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Evaluation of carboxyl beads based latex agglutination test for rapid sero-diagnosis of Japanese encephalitis
Biologicals 62 (2019) 72–76
Contents lists available at ScienceDirect
Biologicals journal homepage: www.elsevier.com/locate/biologicals
Evaluation of carboxyl beads based latex agglutination test for rapid serodiagnosis of Japanese encephalitis
T
M.R. Gracea,1, Jyotsana Chauhana,1, M. Suman Kumarb, Ashok Kumarc, K.N. Bhilegaonkara, Mithilesh Singha, Himani Dhanzea,∗ a
ICAR- Indian Veterinary Research Institute, India ICAR-Central Institute of Research on Goats, India c Indian Council of Agricultural Research, New Delhi, India b
A R T I C LE I N FO
A B S T R A C T
Keywords: Latex agglutination test Covalent coupling Japanese encephalitis
Japanese encephalitis (JE) is a major public health problem in the South Asian countries including India. Pigs serve as a relevant sentinel model, the surveillance of which could predict a potential JE outbreak in human population nearby. However, existing serological detection methods like Enzyme-Linked Immuno Sorbent Assay (ELISA), virus neutralization test (VNT) and Haemagglutination Inhibition (HI) require elaborative laboratory facilities which are invariably not available in field conditions. Recognizing the lacunae, attempts were made to develop recombinant antigen (rNS1) based latex agglutination test (LAT) as a rapid on-site test using covalent coupling method. Four different formats were evaluated using different coupling buffers, blocking buffers and reaction conditions. The format in which borate buffer at alkaline pH (8.5) was used for coupling of antigen with carboxylated beads followed by blocking with skimmed milk powder was found to be the best amongst all. Developed latex based test was used for screening of 207 pig serum samples for JE which revealed relative diagnostic sensitivity and specificity of 80.2% and 95.2%, respectively in comparison with indirect IgG ELISA. Hence, the present study demonstrated that covalently coupled recombinant antigen based LAT could be used as a reliable screening test for surveillance of JE in pigs under field conditions.
1. Introduction Japanese encephalitis (JE) is a mosquito-borne flaviviral zoonotic disease, which is one of the leading forms of viral encephalitis worldwide. Pigs are the amplifying host of JE virus (JEV) and in developing countries domestic pig rearing is an important risk factor for the transmission of JEV to humans. There are many unorganized piggeries and the rural populations who rear pig lives in close proximity with these animals, which has created an enabling ecosystem for transmission of JE. Pigs also serve as a relevant sentinel model, the surveillance of which could predict a potential JE outbreak in human population nearby. Hence, there is need for the development of a screening test that may be suitable for surveillance of JE in pig population. Sero-diagnosis is generally preferred over the other methods for diagnosis of JE. Even though there are many established serological methods such as ELISA, the application of these assays in field conditions is limited due to requirements of laboratory operations, skilled technicians and special equipment/facilities. Thus, the development of
an on-site, rapid and specific assay is essentially required for the surveillance of JEV infection in pig population. Latex agglutination test (LAT) is simple, rapid, cost-effective and easy to perform test, which has been widely employed as rapid on-site diagnostic assay for several diseases both in veterinary and medical field [1–5]. Earlier, we developed LAT based on physical adsorption for sero-diagnosis of JE in pigs but the shelf life of coupled beads was found to be less than 40 days [6]. Other researchers have also reported reduction in the activity of latex beads sensitized by physical adsorption due to the partial desorption of adsorbed protein that normally occurs during its storage [1]. Covalent coupling of LAT is an alternative method and different authors have cited advantages of covalent coupling over physical adsorption, the major one being the permanent nature of covalent attachment thus increasing the shelf life of the beads and also reducing the false positive results [1,4,7]. Considering the advantages of covalent coupling over physical adsorption, efforts were made to develop carboxyl beads based LAT which can be easily applied in field settings for sero-surveillance of JEV in pig population.
∗
Corresponding author. E-mail address: [email protected] (H. Dhanze). 1 Both authors have contributed equally to the work done. https://doi.org/10.1016/j.biologicals.2019.09.003 Received 30 July 2019; Received in revised form 4 September 2019; Accepted 5 September 2019 Available online 10 September 2019 1045-1056/ © 2019 International Alliance for Biological Standardization. Published by Elsevier Ltd. All rights reserved.
Biologicals 62 (2019) 72–76
M.R. Grace, et al.
2.2. Preparation of the antigen
Format IV
Standardization and optimization of reaction conditions for labeling of rNS1 protein with latex beads was done using covalent coupling in four different formats (Table 1).
73
Format I
MES pH 5.0 1. 1% Bovine serum albumin (BSA) in PBS 2. 5% Skimmed Milk Powder (SMP) in PBS-T Activation of beads followed by incubation of protein Not used Coupling buffer along with pH Blocking buffer
2.4.2. Format II In this format, the protein was pre-adsorbed on latex beads using carbonate buffer, followed by the activation of latex beads using EDAC in MES buffer. Briefly, the carboxylate modified latex beads were washed and resuspended in carbonate buffer (pH 9.5). The beads were pre adsorbed with rNS1 antigen in different concentrations (60 μg per 2.5 mg bead and 120 μg per 2.5 mg bead) using PBS. Further, the protein-bead mixture was washed and resuspended using MES buffer and the carboxyl groups on the beads were activated by the addition of EDAC in MES buffer in different concentrations (as mentioned in format I) and incubated. The incubated beads were washed and resuspended using 1% glycine in PBS as blocking and storage buffer and stored at 4 °C after sonication.
