetone in combination or alone. Of the 73 serum samples tested, removal of non-specific inhibitors by aerosil alone occurred in up to 54.79% of the samples, whereas using acetone-aerosil treatment followed by adsorption with goose erythrocytes, the inhibitors were removed in 98.67% of the samples to a level that was undetectable at the 1:4 starting dilution in the HAI test.     

Detection of Foot-and-Mouth Disease Virus Antibodies: I. “Passive” Hemagglutination Test

A passive hemagglutination test has been developed to detect and measure foot-and-mouth disease virus (FMDV) antibody by using glutaraldehyde as a coupling reagent. An optimal concentration of 10 to 40 mug of virus per ml with 0.25% glutaraldehyde at 25 C for 1 hr was established for the sensitization of sheep erythrocytes. A reaction time of 18 hr at 4 C or 2 hr at 37 C induced good agglutination in the presence of specific antibody. Sensitization was carried out in phosphate buffer, whereas agglutination and preadsorption of nonspecific agglutinins from sera were performed in gelatin (0.1%, w/v)-stabilized, phosphate-buffered saline. An optimal pH of 7.2 was also established for all reactions. Antibodies derived from guinea pigs hyperimmunized by infecting with FMDV, types A, O, and C were both virus-and type-specific. Preliminary experiments showed that strain A-119 and strain A-24 Cruzeiro could also be distinguished by hemagglutination. Parallel hemagglutination and complement-fixation tests showed the former to be two to four times more sensitive than the latter.     

The quantitation of rabies-specific antibodies. III. A comparative evaluation of modified counter immunoelectrophoresis, haemagglutination inhibition and serum neutralization titres of human sera

The rabies-specific antibodies of 73 serum samples from vaccinated humans were determined by the modified counter immunoelectrophoresis (MCIE), and the haemagglutination inhibition test (HAI) by using the conventional serum neutralization test (SN) as a yard-stick. Both MCIE and HAI were found to be sensitive and specific for the estimation of rabies antibodies. In general, the unitages obtained by the MCIE and SN showed statistically insignificant differences (P greater than 0.05) and the correlation coefficient between the two methods was 0.697 (P less than 0.05). Although the unitage of the sera detected by HAI tests was lower by a factor of 0.155 from the unitage of SN tests, there was statistically insignificant differences between the two techniques (P greater than 0.05) with a correlation coefficient of 0.556 (P less than 0.05).     

Trypsinized Human Group O Erythrocytes as an Alternative Hemagglutinating Agent for Japanese Encephalitis Virus

Trypsinized human group O erythrocytes were found to be a suitable alternative to gander cells in hemagglutination (HA) and hemagglutination inhibition (HAI) tests for Japanese encephalitis (JE) virus. In the HAI test, no cross-reactions against JE virus were observed with immune sera containing antibody to taxonomically related or unrelated viruses, with mouse brain antigen, or with nonantibody serum inhibitors; specific antibody rise could be detected in an immunized rabbit. Gander and trypsinized human group O cells gave comparable titers in the HAI test, but the latter were preferable since (i) they required less challenging HA antigen, being more sensitive to agglutination by JE virus, and (ii) all human and some animal sera investigated were devoid of natural agglutinins for these cells, thereby eliminating or reducing the need for prior adsorption with packed cells.     

Assay sensitivity and differentiation of monoclonal antibody specificity in ELISA with different coating buffers

Buffers of different pH and ionic strength were employed as coating buffers for antigen adsorption to microtitre plates. Their efficiency for coating plates with rinderpest virus (RPV) and foot-and-mouth disease virus (FMDV) antigens was studied by ELISA with polyclonal and monoclonal antibody preparations. While the adsorption and detection of RPV antigen with polyclonal antiserum was highly dependent on the ionic strength and pH of coating buffer, adsorption of antigenically active FMDV antigen was relatively unaffected by the buffering conditions. Both antigens were adsorbed optimally in 0.01 M phosphate buffer, pH 8.0. When monoclonal antibodies were used to detect antigen, there was a greater degree of dependence on the coating buffer than that found with polyclonal antisera. Moreover, when they were used to detect antigen adsorbed under several buffering conditions, monoclonal antibodies showed a variety of preferred buffers. The usefulness of this differential reactivity in distinguishing epitope specificity is demonstrated.     

Evaluation of a trapping ELISA for the differentiation of foot-and-mouth disease virus strains using monoclonal antibodies.

