Monoclonal antibodies to equine IgM improve the sensitivity of West Nile virus-specific IgM detection in horses

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Veterinary Immunology and Immunopathology 122 (2008) 46–56 www.elsevier.com/locate/vetimm

Monoclonal antibodies to equine IgM improve the sensitivity of West Nile virus-specific IgM detection in horses Bettina Wagner *, Amy Glaser, Julia M. Hillegas, Hollis Erb, Carvel Gold, Heather Freer Department of Population Medicine and Diagnostic Sciences, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853, USA Received 7 July 2007; received in revised form 9 September 2007; accepted 23 October 2007

Abstract West Nile virus (WNV) is a zoonotic pathogen of global importance. In horses with neurological signs, detection of WNVspecific immunoglobulin M (IgM) in serum is widely used to identify clinical cases of WNV encephalitis. Here, we describe the development of two monoclonal antibodies (mAbs) to equine IgM which were used in a WNV IgM-specific enzyme-linked immunosorbent assay (ELISA). Their performance was compared to an established assay based on polyclonal anti-IgM. Check test serum samples from the National Veterinary Service Laboratory (NVSL) were used to evaluate the performance of the three antiIgM antibodies. The anti-IgM 1-22 mAb correctly identified all NVSL samples. Both the polyclonal antibody and monoclonal antiIgM 2B-63 identified eight out of ten samples correctly. The three assays were then compared using serum samples from clinically healthy animals (n = 33) and horses with neurological signs (n = 21). High Spearman rank correlations (0.76–0.86) were found among the ELISA results. Inter-test agreements (weighted kappa) for assay interpretation resulted in strong agreement (0.95) of the results obtained by the mAbs and moderate agreements when monoclonal and polyclonal anti-IgM-based assays were compared. To determine the analytical sensitivities of anti-WNV IgM detection, serial dilutions of WNV IgM-positive serum samples were analyzed. The highest sensitivity was obtained by using the anti-IgM 1-22 mAb to capture IgM from equine serum. In conclusion, the use of monoclonal anti-IgM antibodies can improve the sensitivity of IgM detection in the acute phase of WN disease. # 2007 Elsevier B.V. All rights reserved. Keywords: West Nile virus; Horse; IgM; ELISA; Monoclonal antibody

1. Introduction West Nile virus (WNV) has become endemic in the United States since the first outbreak in 1999 (Lancotti et al., 1999). The virus is transmitted in cycles between mosquitoes (Culex species) and birds but can also cause disease in humans and horses, as well as other domestic and wild mammals (Ludwig et al., 2002; Komar, 2003;

* Corresponding author. Tel.: +1 607 253 3813; fax: +1 607 253 3440. E-mail address: [email protected] (B. Wagner). 0165-2427/$ – see front matter # 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.vetimm.2007.10.013

Castillo-Olivares and Wood, 2004). Compared to earlier outbreaks reported in the 1960s in Africa, Europe and the Middle East (Murgue et al., 2002), recent cases of WNV infection in the United States indicated an increase in severity of the disease in horses and a higher mortality in birds (Blitvitch et al., 2003). Clinical signs in the horse are characterized by neurological symptoms (encephalomyelitis) reported in approximately 10–12% of horses infected with WNV (Ostlund et al., 2000; Peterson and Roehrig, 2001; Bunning et al., 2002), with a mortality of 30% in animals that develop neurological symptoms. In all other cases of equine WNV infection clinical symptoms generally do not

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develop, although a transient fever can occur after infection. In horses, detection of immunoglobulin M (IgM) antibodies to WNV in serum is widely used to diagnose acute infection with the pathogen (Castillo-Olivares and Wood, 2004; Hogrefe et al., 2004; Porter et al., 2004; Long et al., 2006). During acute infection, serological WNV IgM antibody detection was found to be of higher diagnostic sensitivity than viral RNA determination by RT-PCR (Castillo-Olivares and Wood, 2004; Kleiboecker et al., 2004). WNV-specific IgM can be found in the circulation by 6–7 days after infection (Bunning et al., 2002) and can be detected by IgM-capture ELISA (Castillo-Olivares and Wood, 2004; Long et al., 2006). The assay uses a polyclonal anti-equine IgM antibody to capture IgM antibodies from equine serum. In this study, we generated mAbs to equine IgM. The antibodies were used in the WNV IgM-capture ELISA and the results were compared to those obtained by the established assay using polyclonal anti-IgM to determine whether the anti-IgM mAbs could enhance the analytical sensitivity of the assay. 2. Material and methods 2.1. Purification of IgM from horse serum IgM was purified from serum of an adult, healthy ¨ KTA Thoroughbred horse by gel filtration using an A FPLC instrument (GE Healthcare, Piscataway, NJ). A total of 4 ml serum was diluted 1:3 in phosphate buffer (0.05 M sodium phosphate, 0.15 M NaCl, pH 7.2) and separated on a Sephacryl S200 column. The first flow through peak containing IgM was collected and subsequently run over a Protein G column (GE Healthcare, Piscataway, NJ) to deplete remaining IgG antibodies. The protein concentration of the purified IgM fraction was determined using the BCA protein assay (Pierce, Rockford, IL). 2.2. Generation of mAbs to equine IgM The procedure to generate mAbs was described previously (Wagner et al., 2003). Briefly, the purified equine IgM was used to immunize a female BALB/C mouse. Spleen cells from this mouse were fused to X63Ag8.653 mouse myeloma cells. Hybridomas were screened by ELISA for equine IgM detection as described below. Positive cultures were cloned by limiting dilution until they (i) secreted only a single murine antibody isotype, as tested by ELISA using mouse antibody isotyping reagents (Sigma, St. Louis,