Reaction conditions
Table 1 Reaction conditions used in different LAT formats.
Format II
2.4.1. Format I Briefly, carboxylate modified 1 μ sized 2.5% blue dyed latex beads (Polyscience, Inc) were washed and resuspended in MES buffer (pH 5.0). The carboxyl groups on the beads were activated by the addition of N-(3-Dimethyl amino propyl)-Ń- ethylcarbodiimide hydrochloride (EDAC, Sigma) in MES buffer in different concentrations (50 mg/ml, 70 mg/ml and 100 mg/ml). Further, the beads were coated with rNS1 antigen in two different concentrations of 60 μg per 2.5 mg bead and 120 μg per 2.5 mg bead using PBS. The protein was incubated with the beads at 4 °C for two different time periods, 6 h and overnight. Further, the protein-bead mixture was centrifuged and washed. For finding the ideal blocking agent, the pelleted beads were re-suspended in different blocking buffers, 1% bovine serum albumin (BSA) in PBS and 5% Skimmed Milk Powder (SMP) in PBS-T (0.05% tween-20). Two different blocking conditions including overnight incubation with continuous mixing at 4 °C and 1 h incubation at room temperature with continuous mixing were tried. Latex beads were centrifuged and resuspended in storage buffer and stored at 4 °C until used. The storage solution used was either 0.1% BSA in PBS or 0.5% SMP in 0.01% PBST in accordance with the blocking agent used.
Use of quenching agent
2.4. Standardization of latex agglutination test
Carbonate Buffer pH 9.5 1% glycine in PBS
Format III
The hyperimmune serum was raised in rabbits against rNS1protein after getting due permission from the Institute Animal Ethics Committee.
Carbonate Buffer pH 9.5 5% SMP in PBST
2.3. Hyperimmune serum
Order of reaction
JEV non-structural recombinant protein (rNS1) previously expressed and purified in the Division of Veterinary Public Health, ICARIndian Veterinary Research Institute [8] was selected as antigen for coating onto the latex beads. Recombinant NS1 protein was produced in bulk and purified by nickel chelating affinity chromatography using imidazole gradient method. The purified protein was concentrated using Merck Millipore 30 K centrifugal filters and the concentration was estimated to be 750 μg/ml by Bradford reagent. The protein was stored at −80 °C till further use.
Pre adsorption of protein followed by activation 40 mM ethanolamine
A total of 207 pig serum samples collected from endemic areas of India were used in the present study.
Pre adsorption of protein followed by activation Not used
2.1. Serum samples
Borate Buffer pH 8.5 & 6.2 1. BSA in 0.2 M Borate buffer (pH 8.5 and 6.2) 2. SMP in 0.2 M Borate buffer (pH 8.5 and 6.2) Activation of beads followed by incubation of protein 40 mM ethanolamine
2. Materials and methods
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but fluid is slightly opaque was graded as moderately positive (++); reactions wherein about 50% latex beads agglutinate but the liquid is opaque was graded as weak positive (+) and reactions where the mixture remains homogenous was graded as negative.
2.4.3. Format III In this format the protein was pre-adsorbed on latex beads using carbonate buffer followed by the activation of latex beads using EDAC in MES buffer as in format II, followed by quenching and blocking. Quenching was done to remove the unused active carboxyl sites by adding a quenching solution of 40 mM ethanolamine to the pelleted beads. Blocking was done using 5% SMP in PBST and beads were stored in storage buffer (0.5% SMP in PBS-T) at 4 °C after sonication.
2.6. Diagnostic efficacy of LAT A total of 207 field pig serum samples were tested by in-house rNS1 protein based indirect IgG ELISA [8] and the results were compared with the findings of LAT to assess the diagnostic efficacy of LAT and its applicability in field conditions. The relative diagnostic sensitivity, specificity and predictive value of LAT in comparison with ELISA were calculated as described earlier by Thrusfield [10].
