Development of monoclonal antibodies against spores of Nosema ceranae for the diagnosis of nosemosis

Abstract Type C nosemosis caused by the microsporidian fungus Nosema ceranae is a major disease that affects the beekeeping industry worldwide. Nosemosis is very difficult to diagnose as it lacks characteristic symptoms, and any symptoms, presented by a Nosema spp. infections are indistinguishable from those of other diseases seen in honey bees. For these reasons, a novel method was developed for the rapid and accurate diagnosis of nosemosis. Three monoclonal antibodies (mAbs) were developed from three different monoclones (9A4, 14A8, and 19B2) and were found to be reactive to the whole spores during ELISA. The 9A4-, 14A8-, and 19B2-derived mAbs were suitable for western blot analysis and classified as IgG1, IgG2b, and IgG3, respectively. mAbs manufactured in this study have practical applications for nosemosis rapid diagnostic tests. These novel methods, and the production of such kits, may aid the early detection and treatment of nosemosis, help prevent the indiscriminate and needless use of toxic medicines, and prevent hive collapse and monetary losses for beekeepers.


Introduction
Nosemosis was discovered in 1909 and has since been regarded as a seasonal disease with deleterious effects on beekeeping profits (Fries, 1993;Zander, 1909).Nosema spp.are microsporidian (Corradi & Keeling, 2009;Lee et al., 2008).Nosema apis and Nosema ceranae are parasites of adult honey bees that cause severe colony losses worldwide (Fries et al., 1996;Zander, 1909).Nosemosis caused by N. ceranae is more harmful to honey bee health than that caused by N. apis (Higes et al., 2008).The recently published paper reported that N. apis and N. ceranae are classified in the genera Vairimorpha (Tokarev et al., 2020).
Nosema spp.spores are found in common honey bee colonies which are considered a normal condition when the spores are in small numbers.Due to the prevalence of this pathogen, infection intensity can increase rapidly when the honey bee breeding environment is poor, resulting in a reduced rate of honey production and, potentially, a colony collapse.By the time nosemosis is noticeably severe, the entire hive has already been exposed to large amounts of Nosema spp.spores.The consequences of nosemosis infections may be significantly less severe with the development of a dedicated treatment and diagnostic method.
The diagnosis of nosemosis is difficult as it does not present any characteristic symptoms.Diarrhea is the initial symptom following a Nosema apis infection (Michalczyk & Sokot, 2014).Therefore, beekeepers use remedies against nosemosis when diarrhea is observed.Antimicrobial agent, fumagillin, is used for treating nosemosis (Higes et al., 2010;Johnson et al., 2013).
Various methods have been developed for identifying infected honey bees, such as microscopy for the direct observation of Nosema spp.spores and polymerase chain reaction (PCR) for the detection of Nosema spp.genes.In general, the direct observation method using a microscope is preferred, because it is easier and more convenient (Cantwell, 1970;Kim et al., 2016).The accuracy of microscopic diagnosis is dependent on the quality of the separation process of pure Nosema spp.spores via centrifugation.When separating the Nosema spp.spores from the honey bee, possible contamination with dust or intestinal yeast necessitates that samples undergo several rounds of centrifugation at various rpm settings so that only Nosema spp.spores are isolated and observed.PCR is used for the diagnosis of nosemosis by detecting genes specific to Nosema spp.(Klee et al., 2006;Klee et al., 2007).However, this method is time-consuming, and the accuracy of this test varies depending on the experimental environment (Carletto et al., 2013).
The aim of this study was to develop a rapid and accurate diagnostic method for nosemosis caused by N. ceranae using monoclonal antibodies (mAbs) specific to N. ceranae spores proteins.If our diagnostic method is successfully established, it is expected that our method, which is faster than PCR and is economical because it does not require expensive equipment, can be applied for the rapid diagnosis of nosemosis.

Materials and methods
Isolation and identification of Nosema spp.spores N. ceranae-infected honey bees were purchased from an apiary in Chungbuk province, Republic of Korea.Nosema spp.spores were isolated using procedures described previously (Gisder et al., 2011).Following this method, the midgut of honey bees was rinsed with phosphate-buffered saline (PBS) and homogenized using a tissue grinder.Large particles were removed using a 70 lm mesh filter.The filtered suspension was centrifuged at 250 and 400 rcf for 10 min each.Spores species were identified using previously described PCR methods (Genersch et al., 2010;Gisder et al., 2010;Kim et al., 2016) with specific primers for N. ceranae (sense strand: 5 0 -CGGATAAAAGAGTCCGTTACC-3 0 , antisense strand: 5 0 -TGAGCAGGGTTCTAGGGAT-3 0 ) (Bioneer Co., Daejeon, Republic of Korea).Each PCR was preheated to 94 C for 2 min, followed by 94 C for 15 s, 60 C for 30 s, and 72 C for 45 s, with a final extension phase at 72 C for 7 min.A variable number of cycles were used to ensure that amplification occurred in the linear phase.PCR products were separated on a 1.5% agarose gel and visualized by ethidium bromide staining and UV irradiation.