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MO) and (ii) showed a uniform, immunoglobulin positive cell population by flow cytometry after staining with a goat anti-mouse IgG(H + L)-FITC antibody (Jackson ImmunoResearch, West Grove, PA). Monoclonal cell lines were then grown in serum-free largescale cultures and antibodies were purified from the cell culture supernatants using a Protein G column as previously described. 2.3. ELISA to detect mAbs to equine IgM Polyclonal goat anti-horse IgG(H + L) antibody (Jackson ImmunoResearch, West Grove, PA) was used to coat ELISA plates at a concentration of 4 mg/ml. Plates were coated overnight at 4 8C. After washing the plates three times, purified IgM was applied and the plates were incubated for 1 h at room temperature. The plates were washed again and supernatants obtained from the cell fusion were applied and incubated for another hour. After another wash, a secondary peroxidase conjugated goat anti-mouse IgG(H + L) antibody (Jackson ImmunoResearch, West Grove, PA) was used to detect bound antiIgM antibodies. After 45 min of incubation the plates were washed for the last time and substrate was applied and incubated for 20 min in the dark. All buffers, the substrate solution, the washing and incubation steps, and the reading of the plates were composed or performed as previously described (Wagner et al., 2003). This assay was used for screening of the fusion supernatants and throughout the limiting dilution procedure. 2.4. ELISA to evaluate specificity of antibodies to equine IgM The ELISA described above was modified to test whether the anti-IgM antibodies detect other equine isotypes than IgM. Two anti-IgM mAbs (anti-IgM 1-22 and 2B-63) and a polyclonal goat anti-equine IgM antibody (VMRD Inc., Pullman, WA) were tested by this assay. Throughout the text abbreviations were used for these three antibodies; polyclonal anti-IgM = polyclonal goat anti-equine IgM antibody, M1-22 = monoclonal anti-IgM 1-22, and M2B-63 = monoclonal antiIgM 2B-63. Instead of purified IgM, heterohybridoma supernatants containing equine IgM, IgG1, IgG4 or light chains (Wagner et al., 1995) were applied to the plates. Three equine serum samples were also used as positive controls in this assay. To detect the binding of the polyclonal antibody, a peroxidase conjugated mouse anti-goat IgG(H + L) antibody (Jackson ImmunoResearch, West Grove, PA) was used. All other steps of the assay remained the same as described above.

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2.5. SDS-PAGE and Western blotting SDS-Page and Western blotting were performed as described (Wagner et al., 1995). In brief, 8–16% gradient gels (Bio-Rad, Hercules, CA, USA) were used to separate purified IgM or equine serum. The samples were run under either non-reducing or reducing conditions using 2-mercaptoethanol (ME). The proteins were transferred to PVDF membranes (Bio-Rad, Hercules, CA, USA) by Western blotting. After the transfer, free binding spaces on the membranes were blocked by incubation in Tris-buffered saline (TBS) containing 5% (w/v) non-fat dry milk, 0.05% (v/v) Tween 20 (blocking solution) for 1 h. Then, membranes were incubated with supernatants of the newly generated mAbs to equine IgM in dilutions of 1:4 (M2B-63) or 1:8 (M1-22) in blocking solution. The membranes were washed three times with TBS containing 0.05% (v/v) Tween 20 and incubated with a secondary peroxidase conjugated goat anti-mouse IgG(H + L) antibody (Jackson ImmunoResearch, West Grove, PA) in a dilution of 1:5000. All antibody incubation steps were performed for 1 h at room temperature. The membranes were washed another three times before antibody binding was visualized by the ECL chemiluminescence method (Amersham Bioscience, Piscataway, NJ, USA). 2.6. Serum samples The National Veterinary Service Laboratory (NVSL) check test panel set #18 for the 2006 inter-laboratory comparison of equine IgM-capture ELISA for WNV antibodies (USDA, Ames, IA) was used as standardized, control serum panel. A total of 10 NVSL check test-panel sera were measured using three anti-IgM antibodies to define the ranges for interpretation (negative, weak-positive, positive) for each of the anti-IgM reagents. For further evaluation of the IgMcapture ELISA using the different anti-IgM antibodies, 21 serum samples from horses with neurological signs were used. All samples were submitted to the Animal Health Diagnostic Center at Cornell University in 2002. Neurological signs included tremor, muscle fasciculation, depression, weakness in the anterior and/or posterior limbs, ataxia and recumbence. High fever was reported in several cases. At least three of the horses were euthanized after disease progressed. In addition, 33 control serum samples from clinically healthy horses of the herds of the Baker Institute for Animal Health and the Equine Park at Cornell University were compared. The control group included 17 horses which had never