A trapping enzyme-linked immunosorbent assay (ELISA) has been evaluated for the differentiation of foot-and-mouth disease virus (FMDV) strains using a panel of seven anti-serotype O monoclonal antibodies (MAbs). The variation of results within and between tests performed on the same day and on different days was examined using three strains of FMDV. Criteria for establishing antigenic differences between the strains as defined by the individual MAbs are proposed based on the variability measured, which can be used as standards by workers performing this test with other MAbs and FMDV strains.     

Analysis of foot-and-mouth disease virus-neutralizing idiotypes from immune bovine and swine with anti-murine idiotype antibody probes

Rabbit anti-idiotypic antibodies (a-IdAb) induced by foot-and-mouth disease virus (FMDV) neutralizing mAb were used as probes to identify anti-FMDV Id in immune serum from bovine and swine. In a competitive RIA, at least two of the a-IdAb exhibited a dose-dependent capacity to compete with labeled virus for anti-FMDV antibodies from a convalescent bovine serum. These a-IdAb were immobilized on activated Sepharose and used to isolate anti-viral Id from bovine, swine, and murine FMDV immune sera. Both the bovine and swine antibodies recovered from the a-IdAb/Sepharose columns reacted with virus, and to a lesser extent with corresponding mAb-resistant virus variants. The binding of affinity isolated bovine and swine antibodies to virus was specifically inhibited by the homologous a-IdAb, and in addition, both were capable of neutralizing FMDV in suckling mouse protection and plaque reduction neutralization assays. Therefore, by means of a-IdAb probes generated against FMDV murine Id, two neutralizing Id were identified in bovine and swine. These results suggest that FMDV-neutralizing epitopes recognized by murine systems play a role in the overall immunity of foot-and-mouth disease-susceptible animals.     

The use of the single radial haemolysis test for assessing antibody response and protective antibody levels in an influenza B vaccine study

Single radial haemolysis (SRH) and HAI tests were used to determine the levels of antibody in sera obtained from 118 volunteers taking part in a study of influenza B/Hong Kong/73 subunit vaccine. The two tests gave similar overall results, and showed good correlation, but SRH appeared to be more sensitive for the detection of low concentrations of antibody. A greater than or equal to 45% increase in SRH zone area was found to be equivalent to a significant increase in antibody titre and on the basis of this value, the HAI and SRH tests gave similar results for responses to immunization. The concentration of antibody equivalent to a 50% protective level against a challenge virus infection was a zone area of 45 mm2 for the SRH test; this was equivalent to an HAI titre of 1:20 which has previously been established as the 50% protective level.     

Detection of foot-and-mouth virus antibodies using a purified protein from the high-level expression of codon-optimized, foot-and-mouth disease virus complex epitopes in Escherichia coli

A codon optimized DNA sequence coding for foot-and-mouth disease virus (FMDV) capsid protein complex epitopes of VP1 amino acid residues 21-40, 135-160, and 200-213 was genetically fused to the C-terminal end of a glutathione-S-transferase (GST) gene in pGEX-6P-1 vector with the synonymous codons preferred by Escherichia coli . The gene was synthesized using PCR and subsequently expressed in E. coli producing an intracellular, soluble fusion protein that retained antigenicity associated with FMDV antibodies by western blot analysis. The chimera was purified from bacterial lysates by affinity chromatography and could be used in ELISA tests for antibodies against FMDV.     

Variation in the biological function of envelope protein epitopes of yellow fever vaccine viruses detected with monoclonal antibodies.

Three different virus strains (17D-204, 17DD and the French neurotropic vaccine) have been used as live attenuated yellow fever (YF) vaccines and are manufactured in different centres around the world. The envelope proteins of these vaccine viruses were examined and compared using mouse monoclonal antibodies (MAbs) in haemagglutination inhibition (HAI) and neutralization (N) tests. The epitopes eliciting HAI and/or N were found to vary depending on the virus examined. Such variation was also found between vaccine viruses of the same strain manufactured in different centres. These data were confirmed by the use of mouse polyclonal antisera. On the basis of the MAb results in HAI tests a dendrogram of the similarity coefficients between the viruses was constructed and showed that the viruses could be placed into three major groups. Thus, it is concluded that YF vaccines manufactured in different centres are antigenically distinct as recognized by the mouse immune system.     