Preparation of N. ceranae spores antigens
The spores of N. ceranae were isolated, counted under a microscope, and heat inactivated (95 C for 1 h).For the preparation of antigens, PBS suspended spores were stored on ice until further use.

Immunization of mice
Immunization of mice (n ¼ 4) for mAbs is described in Online Supplementary Data (Supplementary Table S1).BALB/c (6-8 weeks old, female) mice were intraperitoneally (250 lL) and subcutaneously (250 lL) injected with 1 Â 10 9 N. ceranae spores emulsified in Freund's complete adjuvant (Sigma-Aldrich, MO, USA).After a 2-week interval, the mice were boosted with 1 Â 10 9 spores emulsified in Freund's incomplete adjuvant.After 3 and 5 weeks, the serum antibody titers were measured using enzyme-linked immunosorbent assay (ELISA), through antigen detection.When diluted sera (10 À3 ) reacted positively with the whole spores antigen in the ELISA, the mice exhibiting an OD > 2 were selected for fusion.Three days before fusion, selected mice were boosted again with 1 Â 10 9 spores through intraperitoneal injections.All procedures were approved by the Ethical Committee of Chungbuk National University (CBNUA-1016-16-01).

Cell preparation and fusion
Spleens were obtained from selected mice and transferred into Roswell Park Memorial Institute medium (Hyclone Laboratories, UT, USA) supplemented with 10% fetal bovine serum (FBS Hyclone Laboratories), 100 U/mL penicillin (Gibco BRL, NY, USA), and 100 mg/mL streptomycin (Gibco BRL).Spleen cells were isolated by gently pressing the excised spleen through a cell strainer (70 mm) and collected by rinsing the cell strainer with the media described above.Sp2/0-Ag14 cells (myeloma cells) were cultured in Dulbecco's Modified Eagle's Medium (Hyclone Laboratories, UT) supplemented with 10% FBS, 100 U/mL penicillin, and 100 mg/mL streptomycin and used for fusion.
Isolated spleen cells of immunized mice and Sp2/ 0-Ag14 cells were fused at a 10:1 ratio in the presence of polyethylene glycol (MW.1450, Sigma-Aldrich).Fused cells were cultured at 37 ± 0.5 C under 5% CO 2 conditions in hypoxanthine-aminopterin-thymidine medium (2 mM of 2% L-glutamine, 20% FBS, 100 U/mL penicillin, and 100 lg/mL of streptomycin).After 10 days, clones were identified using an inverted microscope (CKX41, Olympus, Tokyo, Japan).Individual clones were suspended in culture media and divided among 96-well, round bottom, plates so that there was $1 cell and 150 lL per well.Samples were incubated at 37 C.After 2 days, ELISA was performed on immunoglobulin G (IgG) antibodies using the supernatant of 985 wells that contained clones.mAb-producing clones were then isolated.

Reactivity of mAbs
The reactivity of mAbs against N. ceranae antigens was analyzed according to the ELISA methodology of Anuracpreeda et al. (Anuracpreeda et al., 2009).Wells of microtiter plates (Sigma-Aldrich) were coated with whole spores or spores lysate of the N. ceranae antigen, diluted in carbonate/bicarbonate coating buffer (15 mM Na 2 CO 3 , 35 mM NaHCO 3 , pH 9.6).After a 2 h incubation period at 37 C, the microtiter plate was washed three times with ELISA buffer (0.05% Tween-20 in PBS).Next, 100 lL of blocking buffer, consisting of the ELISA buffer and 0.5% bovine serum albumin (Sigma-Aldrich), was added to each well and incubated (37 C for 1 h).After washing, 50 lL mAb (or undiluted culture supernatants of hybridoma) was added and incubated (37 C for 2 h).The plate was washed again and 50 lL horseradish peroxidase (HRP) conjugated goat anti-mouse Ig (immunoglobulin) was added and incubated (37 C for 1 h).After rewashing the plate, 50 lL of 3,3 0 ,5,5 0 -tetramethyl benzidine (TMB, KPL, Gaithersburg, USA) was added to each well; the reaction then continued in the dark for 10 min at 25 C.The reaction was halted with 50 lL stop solution (1 N HCl).A microplate reader (450 nm, Bio-Rad Laboratories, Hercules, CA, USA) detected the optical densities (OD).