been vaccinated against WNV and 16 horses with a known vaccination history for WNV. All vaccinated horses were administered a modified-live vaccine (Recombitek, Merial) according to the manufacture’s instructions. The last vaccination was performed within the previous year, but not within the last 3 months (n = 4) or longer than a year before sampling (n = 12). 2.7. Plaque reduction neutralization assay Serum samples were two-fold serially diluted from 1:20 to 1:10,240 in 0.1 ml cell culture medium (CCM) (minimum essential medium with Earle’s salts (MEME, Invitrogen, Grand Island, NY), 10% (v/v) fetal bovine serum (FBS) and 10 mg/ml ciprofloxicin hydrochloride (Bayer, Kankakee, IL). Approximately 200 plaqueforming units of WNV were added to each dilution in a volume of 0.1 ml CCM containing 10% (w/v) guinea pig complement (Colorado Serum Company, Denver, CO) and incubated for 1 h at 37 8C in a 5% CO2 incubator. A total of 0.1 ml of the virus-serum suspension was overlayed onto a confluent monolayer of Vero cells and incubated for another hour under the conditions described above. Cell monolayers were overlayed with CCM containing 2% (v/v) FBS and 1% (w/v) low melting point agarose (Invitrogen, Carlsbad, CA). Assays were incubated for 3 days as described above. Monolayers were stained overnight by adding three drops of CCM containing 3 mg neutral red/ml. Plaques were counted on day 4. Wells were scored positively for neutralization if the number of plaques was less than or equal to the average plaque count at a 1:10 dilution of input virus. Results were reported as seropositive at the corresponding dilution of test serum (1:20–1:10,240). If the number of plaques was greater than the average plaque count at a 1:10 dilution of input virus (indicating that the serum had not neutralized viral plaque formation), wells were scored negatively. 2.8. IgM-capture ELISA to detect WNV For the equine IgM-capture ELISA (Fig. 1), plates were coated with the polyclonal anti-IgM antibody, or the M1-22 or M2B-63 mAbs. The polyclonal anti-IgM was used at a dilution of 1:6000, which corresponds to 9 mg/ml antibody, while the mAbs were used at 4 mg/ ml. Antibodies were diluted in 0.1 M carbonate buffer, pH 9.6 for coating of ELISA plates (Maxisorp Immuno Modules, Nunc, Roskilde, Denmark). Batches of approximately 20 plates were incubated overnight at 4 8C with the coating antibody and were then frozen at 20 8C for at least one night before use. Before the

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the same serum sample using supernatant from noninfected cells. For the polyclonal anti-IgM antibody, samples with P/N ratios <2 were considered negative, P/N ratios of 2–3 were considered weak-positive and P/N >3 were considered positive. For the M1-22 and M2B-63 mAbs, the ranges for assay interpretation were determined by the experiments described in this article. Fig. 1. Flow diagram of the anti-WNV IgM-capture ELISA. Step 1: either monoclonal (M1-22 or M2B-63) or polyclonal anti-IgM antibodies were used for coating of the ELISA plates. Step 2: horse serum was applied and serum IgM was bound to the matrix. Step 3: plates were incubated with WNV antigen which was bound if anti-WNV antibodies were present in the serum. Step 4: WNV antigen was detected by a peroxidase-conjugated antibody to WNV E-protein followed by incubation of a colorimetric substrate (not shown).