Comparison of the sensitivity of the serological latex and ELISA tests

A sensitivity of the serological latex and ELISA tests were compared in carnation mottle virus diagnosis. For the latex test carnation mottle virus (CaMV) antiserum was sensibilized with latex suspension for RF-test. Sensibilized antiserum was used in 1: 200 dilution, as compared with fresh antiserum. For ELISA the γ-globuline fraction of antiserum was conjugated with alkaline phosphatase. The optimal dilution in both, CaMV fraction of antisera for coating of plates and γ-globuline-enzyme conjugate were in the ratio of 1: 500, 2 μg of antibodies in 1 ml. The dilution end point of carnation mottle virus in sap from carnation leaves was 1.6 × 10^?4 to 1.25 × l0^?5 and 1 × 10^?4 to 1.25 × l0^?5, when serological latex and ELISA tests were used. As indicated, ELISA as compared to the latex test was found to be more sensitive for carnation mottle diagnosis. As the latex test is considered to be simpler and cheaper, and in addition, showing the same assurance as the biological test onChenopodium amaranticolor, the latex test is recommended for carnation mottle virus detection.     

Absence of antibodies to foot-and-mouth disease virus in free-ranging roe deer from selected areas of Germany (2001-2002)

Blood samples (n = 223) of free-ranging roe deer (Capreolus capreolus) were collected from selected hunting grounds in Germany between October 2001 and October 2002. Samples originated from Lower Saxony (n = 43) and North-Rhine Westphalia (n = 108) within a 20-km area ("cordon") cordoned off along the border of The Netherlands. This is adjacent to the area of a foot-and-mouth disease outbreak that occurred between 21 March and 22 April 2001 in The Netherlands. Negative control samples were taken from northern Germany (Schleswig-Holstein, n = 72). Two different enzyme-linked immunosorbent assays (ELISAs) were used for the detection of antibodies against foot-and-mouth disease virus (FMDV) serotype O strain Manisa. To confirm ELISA-positive results, a virus neutralization test was performed. All samples tested negative for antibodies against FMDV. These results suggest that FMDV was not transmitted to free-ranging roe deer living in parts of Germany adjacent to the area affected by the 2001 foot-and-mouth disease outbreak in The Netherlands.     

Possible Application of the IHI Test in the Species Identification of Hair

Immunological species differentiation of keratein from hair has previously been demonstrated by the precipitin ring test in tubes (Pillemer et al. 1939) and by the indirect hemagglutination test (Simonsen 1970). In the present study the possibility of species identification of s-carboxymethylkeratein (SGMK) from single hairs was examined. SCMK and rabbit anti-SCMK sera from man, horse, dog and ox were prepared according to methods described by Gillespie (1962) and Simonsen (1970, 1971). Suitable antisera were used for the indirect hemagglutination-inhibition (IHI) test (Stavitsky 1954). The antisera were absorbed with heterologous SCMK and the inhibition test performed using SCMK extracted from 5 cm stretches of hairs by reduction and alkylation in 1 ml fluid volumes. To each vial containing 0.5 ml of antiserum in a serial 2-fold dilution row of the respective antisera was added 0.05 ml of a homologous or a heterologous single-hair SCMK. After incubation at 37 °C for 30 min. SCMK-coated goat erythrocytes were added and the test read after incubation at 20 °C for 18 hrs.     

Antibody response in school children to live rubella vaccine (Cendehill strain)

The efficacy of an attenuated rubella virus vaccine, Cendevax, was tested on 65 school children. Forty-nine of them (75%) had pre-existing antibodies and in these there was no increase in the HAI antibody titres after administration of the vaccine. Sixteen children (25%) had no demonstrable rubella HAI antibody prior to vaccination. From the latter group, postvaccination serum samples were available from only 11, and 10 of these seronegative children showed seroconversion after vaccination. The geometric mean HAI titre was 1:180. Seven of the 10 postvaccination serum samples had complement-fixing antibodies and specific IgM antibodies were detected by the immunofluorescence test in 8. No correlation was observed between the CF and the IgM antibodies.     

The suitability of different microtitre plates for detection of antibody to virus antigens by indirect ELISA

To optimize enzyme linked immunosorbent assays (ELISAs) for the detection of virus-specific antibodies, a range of commercially available microtitre plates was evaluated for their ability to bind virus antigen. Rinderpest virus and foot-and-mouth disease virus were investigated as target antigens. Binding capacity for antigen, binding ratios (attachment of specific antibody versus that of non-immune antibody) and the variation in the results of the tests within and between plates were measured. Binding capacity was found to be greater with rinderpest virus (RPV) antigen than with foot-and-mouth disease virus (FMDV) antigen, although higher binding ratios were obtained with FMDV antigen. Variation within and between plates was generally less with RPV antigen than with FMDV antigen. One plate could not be said to out-perform the other plates in all tests. For our purpose, that is the detection of monoclonal antibody production against a variety of virus antigens, a number of plates were found to be suitable (e.g. Dynatech M129B, Flow 77-172-05 and Nunc 4-39454). The differences in the performances of the microtitre plates with these two virus antigens highlights the need for consideration of the solid phase as part of the standardization procedures for ELISAs.     