Purification and isotyping of mAb
Anti-Nosema mAbs were crudely isolated from culture supernatants (hybridoma or mouse ascites) through 50% saturated ammonium sulphate precipitation.The light chain composition and mAb isotypes were determined with an isotyping kit (Roche Diagnostics, Basel, Switzerland).Affinity chromatography further purified mAb, applying Staphylococcus aureus protein A (Pansorbin Cells; Sigma-Aldrich, and Beckman Instruments, Fullerton, CA, USA) or Sepharose-conjugated anti-mouse IgM (Zymed Laboratories, South San Francisco, CA, USA) columns.

SDS-PAGE and Western blotting
The SDS-PAGE was performed following the Laemmli method (Laemmli, 1970).The gel was stained with Coomassie brilliant blue (Bio-Rad Laboratories).Proteins separated by 12% or 15% acrylamide gel were transferred to polyvinylidene fluoride membranes (Immobilon-P, Millipore, Billerica, MA, USA).The membranes were incubated for 1 h with Tris-buffered saline (10 mM Tris-Cl, pH 7.4), containing 0.5% Tween 20 and 5% non-fat dry milk, which acted as a blocking solution.Membranes were then probed with the primary antibody (or hybridoma culture supernatant) in a blocking solution overnight.The membranes were subsequently washed and incubated with the developing HRP-conjugated second antibody for 1 h.The protein bands were developed by enhanced chemiluminescence (Amersham Pharmacia, Biotech, Piscataway, NJ, USA).Amoeba (Malpighamoeba mellificae) lysate protein was used as a control antigen of mAb to Nosema spp.spores.

Statistical analysis
SPSS (version 17.0; SPSS Inc., Chicago, IL, USA) was used for data analysis.Results are expressed as mean ± standard deviation (SD).Statistical analyses were performed using one-way analysis of variance, and Dunnett's post-hoc test was used to determine the significance.Statistical significance is indicated by Ã or # in the figures.

Comparison of antibody titer in immunized mice
Before the second (Figure 1) and third (Figure 2) boosting, a comparative serum antibody titer was analyzed against whole spores and spores lysate of N. ceranae.The sera of mice reacted more strongly with whole spores (1 Â 10 5 cells) than with the spores lysate (300 ng/mL) (Figure 1).Due to the similar results of each antibody, the antigen binding specificity between antibodies could not be compared.
When compared with mice that received only one booster injection (Figure 1), twice-boosted mice exhibited stronger reactions to the whole spores antigen (Figure 2A).Among all the twice-boosted mice, the lowest dilution (1:100) elicited the highest level of reactivity to the whole spores antigen (Figure 2A).The OD values of all immunized sera reached 2, even at a 1:1000 dilution.Antibody response increased with the number of spores.On the second bleed, all mice (n ¼ 4) showed higher reactivity with whole spores (Figure 2A) than with spores lysate (Figure 2B); Figure 1 further illustrates this point.Antibodies did not react to the lysate antigen (OD 0.6).

Generation of hybridoma and characterization of mAbs
In our study, about 985 hybridomas were screened; 27 hybridomas reacted positively with the antigen.Among these 27, three monoclones (9A4, 14A8, and 19B2) had acceptable optical absorbance; the antibody produced by 14A8 reacted most strongly with the spores antigen of N. ceranae (Supplementary Figure S1).The well H12 of each plate represents the positive control.Selected hybridomas were cloned by limiting dilution culture in 96-well plates and rescreened using ELISA.As shown in Online Supplementary Data (Supplementary Table S2), three monoclones had acceptable optical absorbance at 450 nm.

Mass production and isotyping of mAbs
Cloned hybridoma was grown and injected into the peritoneal cavity of the mouse.After 10 days, mAbs were isolated from ascetic-fluid and the isotypes were confirmed.Table 1 represents the isotype determination of mAbs against all three different types of antibodies, especially those bound only to N. ceranae spores.mAbs 9A4 and 14A8 showed IgG1 and IgG2b sub-class, respectively; 19B2 derived mAb showed IgG3 sub-class.mAbs 9A4 and 14A8 exhibited significantly higher OD values than 19B2.Each mAb had a kappa-type light chain.