2.4.4. Format IV In this format, the latex beads were activated using EDAC and NHydroxysulfosuccinimide sodium salt (sulfo–NHS) in MES buffer followed by binding of the protein in borate buffer at two different pH (pH 8.5 and 6.2), quenching and blocking. Two types of latex beads were used, carboxylated modified 1.0 μ sized blue dyed microspheres (2.5% solids) (Polyscience, Inc.) and 0.9 μ (10% solids) undyed carboxylate modified microspheres (Sigma-Aldrich). Briefly, the latex beads were washed and resuspended using carbonate buffer (pH 9.5). The beads were washed and resuspended again using MES buffer (pH 5.0) to obtain a final concentration of 2.5% for both the beads. Further, the carboxyl groups on the beads were activated by the addition of EDAC and Sulfo-NHS (Sigma-Aldrich) in MES buffer dropwise to the uniformly resuspended beads, followed by incubation at room temperature for 15 min with continuous mixing. Different concentrations of EDAC were used (50 mg/ml, 70 mg/ml and 100 mg/ml) and sulfo-NHS was added at the concentration of 11 mg/ml. The unused EDAC and sulfoNHS were removed by centrifugation and the pelleted beads were washed and resuspended in 0.2 M Borate buffer at two different pH (pH 8.5 and 6.2). The beads were immediately treated with rNS1 antigen in different concentrations (60 μg per 2.5 mg bead, 90 μg per 2.5 mg bead, 120 μg per 2.5 mg bead, 150 μg per 2.5 mg bead, 180 μg per 2.5 mg bead and 300 μg per 2.5 mg bead). The protein was incubated with the beads overnight at 4 °C with continuous mixing. After incubation, the protein-bead mixture was centrifuged as before and the beads were resuspended in 0.2 M Borate buffer (pH 8.5 and 6.2). The coupling chemistry was stopped by adding ethanolamine in concentration of 40 mM at different volume (10 μl, 30 μl, 50 μl, 70 μl and 100 μl) to the pelleted beads and incubated for 30 min with continuous mixing at 4 °C. Further, to determine the ideal blocking agent, the pelleted beads were resuspended in two different blocking buffers, 1% BSA in 0.2 M Borate buffer (pH 8.5 and 6.2) or 5% SMP in 0.2 M Borate buffer (pH 8.5 and 6.2). Latex beads were centrifuged thrice as before using the respective blocking buffer and resuspended as a 2.5% suspension in storage buffer. The storage solutions used were either 0.1% BSA in PBS or 0.5% SMP in 0.01% PBST in accordance with the blocking agent used. Further, the beads were sonicated at 12 μm amplitude for 5 cycles, 2 s each and stored at 4 °C until used.
2.7. Storage life of the latex beads The storage life of the beads was evaluated by storing the beads at 4 °C with periodical testing for sensitivity and specificity using a panel of known positive and negative pig serum samples. 3. Results Latex agglutination test was standardized using rNS1 protein as antigen by covalent coupling onto carboxylate modified latex beads for sero-diagnosis of JE in pigs. 3.1. Format I In this format, wherein the activated beads were incubated with protein at acidic pH for covalent binding, all the combinations of reagents and reaction conditions tried failed to give any agglutination even with the undiluted hyperimmune serum. 3.2. Format II In this format, the protein was pre-adsorbed onto latex beads followed by activation of the beads and storage in glycine buffer. Addition of protein at the rate of 120 μg per 2.5 mg bead and use of EDAC at 50 mg/ml yielded agglutination reaction in both the hyperimmune serum and positive serum. However, the specificity of the format was low as the beads showed agglutination with known negative field pig serum samples also. 3.3. Format III In this format, quenching with 40 mM ethanolamine and blocking using 5% SMP in PBST was tried to minimize the non-specific reactions of format II. But this format failed to give any agglutination reaction even with the undiluted hyperimmune serum.
2.5. Suitability of LAT for field testing
3.4. Format IV
A panel of confirmed JE positive and JE negative serum samples was prepared by testing the collected pig serum samples using virus neutralization test [9]. The LAT was performed on glass slides using serial dilutions of hyperimmune serum, the panel of confirmed JE positive pig serum, JE negative pig serum and PBS. Three different reaction volume of rNS1 coupled beads and serum (5 μl beads and 5 μl serum; 5 μl beads and 10 μl serum and 10 μl beads and 10 μl serum) were tried. The slide was rocked briefly for 2 min to mix the coated beads and serum samples. The reaction was read at three different time periods (5, 10 and 15 min) and the conditions giving best visual perception of agglutination were selected. The result of the test was graded according to the degree of agglutination reaction. The reactions where the mixture became completely transparent with strongly agglutinated particles, which tend to settle at the edge of the beads-serum mixtures was graded as strong positive (+ + +); reactions wherein most of the latex particles have agglutinated
This format gave the best results amongst the various formats tried in this study. The reaction conditions which gave the best visual agglutination reaction are described here. Among the different concentrations of EDAC used, the protocol using double the amount of the weight of beads was found to be the best. There was no appreciable change in the sensitivity and specificity due to use of Sulfo-NHS in the present format. Among the different pH of borate buffer used, the protocol using alkaline pH (8.5) gave better agglutination reactions (up to 1:40 dilution of hyperimmune serum) as compared to acidic pH (up to 1:10 dilution). Sensitivity of covalent LAT increased with increasing protein concentration up to 150 μg per 2.5 mg bead. No improvement in the sensitivity was observed when the protein concentration was further increased. Ethanolamine, when used as quenching agent was found to be most effective at 40 mM concentration and 70 μl volume. Among 74