assay was performed, the plates were thawed and washed five times with phosphate-buffered saline (PBS) containing 0.05% (v/v) Tween 20. Test and control serum samples were diluted 1:400 in Trisbuffered saline (TBS) containing 5% (w/v) non-fat dry milk and 0.05% (v/v) Tween 20 and four wells were loaded per sample. The serum was incubated for 1 h at 37 8C. Plates were washed as above and cell culture derived, inactivated WNV antigen (two wells) and antigen control (two wells) were added to the plates. The antigen was prepared from supernatant of WNVinfected Vero cells. Control antigen was obtained from non-infected cells. The supernatants were inactivated with beta-propionolactone (Sigma, St. Louis, MO) overnight at 4 8C and then neutralized with 1/10 volume of 1 M Tris pH 8.0. The antigen solutions were incubated on the ELISA plates overnight at 4 8C. The next day, plates were washed and a peroxidaseconjugated anti-St. Louis Encephalitis mAb (6B6C, kindly provided by the Center for Disease Control (CDC)), with reported cross-reactivity to the WNV Eprotein (Roehrig et al., 1983) was added. The antibody was diluted 1:2000 in TBS containing 0.05% (v/v) Tween 20 and incubated for 1 h at 37 8C. Afterwards, the plates were washed again and substrate solution (TMB Microwell Peroxidase Substrate System, KPL, Gaithersburg, MD) was added. The substrate reaction was stopped after 10 min by adding an equal volume of 1 N H2SO4 solution to the plates. The reactions were measured in an automated microplate ELISA Reader (BioTek instruments, Winooski, VE) at a wavelength of 450 nm. Positive/negative (P/N) ratios for all serum samples were calculated. The P/N ratio is the mean optical density (OD) of a serum sample incubated with WNV-positive supernatant, divided by the mean OD of

2.9. Statistical analysis The statistical analysis was performed using the MedCal software, version 9.0.1.0 by F. Schoonjas. To describe the consistency in ranking and decisions across the three tests, scatter diagrams of P/N ratios were plotted and rank correlations were calculated for P/N ratios. In addition, the weighted kappa concordance coefficients for the categorization of the P/N ratios were calculated. The kappa describes the percentage of agreement beyond that expected just by change. The weighted kappa gives partial weight to ‘‘near misses’’ (such as 0 versus 1) as opposed to ‘‘father misses’’ (such as 0 versus 2). The data were categorized according to the P/N ratio in negative, weak-positive, and positive, and weighted kappa values were calculated. Weighted kappa values >0.60 were considered important (Dohoo et al., 2003). 3. Results 3.1. Characterization of anti-IgM mAbs IgM was purified from equine serum and was tested by ELISA confirming a high content of equine IgM. IgG antibodies could not be detected in the purified IgM fraction (data not shown). The purified IgM was used to immunize a BALB/c mouse and to produce mAbs to equine IgM. Two hybridoma cell lines secreting antibodies to equine IgM (M1-22 and M2B-63) were established. The mAbs were tested by SDS-PAGE and Western blotting using purified equine IgM and equine serum (Fig. 2). Both mAbs detected IgM under nonreducing conditions indicated by a band at high molecular weight (200 kDa) in equine serum and purified IgM. Reduction of the samples by mercaptoethanol destroyed disulfide bonds and induced the separation of immunoglobulin heavy and light chains. Under reducing conditions, both antibodies detected the reduced IgM heavy chain of approximately 85 kDa. The murine isotypes of the anti-IgM mAbs were determined using a mouse isotyping ELISA. Both mAbs are murine IgG1 isotypes.

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Fig. 2. SDS-Page and Western blot using anti-IgM mAbs. Purified equine IgM (1) and equine serum (2) were separated in 8–16% gradient gels under reducing (+ME) and non-reducing ( ME) conditions. The gels were blotted to PVDF membranes and the membranes were incubated with monoclonal anti-IgM 1-22 (A) or anti-IgM 2B-63 (B).

3.2. Equine immunoglobulin isotype specificity of the three anti-IgM antibodies The M1-22 and M2B-63 mAbs were tested for their specificity to equine IgM by ELISA using heterohybridoma-derived equine IgM, IgG and light chains. Heterohybridoma cell line-derived immunoglobulins are pure reagents and free of other equine isotypes. A polyclonal anti-IgM was also included in the test for comparison. Both M1-22 and M2B-63 detected equine IgM only. The polyclonal anti-IgM detected equine IgM and also cross-reacted with equine IgG4 (Fig. 3). Taking into account that the concentrations of all heterohy-

bridoma immunoglobulins were similar, the optical density of the IgG4 detection was clearly decreased compared to the optical density obtained with IgM and the polyclonal antibody. This suggested that the polyclonal anti-IgM predominately detected equine IgM and to a lesser extent IgG4 in equine serum. Before anti-IgM mAbs were developed, this polyclonal anti-IgM reagent was used as a capture reagent in an assay to detect WNV-specific IgM in horse serum. The following experiments were performed to evaluate the new M1-22 and M2B-63 mAbs for this assay and to compare them to the established test using polyclonal anti-IgM.