Confirmation of antigen and immunoglobulin protein using SDS-PAGE
N. ceranae spores lysate analyzed by 12% acrylamide gel SDS-PAGE showed a prominent protein $75 kDa (Figure 3A).In contrast, proteins with molecular weights <10 kDa were detected when analyzed with 15% acrylamide gel SDS-PAGE (Figure 3B).The hybridoma cells 9A4, 14A8, and 19B2 exhibited a $50 kDa heavy chain fragment and a $25 kDa light chain fragment (Figure 3C); this is similar to what you would expect from an IgG subclass.

Evaluation of mAbs suitability for Western blot analysis
Among the three selected mAbs, 9A4 derived mAb recognized antigens of $75 kDa; however, 14A8 and 19B2 derived mAbs recognized antigens with molecular weights $15 kDa (Figure 3D).
To demonstrate the specificity of mAb to N. ceranae spores, amoeba (Malpighamoeba mellificae) lysates were used as a similar antigen of Nosema spp.All three different antibodies (9A4, 14A8, and 19B2) are bound solely to N. ceranae antigens (Figure 3E).

Discussion
Nosemosis is widespread among honey bees; however, it is rarely diagnosed accurately by beekeepers before administering an anti-Nosema drug in countries where anti-Nosema drugs are legal (Klee et al., 2007;Michalczyk & Sokot, 2014).Therefore, this study was designed to develop a more accurate and practical diagnostic method for N. ceranae infection.
In this study, a diagnostic method using mAbs was developed for N. ceranae infection.The mAb production technique developed by Kohler and  The serum antibody titers of blood were measured using ELISA against multiple concentrations of whole spores and spores lysate, where (A) represents 1 Â 10 4 (white) and 1 Â 10 5 (grey), and (B) represents 100 ng (white), and 300 ng (grey).The data (mean OD ± SD) are collected from three, independent, experiments, each performed in triplicate.Ã p < 0.05, ÃÃ p < 0.01 as compared to the result of 1 Â 10 4 spores.# p < 0.05, ## p < 0.01 as compared to the result of 100 ng.
Milstein has greatly improved our ability to identify microsporidian species and rapidly diagnose pathogens (Kohler & Milstein, 1975).Among the 985 hybridomas, three monoclones (9A4, 14A8, and 19B2) exhibited acceptable reactivity to the antigens, while the mAbs produced from these monoclones exhibited a strong reactivity.These three different mAbs against N. ceranae spores were developed and assessed for the identification of N. ceranae using ELISA and western blotting.
An ELISA method using mAbs for detecting whole spores of Nosema bombycis (N.bombycis) has been previously developed (Greenstone, 1983;Qian et al., 1986); however, the level of sensitivity for these antigens was very low.Kawarabata and Hayasaka also developed a more sensitive indirect ELISA method that can detect as little as 600 ng of soluble proteins of N. bombycis spores (Kawarabata & Hayasaka, 1987).Aronstein et al. (2013) reported an ELISAbased method to detect the SWP-32 protein of N. ceranae using polyclonal antibody and also compared its sensitivity to that of the PCR-based method.Although the ELISA method using the polyclonal antibody for SWP-32 is not clearly superior to the PCR method in terms of diagnosing nosemosis, it simplified the process of sample preparation.mAbs have a higher specificity for antigen detection than polyclonal antibodies; hence, the probability of false positives is very low when used to diagnose diseases.The mAbs produced in this study detected 1 Â 10 4 N. ceranae spores in the ELISA.In the future, we aim to develop a sandwich ELISA-based rapid test kit for N. ceranae using the antibodies developed in this study for more sensitive detection.Sandwich ELISA is method for quantification of antigens between two layers of antibodies (i.e., capture and detection antibody).
In western blotting, the 9A4 derived mAb recognized antigens around 75 kDa; however, the 14A8 and 19B2 derived mAbs recognized antigens around 15 kDa (Figure 3D).This difference can be attributed to each antibody recognizing different antigens because the whole spores were used for immunization in mice.The mAbs (9A4, 14A8, and 19B2) enabled the detection of <1 lg of N. ceranae spores lysate via western blotting.The total amount of protein that can be obtained from <1 Â 10 4 spores is 1 lg.Considering that an infected bee contains >5 Â 10 6 spores, a 1 lg detection sensitivity will enable detection of a low level of N. ceranae infection (Aronstein et al., 2013).The binding specificity of the antibody to N. ceranae was confirmed using a honey bee-targeting pathogen (amoeba) as a negative control.Other Nosema spp., such as N. apis, would have made for a good comparison; however, the other Nosema spp.are rarely found in the beehives of the Republic of Korea.
Microscopic observation of Nosema spp.spores or PCR detection of Nosema spp.genes are the common methods employed for the diagnosis of Nosema spp.infection.In this study, microscopic observation was used as the preliminary method to identify the severity of N. ceranae infection.The severity of a colony's Nosema spp.infection is commonly determined by the average number of spores per honey bee in pooled samples (Cantwell, 1970;Kim et al., 2016).The infection intensity may also be determined through the ratio of infected honey bees to the total amount of honey bees in the colony (Botias et al., 2012;Doull, 1965;Higes et al., 2008;L'Arrivee, 1963).Although these methods each have their uses (Fries et al., 1984), neither of these detection methods can detect spores in small numbers (Higes et al., 2008).The mAbs for N. ceranae in this study enabled the detection of 1 Â 10 4 N. ceranae spores via ELISA and 1 lg/lane of N. ceranae spores lysate via western blotting.The detection limits of these antibodies need further assessment.
Nosemosis control is difficult because less sensitive diagnostic methods diagnose weakly infected colonies as uninfected.Several stronger molecular methods have been developed to increase the sensitivity and accuracy of these diagnostic tests (Klee et al., 2006(Klee et al., , 2007)).Molecular methods can detect asymptomatic infection, caused by an infection of only a small number of spores.As a result, the presence of Nosema spp.spores in honey bees can be confirmed by PCR even if they have not been observed under a microscope.However, PCR is greatly influenced by the experimental conditions and the environment.Low-sensitivity primers can result in false positives due to false amplification reactions with template genes.In addition, PCR efficiencies may differ due to PCR conditions (e.g., temperatures) and chemicals used (Derveaux et al., 2010).For these reasons, it is recommended that each laboratory optimizes and adjusts the PCR protocol to suit their laboratory's conditions.However, compared to that of the PCR method, the efficiency of diagnostic methods using mAbs is less influenced by the experimental environment (Aronstein et al., 2013).
In summary, our results suggest a new diagnostic method for nosemosis using mAbs.The strong reactivity of these mAbs against the antigens was demonstrated using ELISA and western blotting.We expect to develop a rapid detection kit for nosemosis using the antibodies evaluated in this study.This will aid in the early detection and treatment of nosemosis, prevent the indiscriminate and unnecessary use of medicines toxic to humans, prevent hive collapse, and alleviate monetary losses for beekeepers.