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covalent chemistry was developed for sero-diagnosis of JE in pigs. Covalent coupling employs specialized beads having functional groups incorporated on the bead surface, which undergoes covalent binding with the protein. Though various reactive groups are employed, the most commonly used is carboxylated beads [7]. In general, for visual slide agglutination the particle diameter range is 0.2–0.9 μm [7]. After perusal of literature, carboxylated latex beads of diameter 0.9 μm and 1 μm were employed in the present study. In principle, the amine groups of proteins can directly be coupled onto the carboxyl groups on the latex bead surface. Since such reactions are too slow, it is convenient to transform the carboxyl groups into the faster acylureas by addition of an activator [11]. Hence, in the present study N-(3-Dimethyl amino propyl)–Ń- ethylcarbodiimide hydrochloride (EDAC) was employed. This activation occurs at a slightly acidic pH and thus 100 mM MES buffer (pH 5) was opted for the same. In the format I, beads failed to react even with undiluted hyperimmune serum which might be due to the acidic pH of the coupling buffer. The sensitized beads in the format II gave agglutination up to 1:10 dilution of hyperimmune serum. But the specificity of the test was low with a high proportion of false positive reactions. Though glycine has been reported as the best blocking agent by other researchers [1], in the present study it was found to cause non specific agglutination. Format III was modified to reduce the number of false positives by using quenching and blocking agents. However, this modified format failed to give agglutination reaction even with the undiluted hyperimmune serum. It was concluded that the positive agglutination reactions in format II might be due to the activity of glycine. When SMP was used instead of glycine the beads failed to agglutinate even with undiluted hyperimmune serum. In format IV, the activated latex beads were incubated with rNS1 protein in borate buffer at two different pH (pH 8.5 and 6.2), followed by quenching and blocking. We observed that the covalent coupling was better at alkaline pH (8.5). This is in accordance with the findings of other researchers [12], who concluded that the covalent coupling of protein to carboxylated latexes using carbodiimide chemistry should be performed at basic pH, where a good colloidal stability can be expected as carbodiimide latex complexes have low colloidal stability at pH near to isoelectric point of protein. Addition of Sulfo-NHS stabilizes the amine-reactive intermediate by converting it to an amine-reactive Sulfo-NHS ester [13]. Thus, Sulfo-NHS was also tried at the concentration of 11 mg/ml in this format but there was no appreciable change in the sensitivity and specificity. There was no improvement in the sensitivity when protein concentration was increased from 150 μg per 2.5 mg bead to 180 μg and 300 μg per 2.5 mg bead. This might be due to the fact that the ratio between the total-linked protein and the total added protein decreases when increased amount of protein is added, owing to the saturation of particle surface [2]. The number of false positive reactions was more when 1% BSA in 0.2 M borate buffer was used as blocking agent as compared to 5% SMP in 0.2 M Borate buffer. This has similarity with an earlier report [3] wherein the BSA-blocked latex beads caused non-specific reactions. Since BSA is a serum protein, in certain circumstances it could cause non-specific signals [14]. One of the major disadvantages of EDAC is that it causes non specific cross linking resulting in clump formation which persisted even after vigorous pipetting and vortexing. Therefore, a brief sonication of the beads prior to storage was done for uniformly dispersing the latex beads and thus reduced the difficulty in interpreting the results. The ideal reaction condition was the one in which 5 μl beads at 2.5% concentration and 10 μl serum were reacted for a period of 5 min. When the reaction time was increased, there was difficulty in interpreting the result due to settling of the particles and drying of the mixture leading to false positive interpretations. The immuno-agglutination assay does not reach an end point and hence reaction time analysis is an important factor to consider when optimizing an assay. Optimizing concentration of beads is also an important factor in optimizing the latex
Fig. 1. LAT by covalent coupling. (a) Positive control. (b) Negative control.
the various blocking agents tried, 5% SMP in 0.2 M borate buffer (pH 8.5) at room temperature gave the least number of false positive reactions. The specificity of the test increased when blocking time was increased from 30 min to 2 h at room temperature. The best visual agglutination was observed when 5 μl beads and 10 μl serum was reacted (Fig. 1). The reaction time up to 5 min was found to be optimum, with longer time leading to settling and drying of the mixture. Carboxylated latex beads of 0.9 μm mean particle size (10% solids) (Sigma-Aldrich) were tried to improve the sensitivity of the test. Since the beads were colorless, the visibility of agglutination reaction was poor. Hence, it was concluded that carboxylate modified 1.0 μ sized blue dyed microspheres (Polyscience, Inc.) are better for visualization of the reaction. 3.5. Suitability of LAT for field testing Based on the results of the study, format IV was found to be the most ideal one and it was used to screen the field samples (Fig. 2). A total of 207 pig serum samples were tested using format IV and the seropositivity for JE was found to be 41.5% (86/207). 3.6. Diagnostic efficacy of LAT Diagnostic sensitivity, specificity, efficiency, positive predictive value and negative predictive value of LAT in comparison with in-house rNS1 protein based indirect IgG ELISA were 80.2%, 95.2%, 88.3%, 94.2% and 83.5%, respectively. 3.7. Storage life of the latex beads The storage life of the beads was evaluated by keeping the beads at 4 °C and checking at periodic intervals. There was no appreciable change in sensitivity and specificity when the beads were reacted after 60 days interval. 4. Discussion In present study, rNS1 based latex agglutination test utilizing
Fig. 2. Screening of field samples using LAT. 1,2,4–8: Negative samples. 3: Positive sample. 75
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Outreach Program on Zoonotic Diseases.
agglutination assay. At low particle concentration, the naturally occurring proteins found in serum samples can interfere with immunoassays while at high particle concentration there is decreased formation of immunocomplexes due to imbalance between antigen and antibody concentrations or due to the increased steric hindrance or non-specific agglutination [1]. The relative diagnostic sensitivity and specificity of carboxyl beads based latex agglutination test for sero-diagnosis of JE in pigs was 80.2% and 95.2%, respectively. The sensitivity of LAT in comparison to ELISA was low for which we do not have any plausible explanation but the other advantages of LAT like it give results in 5 min, compared to 3 h of ELISA and non requirement of any equipment, make this assay a suitable screening test for use in field conditions. Further, it is warranted to validate this assay including the production procedure at wider scale before recommending its direct use in field conditions.