Fig. 3. Equine immunoglobulin isotypes were detected using three antibodies to equine IgM by ELISA. Equine IgM, IgG1, IgG4 and light chains (EqL1, EqL2, EqL4) were obtained from heterohybridoma cell lines. All of them represent monoclonal standards for the respective isotypes. Sera from three horses were included in the measurement for positive control. PBS served as negative control. The bars represent means and standard deviations obtained from triplicates of each sample. Data from one assay are shown. The assay has been repeated three times with identical results for antibody specificity.

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Table 1 Comparison of positive/negative (P/N) ratios of NVSL check test samples for West Nile virus IgM-capture ELISA using three different anti-equine IgM antibodies NVSL code

Expected outcome

P/N ratioa Polyclonal anti-IgM

mAb anti-IgM 1-22

mAb anti-IgM 2B-63

2.1 3.7 17.3 8.3 1.4 2.2 1.0 0.9 1.4 0.9

7.3 14.9 33.1 15.7 2.6 2.9 1.2 1.2 1.8 1.5

5.6 8.8 25.7 15.0 4.4 3.6 1.3 1.3 1.7 1.3

Positive control Negative control

16.0 1.1

30.3 1.2

32.9 1.6

Correct outcome (NVSL samples)

8/10

10/10

8/10

#1 #3 #5 #10 #4 #9 #2 #6 #7 #8

Positive Positive Positive Positive Weak-positive Weak-positive Negative Negative Negative Negative

a

For the currently established IgM-capture ELISA using a polyclonal anti-IgM antibody the results are interpreted as follows: P/N ratio <2 = negative, P/N ratio 2–3 = weak-positive, P/N ratio >3 = positive. The results that are not according to the expected outcome are written in bold.

3.3. Detection of WNV-specific IgM by IgM-capture ELISA using reference sera The M1-22 and M2B-63 antibodies were used in a WNV IgM-capture ELISA to identify anti-WNV IgM antibodies in standardized NVSL check test horse serum samples and compared to results obtained using the polyclonal anti-IgM reagent. A total of 12 sera were used for the comparison, 10 of which were NVSL samples and one positive and one negative control that were used routinely for the current assay (Table 1). Polyclonal anti-IgM identified eight out of ten NVSL samples correctly. For the remaining two samples, P/N ratios lower than expected were obtained and resulted in one NVSL positive sample being classified as ‘‘weakpositive’’, and one weak-positive being classified as ‘‘negative’’. All ten NVSL samples were correctly identified by M1-22. The M2B-63 mAb correctly identified all positive and negative sera (8/10). Both weak-positive samples were classified as ‘‘positive’’ by M2B-63. However, both weak-positive samples had lower P/N ratios than all expected positive samples, suggesting that the cut-off between weak-positive and positive samples could be shifted to a P/N ratio of >5 rather than >3 for this antibody. This adjustment would result in the correct identification of all NVSL samples by M2B-63. Compared to the polyclonal anti-IgM, both mAbs resulted in higher P/N ratios for all positive NVSL samples. In contrast, the P/N ratios for the negative

samples were not different between the antibodies. To further investigate this finding, additional serum samples were compared using all three antibodies for WNV-specific IgM determination. 3.4. Comparison of polyclonal and monoclonal anti-IgM antibodies in the anti-WNV IgM-capture ELISA A total of 54 serum samples from horses with neurological signs (n = 21), clinically healthy horses without a history of WNV vaccination (n = 17), and clinically healthy horses with known vaccination history (n = 16) were tested by the WNV IgM ELISA using polyclonal anti-IgM, and the M1-22 or M2B-63 mAbs. In addition, plaque reduction serum neutralizing antibody titers to WNV were determined for these samples. All 17 healthy, non-vaccinated horses had no detectable PRN titers (negative at a serum dilution of 1:20), and their P/N ratios obtained by WNV IgM ELISA ranged from 0.8 to 1.4 for the assay using polyclonal antiIgM, from 1.0 to 1.4 (M1-22), and from 1.0 to 1.6 (M2B63). Because antibodies to WNV were not detected in any of the assays, these horses were considered not exposed to WNV. The data obtained from sera of horses with neurological signs and clinically healthy, vaccinated horses are shown in Table 2. Horses with neurological signs separated into two groups: (i) animals with positive PRN titer (n = 14) which were considered to be exposed to and suffering from WNV, and (ii) horses with no

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Table 2 P/N ratios obtained from serum samples of horses with neurological signs and clinical healthy horses vaccinated against WNV by comparing three anti-IgM antibodies for coating in the anti-WNV IgM-capture ELISA Sample