Figure 1 .
Figure1.Comparative titration of serum antibody levels in BALB/c (6-8 weeks old, female) mice (n ¼ 4) after the first boosting injection (First screening of antibody titer in serums of immunized mice).The serum antibody titers of blood, collected 1 week after the first boost, were measured using ELISA against whole spores (white) and spores lysate (grey).The data (mean OD ± SD) are collected from three, independent, experiments, each performed in triplicate.Ã p < 0.05, ÃÃ p < 0.01 as compared to the result of whole spores.

Figure 2 .
Figure 2. Comparative titration of serum antibody levels in BALB/c (6-8 weeks old, female) mice (n ¼ 4) following the second boosting injection.(Second screening of antibody titer in the serum of immunized mice).The serum antibody titers of blood were measured using ELISA against multiple concentrations of whole spores and spores lysate, where (A) represents 1 Â 10 4 (white) and 1 Â 10 5 (grey), and (B) represents 100 ng (white), and 300 ng (grey).The data (mean OD ± SD) are collected from three, independent, experiments, each performed in triplicate.Ã p < 0.05, ÃÃ p < 0.01 as compared to the result of 1 Â 10 4 spores.# p < 0.05, ## p < 0.01 as compared to the result of 100 ng.

Figure 3 .
Figure 3. Confirmation of antigen, immunoglobulin proteins, and suitability of mAbs.Spores lysate were separated by 12 (A) and 15% (B) acrylamide gel.Three different hybridoma cells (9A4, 14A8, 19B2) derived mAbs were analyzed by SDS-PAGE using 12% acrylamide gel (C).Each acrylamide gel was stained with Coomassie brilliant blue.Purified immunoglobulin (1 lg/mL) derived from ascites were evaluated as a primary antibody, and HRP-conjugated anti-mouse IgG (1:1000 dilution) were used as a second antibody (D and E).All the experiments were repeated more than three times and representative data are presented.

Table 1 .
Comparison of antibody titer and isotyping of selected clones.Data shown are representative of three independent experiments.Bold character values indicate meaningful results.