References [1] Garcia VS, Gonzalez VDG, Marcipar IS, Vega JR Gugliotta LM. Optimisation and standardisation of an immunoagglutination assay for the diagnosis of Trypanosoma cruzi infection based on latex- (recombinant antigen) complexes. Trop Med Int Health 2014;19(1):37–46. [2] Gonzalez VD, Garcia VS, Vega JR, Marcipar IS, Meira GR, Gugliotta LM. Immunodiagnosis of Chagas disease: synthesis of three latex–protein complexes containing different antigens of Trypanosoma cruzi. Colloids Surfaces B Biointerfaces 2010;77(1):12–7. [3] Horie M, Ogawa H, Yamada K, Hara A, Bui VN, Awad SS, et al. A latex agglutination test using a recombinant nucleoprotein for detection of antibodies against avian influenza virus. J Virol Methods 2009;161(2):259–64. [4] Inzana TJ. Simplified procedure for preparation of sensitized latex particles to detect capsular polysaccharides: application to typing and diagnosis of Actinobacillus pleuropneumoniae. J Clin Microbiol 1995;33(9). 2297-230. [5] Yu Z, Jin M, Xu X, Zhang R, Zhou H, Hu Q, et al. Development of a specific latex agglutination test based on a recombinant hemagglutinin protein to detect antibodies to H5 avian influenza viruses. Avian Dis 2006;50(2):264–8. [6] Grace MR, Dhanze H, Pantwane P, Sivakumar M, Gulati BR, Kumar A. Latex agglutination test for rapid on-site serodiagnosis of Japanese encephalitis in pigs using recombinant NS1 antigen. J Vector Borne Dis 2019;56:126–31. [7] Molina-Bolivar JA, Galisteo-Gonzalez F. Latex immunoagglutination assays. J Macromol Sci C Polym Rev 2005;45(1):59–98. [8] Dhanze H, Bhilegaonkar KN, Rawat S, Chethan Kumar HB, Kumar A, Gulati BR, et al. Development of recombinant nonstructural 1 protein based indirect enzyme linked immunosorbent assay for sero-surveillance of Japanese encephalitis in swine. J Virol Methods 2019https://doi.org/10.1016/j.jviromet.2019.113705. [9] Grace MR, Dhanze H, Pantawane PB, Sivakumar M, Kumar A. Sero-positivity of Japanese encephalitis virus in swine using virus neutralization test. J Vet Public Health 2016;14(1):9–12. [10] Veterinary Thrusfield. epidemiology. third ed. London, UK: Blackwell Science Ltd.; 2005. [11] Joullié MM, Lassen KM. Evolution of amide bond formation. Arkivoc 2010;8:189–250. [12] Bastos-Gonzalez D, Ortega-Vinuesa JL, De Fj L, Hidalgo-Alvarez R. Carboxylated latexes for covalent coupling antibodies. I. J Colloid Interface Sci 1995;176(1):232–9. [13] Jang LS, Keng HK. Modified fabrication process of protein chips using a short-chain self-assembled monolayer. Biomed Microdevices 2008;10(2):203–11. [14] Xiao Y, Isaacs SN. Enzyme-linked immunosorbent assay (ELISA) and blocking with bovine serum albumin (BSA)-not all BSAs are alike. J Immunol Methods 2012;384(1):148–51.
5. Conclusion Latex agglutination test developed in the present study can be used as rapid on-site screening assay for sero-surveillance of JE in pig population. Timely detection of JEV antibodies in pig population will aid in taking adequate preventive measures to control outbreak in humans. Since this test does not require any specific secondary antibody, theoretically it can also be used to screen human and equine sera samples for JEV specific antibodies. The novel approach adopted in present study for covalent coupling of the recombinant protein onto the latex beads may be adopted for the development of rapid on site diagnostics assays for other infectious diseases as well. Conflicts of interest None. Funding This study was funded by Indian Council of Agricultural Research-
76
Contents lists available at ScienceDirect
Biologicals journal homepage: www.elsevier.com/locate/biologicals
Evaluation of carboxyl beads based latex agglutination test for rapid serodiagnosis of Japanese encephalitis
T
M.R. Gracea,1, Jyotsana Chauhana,1, M. Suman Kumarb, Ashok Kumarc, K.N. Bhilegaonkara, Mithilesh Singha, Himani Dhanzea,∗ a
ICAR- Indian Veterinary Research Institute, India ICAR-Central Institute of Research on Goats, India c Indian Council of Agricultural Research, New Delhi, India b
A R T I C LE I N FO
A B S T R A C T
Keywords: Latex agglutination test Covalent coupling Japanese encephalitis
Japanese encephalitis (JE) is a major public health problem in the South Asian countries including India. Pigs serve as a relevant sentinel model, the surveillance of which could predict a potential JE outbreak in human population nearby. However, existing serological detection methods like Enzyme-Linked Immuno Sorbent Assay (ELISA), virus neutralization test (VNT) and Haemagglutination Inhibition (HI) require elaborative laboratory facilities which are invariably not available in field conditions. Recognizing the lacunae, attempts were made to develop recombinant antigen (rNS1) based latex agglutination test (LAT) as a rapid on-site test using covalent coupling method. Four different formats were evaluated using different coupling buffers, blocking buffers and reaction conditions. The format in which borate buffer at alkaline pH (8.5) was used for coupling of antigen with carboxylated beads followed by blocking with skimmed milk powder was found to be the best amongst all. Developed latex based test was used for screening of 207 pig serum samples for JE which revealed relative diagnostic sensitivity and specificity of 80.2% and 95.2%, respectively in comparison with indirect IgG ELISA. Hence, the present study demonstrated that covalently coupled recombinant antigen based LAT could be used as a reliable screening test for surveillance of JE in pigs under field conditions.