Group

PRN titera

P/N ratiob Polyclonal anti-IgM

Anti-IgM 1–22

Anti-IgM 2B-63

1 2 3 4 5 6 7 8 9 10 11 12 13 14

Neurological Neurological Neurological Neurological Neurological Neurological Neurological Neurological Neurological Neurological Neurological Neurological Neurological Neurological

320 >10,240 2560 2560 1280 640 640 640 640 2560 1280 80 160 20

2.9 3.7 3.4 4.5 2.9 2.2 3.0 2.4 1.7 2.3 2.2 1.3 1.2 1.2

17.6 17.5 16.8 16.3 14.5 14.4 13.3 13.1 13.1 11.9 11.0 4.2 3.8 2.6

15.6 18.7 10.6 15.2 12.5 9.7 13.7 7.3 5.3 14.7 7.1 6.5 2.4 1.4

15 16 17 18 19 20 21

Neurological Neurological Neurological Neurological Neurological Neurological Neurological

Negative Negative Negative Negative Negative Negative Negative

1.4 1.3 1.3 0.8 1.2 1.0 0.9

1.4 1.4 1.3 1.2 1.1 1.1 1.1

1.6 1.8 1.6 1.1 1.2 0.9 1.2

22 23 24 25 26 27 28 29 30

Vaccinated Vaccinated Vaccinated Vaccinated Vaccinated Vaccinated Vaccinated Vaccinated Vaccinated

1280 640 160 160 160 160 40 40 40

1.2 1.2 1.0 1.0 0.9 1.1 1.0 1.0 1.2

1.5 1.4 1.1 1.4 1.2 1.3 1.2 1.2 1.1

1.6 1.8 1.1 1.3 1.2 1.3 1.2 1.5 1.3

31 32 33 34 35 36 37

Vaccinated Vaccinated Vaccinated Vaccinated Vaccinated Vaccinated Vaccinated

Negative Negative Negative Negative Negative Negative Negative

1.5 1.1 1.1 1.1 1.3 1.1 1.1

1.5 1.3 1.4 1.2 1.9 1.1 1.1

1.3 1.1 1.5 1.4 1.5 1.1 1.1

a b

A negative plaque reduction neutralization result indicates that viral plaque formation was not inhibited at a serum dilution of 1:20. Results were obtained at a serum dilution of 1:400. Samples with P/N ratios <2.0 were interpreted as negative for anti-WNV IgM antibodies.

detectable neutralizing antibodies to WNV (n = 7). AntiWNV IgM antibodies (P/N ratio 2.0) were detected in all horses with neurological signs and positive PRN antibody titer using M1-22 and in all but one horse using M2B-63. The ELISA based on polyclonal anti-IgM identified anti-WNV IgM in 10 out of 14 animals. The horses with neurological signs and negative PRN titers had P/N ratios <2.0 when sera were measured in the WNV IgM ELISA independent of the anti-IgM antibody used. These animals were considered not exposed to WNV and suffering from other

neurological disorders. All 16 clinical healthy horses with known WNV vaccination history (last vaccination at least 3 months ago) had no detectable WNV IgM antibodies by ELISA. Nine of them had detectable PRN titers. Spearman’s rank correlations (rsp) between the P/N ratios of all 54 samples obtained by the WNV IgM ELISAs indicated high consistency between P/N rankings with rsp = 0.76–0.86 (Fig. 4). Similarly, there was a strong agreement beyond chance between the ELISAs using anti-IgM mAbs when the results were

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Table 3 Concordance beyond change (weighted kappa) between anti-WNV IgM-capture ELISA using two anti-IgM mAbs for testing of horse serum (n = 54) Anti-IgM 2B-63 (P/N ratio)

Anti-IgM 1–22 (P/N ratio) <2 (neg.)

2–3 (wk pos.)

>3 (pos.)

<2 (neg.) 2–5 (wk pos.) >5 (pos.)

40 0 0

1 0 0

0 1 12

Weighted kappa

0.95

Standard error

0.13

neg. = negative, wk pos. = weak-positive, pos. = positive.