Selection of influenza A virus adsorptive mutants by growth in the presence of a mixture of monoclonal antihemagglutinin antibodies.

The influenza virus hemagglutinin contains four major regions that are recognized by antibodies able to neutralize viral infectivity. To investigate the effect of an antibody response directed against each of these sites on viral evolution, influenza virus A/PR/8/34 (H1N1) was grown in allantois-on-shell cultures in the presence of a mixture of monoclonal antihemagglutinin antibodies. This selection mixture contained antibodies (two or three antibodies per antigenic site) whose concentrations were adjusted to achieve equal neutralization titers against each of the four antigenic sites. By varying the ratio of input virus to selection mixture concentration, we observed that variant viruses emerged under conditions of partial neutralization. Each of the four variants characterized in detail differed from the parental virus in its interaction with cellular receptors and exhibited minimal changes in antigenicity. Thus, these variants were virtually indistinguishable from wild-type viruses, as assessed by the binding of 103 monoclonal antihemagglutinin antibodies in an indirect radioimmunoassay. Despite this, many of the same antibodies demonstrated decreased titers to the variants in hemagglutination inhibition tests. The magnitude of the differences depended on the indicator erythrocytes used (much greater differences were detected with chicken erythrocytes than with human erythrocytes). Hemagglutination mediated by the variants was more resistant to neuraminidase treatment of erythrocytes than hemagglutination mediated by the parental virus. These findings are consistent with the idea that the variants were initially selected by virtue of their increased avidity for host cell receptors. Sequencing of viral RNA revealed that each of the variants differed from the parental virus by a single amino acid alteration in its HA1 subunit. Two of the changes were close to the proposed receptor binding site on hemagglutinin and could directly alter receptor binding, while a third was located near the trimer interface and may have increased receptor binding by altering monomer-monomer interactions.     

Validation and use of an indirect enzyme-linked immunosorbent assay for detection of antibodies to West Nile virus in American Alligators (Alligator mississippiensis) in Florida

In October 2002, West Nile virus (WNV) was identified in farmed American alligators (Alligator mississippiensis) in Florida showing clinical signs and having microscopic lesions indicative of central nervous system disease. To perform seroepidemiologic studies, an indirect enzyme-linked immunosorbent assay (ELISA) was developed to determine exposure of captive and wild alligators to WNV. To validate the test, a group of WNV-seropositive and -seronegative alligators were identified at the affected farm using hemagglutination inhibition (HAI) and the plaque reduction neutralization test (PRNT). The indirect ELISA utilized a rabbit anti-alligator immunoglobulins polyclonal antibody as the secondary antibody, and inactivated WNV-infected Vero cells were used as the coating antigen. For all samples (n=58), the results of the ELISA were consistent with the HAI and PRNT findings. Plasma was collected from 669 free-ranging alligators from 21 sites across Florida in April and October 2003. Four samples collected in April and six in October were positive for WNV antibodies using HAI, PRNT, and the indirect ELISA. This indicated that wild alligators in Florida have been exposed to WNV. These findings can be used as a baseline for future surveys.     

Virus Aggregation as the Cause of the Non-neutralizable Persistent Fraction

The non-neutralizable or persistent fraction of virus populations has been found to be caused by aggregated virus. Detailed investigation was performed with the prototype strain of echovirus type 4 (Pesascek), as this virus is notorious for its large non-neutralizable fraction. When Pesascek virus was clarified by low-speed centrifugation, homologous antiserum hardly neutralized the virus. However, when the virus was filtered through membranes having a porosity only twice the diameter of the virus, monodispersed virus was obtained which was efficiently neutralized. Serum titers were up to 1,000 times higher if the neutralization test was carried out with monodispersed virus. Virus in non-neutralizable aggregates was found to constitute 30% of the infective units of unfiltered Pesascek virus but only 0.1% of the antigenically related DuToit strain. This explains why DuToit strain has been a more satisfactory indicator strain for detecting type 4 antibodies, regardless of the echo 4 strain used for inducing the antibodies. Clarified suspensions and ultrafiltrates of viruses belonging to the picorna-, reo-, myxo-, adeno-, herpes-, and poxvirus groups were studied. Clarified suspensions yielded persistent fractions of 0.005% for poliovirus, of 0.1% for reovirus, of 0.6% for influenza virus, of <0.001% for adenovirus, of 0.06% for herpesvirus, and of 10 to 30% for vaccinia virus. In all cases the persistent fractions were removed by membrane filters which had a pore diameter no larger than twice that of the virus under test, and the high concentration of virus in each ultrafiltrate was completely neutralized by antiserum.