1. Introduction Japanese encephalitis (JE) is a mosquito-borne flaviviral zoonotic disease, which is one of the leading forms of viral encephalitis worldwide. Pigs are the amplifying host of JE virus (JEV) and in developing countries domestic pig rearing is an important risk factor for the transmission of JEV to humans. There are many unorganized piggeries and the rural populations who rear pig lives in close proximity with these animals, which has created an enabling ecosystem for transmission of JE. Pigs also serve as a relevant sentinel model, the surveillance of which could predict a potential JE outbreak in human population nearby. Hence, there is need for the development of a screening test that may be suitable for surveillance of JE in pig population. Sero-diagnosis is generally preferred over the other methods for diagnosis of JE. Even though there are many established serological methods such as ELISA, the application of these assays in field conditions is limited due to requirements of laboratory operations, skilled technicians and special equipment/facilities. Thus, the development of
an on-site, rapid and specific assay is essentially required for the surveillance of JEV infection in pig population. Latex agglutination test (LAT) is simple, rapid, cost-effective and easy to perform test, which has been widely employed as rapid on-site diagnostic assay for several diseases both in veterinary and medical field [1–5]. Earlier, we developed LAT based on physical adsorption for sero-diagnosis of JE in pigs but the shelf life of coupled beads was found to be less than 40 days [6]. Other researchers have also reported reduction in the activity of latex beads sensitized by physical adsorption due to the partial desorption of adsorbed protein that normally occurs during its storage [1]. Covalent coupling of LAT is an alternative method and different authors have cited advantages of covalent coupling over physical adsorption, the major one being the permanent nature of covalent attachment thus increasing the shelf life of the beads and also reducing the false positive results [1,4,7]. Considering the advantages of covalent coupling over physical adsorption, efforts were made to develop carboxyl beads based LAT which can be easily applied in field settings for sero-surveillance of JEV in pig population.
∗
Corresponding author. E-mail address: [email protected] (H. Dhanze). 1 Both authors have contributed equally to the work done. https://doi.org/10.1016/j.biologicals.2019.09.003 Received 30 July 2019; Received in revised form 4 September 2019; Accepted 5 September 2019 Available online 10 September 2019 1045-1056/ © 2019 International Alliance for Biological Standardization. Published by Elsevier Ltd. All rights reserved.
Biologicals 62 (2019) 72–76
M.R. Grace, et al.
2.2. Preparation of the antigen
Format IV
Standardization and optimization of reaction conditions for labeling of rNS1 protein with latex beads was done using covalent coupling in four different formats (Table 1).
73
Format I
MES pH 5.0 1. 1% Bovine serum albumin (BSA) in PBS 2. 5% Skimmed Milk Powder (SMP) in PBS-T Activation of beads followed by incubation of protein Not used Coupling buffer along with pH Blocking buffer
2.4.2. Format II In this format, the protein was pre-adsorbed on latex beads using carbonate buffer, followed by the activation of latex beads using EDAC in MES buffer. Briefly, the carboxylate modified latex beads were washed and resuspended in carbonate buffer (pH 9.5). The beads were pre adsorbed with rNS1 antigen in different concentrations (60 μg per 2.5 mg bead and 120 μg per 2.5 mg bead) using PBS. Further, the protein-bead mixture was washed and resuspended using MES buffer and the carboxyl groups on the beads were activated by the addition of EDAC in MES buffer in different concentrations (as mentioned in format I) and incubated. The incubated beads were washed and resuspended using 1% glycine in PBS as blocking and storage buffer and stored at 4 °C after sonication.
Reaction conditions
Table 1 Reaction conditions used in different LAT formats.
Format II
2.4.1. Format I Briefly, carboxylate modified 1 μ sized 2.5% blue dyed latex beads (Polyscience, Inc) were washed and resuspended in MES buffer (pH 5.0). The carboxyl groups on the beads were activated by the addition of N-(3-Dimethyl amino propyl)-Ń- ethylcarbodiimide hydrochloride (EDAC, Sigma) in MES buffer in different concentrations (50 mg/ml, 70 mg/ml and 100 mg/ml). Further, the beads were coated with rNS1 antigen in two different concentrations of 60 μg per 2.5 mg bead and 120 μg per 2.5 mg bead using PBS. The protein was incubated with the beads at 4 °C for two different time periods, 6 h and overnight. Further, the protein-bead mixture was centrifuged and washed. For finding the ideal blocking agent, the pelleted beads were re-suspended in different blocking buffers, 1% bovine serum albumin (BSA) in PBS and 5% Skimmed Milk Powder (SMP) in PBS-T (0.05% tween-20). Two different blocking conditions including overnight incubation with continuous mixing at 4 °C and 1 h incubation at room temperature with continuous mixing were tried. Latex beads were centrifuged and resuspended in storage buffer and stored at 4 °C until used. The storage solution used was either 0.1% BSA in PBS or 0.5% SMP in 0.01% PBST in accordance with the blocking agent used.