The anti-WNV IgM P/N ratios obtained from samples of horses suffering from disease suggested different assay sensitivities depending on the anti-IgM antibody used for the ELISA. To explore the analytical sensitivities of the three assays, the serum samples from these horses were further diluted and compared by ELISA again (Table 6). An increase in analytical sensitivity, indicated by more samples with detectable anti-WNV IgM antibodies (P/N ratios 2.0) at the first two dilutions, was observed for M1-22 and M2B-63 compared to the polyclonal reagent. In addition, M1-22 detected 8/14 positive horses even at the 1:40,000 dilution. The results suggest that the highest analytical sensitivity of anti-WNV IgM detection was obtained with M1-22. Four randomly selected serum samples which had previously been IgM-negative were included in the serum titration comparison. Independent of the anti-IgM reagent used, the P/N ratios were <2.0 for these samples at all dilutions (data not shown). 4. Discussion In this report, we describe the generation of two mAbs to equine IgM and their use to detect WNV-

Fig. 4. Spearman’s rank correlations (rsp) were calculated for three anti-IgM antibodies used in the WNV IgM-capture ELISA. Positive/ negative (P/N) ratios for WNV-specific IgM antibodies were obtained for 54 serum samples. All sera were measured three times by using each of the anti-IgM antibodies for coating of the plates. The 95% confidence intervals for the correlation (CI(r)) are given in each graph.

categorized (Table 3). Comparison of categorized results between the ELISAs using polyclonal and monoclonal anti-IgM antibodies showed moderate agreements (Tables 4 and 5).

Table 4 Concordance beyond change (weighted kappa) between anti-WNV IgM-capture ELISA using a polyclonal goat-anti-horse IgM or the anti-IgM 1-22 mAb for testing of horse sera (n = 54) Polyclonal anti-IgM (P/N ratio)

Anti-IgM 1–22 (P/N ratio) <2 (neg.)

2–3 (wk pos.)

>3 (pos.)

<2 (neg.) 2–3 (wk pos.) >3 (pos.)

40 0 0

1 0 0

3 6 4

Weighted kappa

0.62

Standard error

0.11

neg. = negative, wk pos. = weak-positive, pos. = positive.

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Table 5 Concordance beyond change (weighted kappa) between anti-WNV IgM-capture ELISA using a polyclonal goat-anti-horse IgM or the anti-IgM 2B-63 mAb for testing of horse serum (n = 54) Polyclonal anti-IgM (P/N ratio)

Anti-IgM 2B-63 (P/N ratio) <2 (neg.)

2–5 (wk pos.)

>5 (pos.)

<2 (neg.) 2–3 (wk pos.) >3 (pos.)

41 0 0

1 0 0

2 6 40

Weighted kappa

0.66

Standard error

0.11

neg. = negative, wk pos. = weak-positive, pos. = positive.

specific IgM antibodies in equine serum. Compared to an established IgM-capture ELISA based on a polyclonal anti-IgM antibody, the mAbs improved the detection of anti-WNV IgM. Although all three ELISAs showed moderate to strong correlations, the assays based on anti-IgM mAbs detected low concentrations of WNV-specific IgM antibodies in serum samples that could not be identified with the established assay. The mAb M1-22 had the highest analytical sensitivity for anti-WNV IgM detection in equine serum. Polyclonal antibodies detect several different epitopes on a protein. MAbs detect only one specific epitope. For detecting IgM in serum samples, the recognition of several epitopes does not seem to be a disadvantage for IgM binding. However, we detected some cross-reactivity of the polyclonal anti-IgM with

equine IgG4 (also known as IgGb) which is a major IgG isotype in horse serum (Sheoran et al., 2000; Wagner, 2006). This suggested that a few antibodies with specificity to IgG4 exist in the polyclonal anti-IgM reagent. These antibodies can bind serum IgG4 which is unlikely to contribute to the WNV-specific antibody pool during early infection with WNV (see below). Nevertheless, these anti-IgG antibodies compete for spaces on the ELISA plate and can decrease the amount of IgM-specific antibodies that can bind to the plates. Consequently, a polyclonal reagent with even small cross-reactivity to IgG antibodies reduces the amount of IgM bound to the plate and can decrease the sensitivity of the assay compared to a more specific reagent, such as monoclonal anti-IgM. Cross-reactivity of serum antibodies produced in response to heterologous flavivirus infections in the WNV IgM assay has been described. For human serum samples, an IgM-capture ELISA using WNV recombinant antigen (preM/E) has been evaluated for crossreactivity to other flaviviruses. Infection with St. Louis encephalitis virus (SLEV) resulted in the highest crossreactivity (31% of the serum samples), with lower cross-reactivities identified for individuals infected with Dengue virus (21%) and Japanese encephalitis virus (5%) using a commercial assay (Hogrefe et al., 2004). Due to the lack of systematic testing in horses, it is unknown whether IgM-positive, WNV neutralizing antibody positive samples can be a result of SLEV infection.

Table 6 Comparison of P/N ratios obtained by titration of WNV-IgM-positive sera (n = 14) using the mAbs to IgM or polyclonal anti-IgM for the ELISA

Grey fields indicate negative ELISA results (P/N ratio <2.0).