Use of quenching agent
2.4. Standardization of latex agglutination test
Carbonate Buffer pH 9.5 1% glycine in PBS
Format III
The hyperimmune serum was raised in rabbits against rNS1protein after getting due permission from the Institute Animal Ethics Committee.
Carbonate Buffer pH 9.5 5% SMP in PBST
2.3. Hyperimmune serum
Order of reaction
JEV non-structural recombinant protein (rNS1) previously expressed and purified in the Division of Veterinary Public Health, ICARIndian Veterinary Research Institute [8] was selected as antigen for coating onto the latex beads. Recombinant NS1 protein was produced in bulk and purified by nickel chelating affinity chromatography using imidazole gradient method. The purified protein was concentrated using Merck Millipore 30 K centrifugal filters and the concentration was estimated to be 750 μg/ml by Bradford reagent. The protein was stored at −80 °C till further use.
Pre adsorption of protein followed by activation 40 mM ethanolamine
A total of 207 pig serum samples collected from endemic areas of India were used in the present study.
Pre adsorption of protein followed by activation Not used
2.1. Serum samples
Borate Buffer pH 8.5 & 6.2 1. BSA in 0.2 M Borate buffer (pH 8.5 and 6.2) 2. SMP in 0.2 M Borate buffer (pH 8.5 and 6.2) Activation of beads followed by incubation of protein 40 mM ethanolamine
2. Materials and methods
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but fluid is slightly opaque was graded as moderately positive (++); reactions wherein about 50% latex beads agglutinate but the liquid is opaque was graded as weak positive (+) and reactions where the mixture remains homogenous was graded as negative.
2.4.3. Format III In this format the protein was pre-adsorbed on latex beads using carbonate buffer followed by the activation of latex beads using EDAC in MES buffer as in format II, followed by quenching and blocking. Quenching was done to remove the unused active carboxyl sites by adding a quenching solution of 40 mM ethanolamine to the pelleted beads. Blocking was done using 5% SMP in PBST and beads were stored in storage buffer (0.5% SMP in PBS-T) at 4 °C after sonication.
2.6. Diagnostic efficacy of LAT A total of 207 field pig serum samples were tested by in-house rNS1 protein based indirect IgG ELISA [8] and the results were compared with the findings of LAT to assess the diagnostic efficacy of LAT and its applicability in field conditions. The relative diagnostic sensitivity, specificity and predictive value of LAT in comparison with ELISA were calculated as described earlier by Thrusfield [10].
2.4.4. Format IV In this format, the latex beads were activated using EDAC and NHydroxysulfosuccinimide sodium salt (sulfo–NHS) in MES buffer followed by binding of the protein in borate buffer at two different pH (pH 8.5 and 6.2), quenching and blocking. Two types of latex beads were used, carboxylated modified 1.0 μ sized blue dyed microspheres (2.5% solids) (Polyscience, Inc.) and 0.9 μ (10% solids) undyed carboxylate modified microspheres (Sigma-Aldrich). Briefly, the latex beads were washed and resuspended using carbonate buffer (pH 9.5). The beads were washed and resuspended again using MES buffer (pH 5.0) to obtain a final concentration of 2.5% for both the beads. Further, the carboxyl groups on the beads were activated by the addition of EDAC and Sulfo-NHS (Sigma-Aldrich) in MES buffer dropwise to the uniformly resuspended beads, followed by incubation at room temperature for 15 min with continuous mixing. Different concentrations of EDAC were used (50 mg/ml, 70 mg/ml and 100 mg/ml) and sulfo-NHS was added at the concentration of 11 mg/ml. The unused EDAC and sulfoNHS were removed by centrifugation and the pelleted beads were washed and resuspended in 0.2 M Borate buffer at two different pH (pH 8.5 and 6.2). The beads were immediately treated with rNS1 antigen in different concentrations (60 μg per 2.5 mg bead, 90 μg per 2.5 mg bead, 120 μg per 2.5 mg bead, 150 μg per 2.5 mg bead, 180 μg per 2.5 mg bead and 300 μg per 2.5 mg bead). The protein was incubated with the beads overnight at 4 °C with continuous mixing. After incubation, the protein-bead mixture was centrifuged as before and the beads were resuspended in 0.2 M Borate buffer (pH 8.5 and 6.2). The coupling chemistry was stopped by adding ethanolamine in concentration of 40 mM at different volume (10 μl, 30 μl, 50 μl, 70 μl and 100 μl) to the pelleted beads and incubated for 30 min with continuous mixing at 4 °C. Further, to determine the ideal blocking agent, the pelleted beads were resuspended in two different blocking buffers, 1% BSA in 0.2 M Borate buffer (pH 8.5 and 6.2) or 5% SMP in 0.2 M Borate buffer (pH 8.5 and 6.2). Latex beads were centrifuged thrice as before using the respective blocking buffer and resuspended as a 2.5% suspension in storage buffer. The storage solutions used were either 0.1% BSA in PBS or 0.5% SMP in 0.01% PBST in accordance with the blocking agent used. Further, the beads were sonicated at 12 μm amplitude for 5 cycles, 2 s each and stored at 4 °C until used.