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The detection of neutralizing antibodies has been considered by some authors to be a specific diagnostic tool for WNV infection in horses (Ostlund et al., 2001; Bunning et al., 2002). However, the detection of neutralizing antibodies is time consuming and for horses with acute neurological disorders, anti-WNV IgM detection by ELISA provides a faster diagnostic alternative for disease. We determined WNV plaque reduction serum neutralization antibodies in all serum samples that were used for the IgM-capture assays. By using the M1-22 mAb, all samples that tested positive in the ELISA had also detectable PRN titers. All samples were submitted in 2002 when WNV emerged in NY State and all of these animals had severe, acute neurological disorders. This strongly suggested that the animals were suffering from WNV infection. All animals with a neurological disorder and no detectable PRN titers tested negative in the IgM ELISA suggesting that the horses had neurological diseases of another origin. Although these data support a strong correlation of neutralizing antibodies and anti-WNV IgM antibodies in horses with acute neurological disease, our data obtained form clinically healthy, vaccinated horses indicate that neutralizing antibodies to WNV cannot generally be used as a gold standard for anti-WNV IgM detection. In most of the previously vaccinated horses, PRN titers could be measured in serum samples in the absence of detectable anti-WNV IgM antibodies. Most likely, IgM and IgG antibodies can both contribute to WNV neutralization titers. IgM is characterized by its rapid onset during acute infection and precedes IgG responses. IgM antibodies to WNV were observed in serum by 1 week after infection (Bunning et al., 2002) and were believed to be detectable for less than 3 months (Ostlund et al., 2000). IgM contributes to the primary antibody-mediated immune response early after infection and most likely also after first vaccination against WNV. Later, IgM antibody titers decrease and the immune response is dominated by long-lasting IgG antibodies. Neutralizing antibody titers do not distinguish between IgM and IgG antibodies and can be found in animals that were previously vaccinated against or infected with WNV. In the absence of clinical signs, neutralizing antibodies only indicate exposure to WNV in the past. Some of the horses in our study were vaccinated longer than a year before sampling and still had detectable neutralizing antibodies against WNV in the circulation. A study by Ostlund et al. also found that neutralizing antibodies, which are characteristic for a secondary immune response dominated by IgG antibodies can last

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for over a year after infection (Ostlund et al., 2001). Another report showed that neutralizing antibody titers in naturally infected horses were maintained for at least 5–7 months, while a decrease in titers was observed at the same time intervals in horses vaccinated with an inactivated vaccine (Davidson et al., 2005). Thus, in the absence of clinical signs, neutralization titers alone can indicate previous exposure by either natural infection or vaccination. Depending on the time point of the sampling, IgM antibodies might already be undetectable in previously exposed horses. Consequently, serum neutralization titers do not necessarily correlate with detectable concentrations of WNV-specific IgM antibodies. In contrast to neutralizing antibodies, antiWNV IgM antibodies provide only a marker for acute infection or recent exposure. Together with neutralizing antibody detection, they are a powerful tool to diagnose WNV disease in horses with acute signs of neurological disorders. In conclusion, anti-equine IgM mAbs improved the sensitivity of the WNV IgM-capture ELISA compared to the established assay using polyclonal anti-IgM. This is of advantage for the detection of low concentrations of WNV-specific IgM antibodies early in infection, monitoring IgM antibodies during the entire period of the primary immune response to WNV, and to identify WNV infection in low responders. Acknowledgements We thank Dr. Douglas Antczak (Baker Institute for Animal Health), Dr. Rodney Page (Department of Clinical Sciences) and Carol Collyer (Equine Park-all at Cornell University) for generously providing the control samples for this study. References Blitvitch, B.J., Bowen, R.A., Marlenee, N.L., Hall, R.A., Bunning, M.L., Beaty, B.J., 2003. Epitope-blocking enzyme-linked immunosorbent assays for detection of West Nile virus antibodies in domestic mammals. J. Clin. Microbiol. 41, 2676–2679. Bunning, M.L., Bowen, R.A., Cropp, C.B., Sullivan, K.G., Davis, B.S., Komar, N., Godsey, M.S., Baker, D., Hettler, D., Holmes, D., Mitchell, C.J., 2002. Experimental infection of horses with West Nile virus and their potential to infect mosquitoes and serve as amplifying host. Emerg. Infect. Dis. 8, 380–386. Castillo-Olivares, J., Wood, J., 2004. West Nile virus infection of horses. Vet. Res. 35, 467–483. Davidson, A.H., Traub-Dargatz, J.L., Rodeheaver, R.M., Ostlund, E.N., Pedersen, D.D., Moorhead, R.G., Stricklin, J.B., Dewell, R.D., Roach, S.D., Long, R.E., Albers, S.J., Callan, R.J., Salman, M.D., 2005. Immunologic responses to West Nile virus in vacci-

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