2.7. Storage life of the latex beads The storage life of the beads was evaluated by storing the beads at 4 °C with periodical testing for sensitivity and specificity using a panel of known positive and negative pig serum samples. 3. Results Latex agglutination test was standardized using rNS1 protein as antigen by covalent coupling onto carboxylate modified latex beads for sero-diagnosis of JE in pigs. 3.1. Format I In this format, wherein the activated beads were incubated with protein at acidic pH for covalent binding, all the combinations of reagents and reaction conditions tried failed to give any agglutination even with the undiluted hyperimmune serum. 3.2. Format II In this format, the protein was pre-adsorbed onto latex beads followed by activation of the beads and storage in glycine buffer. Addition of protein at the rate of 120 μg per 2.5 mg bead and use of EDAC at 50 mg/ml yielded agglutination reaction in both the hyperimmune serum and positive serum. However, the specificity of the format was low as the beads showed agglutination with known negative field pig serum samples also. 3.3. Format III In this format, quenching with 40 mM ethanolamine and blocking using 5% SMP in PBST was tried to minimize the non-specific reactions of format II. But this format failed to give any agglutination reaction even with the undiluted hyperimmune serum.
2.5. Suitability of LAT for field testing
3.4. Format IV
A panel of confirmed JE positive and JE negative serum samples was prepared by testing the collected pig serum samples using virus neutralization test [9]. The LAT was performed on glass slides using serial dilutions of hyperimmune serum, the panel of confirmed JE positive pig serum, JE negative pig serum and PBS. Three different reaction volume of rNS1 coupled beads and serum (5 μl beads and 5 μl serum; 5 μl beads and 10 μl serum and 10 μl beads and 10 μl serum) were tried. The slide was rocked briefly for 2 min to mix the coated beads and serum samples. The reaction was read at three different time periods (5, 10 and 15 min) and the conditions giving best visual perception of agglutination were selected. The result of the test was graded according to the degree of agglutination reaction. The reactions where the mixture became completely transparent with strongly agglutinated particles, which tend to settle at the edge of the beads-serum mixtures was graded as strong positive (+ + +); reactions wherein most of the latex particles have agglutinated
This format gave the best results amongst the various formats tried in this study. The reaction conditions which gave the best visual agglutination reaction are described here. Among the different concentrations of EDAC used, the protocol using double the amount of the weight of beads was found to be the best. There was no appreciable change in the sensitivity and specificity due to use of Sulfo-NHS in the present format. Among the different pH of borate buffer used, the protocol using alkaline pH (8.5) gave better agglutination reactions (up to 1:40 dilution of hyperimmune serum) as compared to acidic pH (up to 1:10 dilution). Sensitivity of covalent LAT increased with increasing protein concentration up to 150 μg per 2.5 mg bead. No improvement in the sensitivity was observed when the protein concentration was further increased. Ethanolamine, when used as quenching agent was found to be most effective at 40 mM concentration and 70 μl volume. Among 74
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covalent chemistry was developed for sero-diagnosis of JE in pigs. Covalent coupling employs specialized beads having functional groups incorporated on the bead surface, which undergoes covalent binding with the protein. Though various reactive groups are employed, the most commonly used is carboxylated beads [7]. In general, for visual slide agglutination the particle diameter range is 0.2–0.9 μm [7]. After perusal of literature, carboxylated latex beads of diameter 0.9 μm and 1 μm were employed in the present study. In principle, the amine groups of proteins can directly be coupled onto the carboxyl groups on the latex bead surface. Since such reactions are too slow, it is convenient to transform the carboxyl groups into the faster acylureas by addition of an activator [11]. Hence, in the present study N-(3-Dimethyl amino propyl)–Ń- ethylcarbodiimide hydrochloride (EDAC) was employed. This activation occurs at a slightly acidic pH and thus 100 mM MES buffer (pH 5) was opted for the same. In the format I, beads failed to react even with undiluted hyperimmune serum which might be due to the acidic pH of the coupling buffer. The sensitized beads in the format II gave agglutination up to 1:10 dilution of hyperimmune serum. But the specificity of the test was low with a high proportion of false positive reactions. Though glycine has been reported as the best blocking agent by other researchers [1], in the present study it was found to cause non specific agglutination. Format III was modified to reduce the number of false positives by using quenching and blocking agents. However, this modified format failed to give agglutination reaction even with the undiluted hyperimmune serum. It was concluded that the positive agglutination reactions in format II might be due to the activity of glycine. When SMP was used instead of glycine the beads failed to agglutinate even with undiluted hyperimmune serum. In format IV, the activated latex beads were incubated with rNS1 protein in borate buffer at two different pH (pH 8.5 and 6.2), followed by quenching and blocking. We observed that the covalent coupling was better at alkaline pH (8.5). This is in accordance with the findings of other researchers [12], who concluded that the covalent coupling of protein to carboxylated latexes using carbodiimide chemistry should be performed at basic pH, where a good colloidal stability can be expected as carbodiimide latex complexes have low colloidal stability at pH near to isoelectric point of protein. Addition of Sulfo-NHS stabilizes the amine-reactive intermediate by converting it to an amine-reactive Sulfo-NHS ester [13]. Thus, Sulfo-NHS was also tried at the concentration of 11 mg/ml in this format but there was no appreciable change in the sensitivity and specificity. There was no improvement in the sensitivity when protein concentration was increased from 150 μg per 2.5 mg bead to 180 μg and 300 μg per 2.5 mg bead. This might be due to the fact that the ratio between the total-linked protein and the total added protein decreases when increased amount of protein is added, owing to the saturation of particle surface [2]. The number of false positive reactions was more when 1% BSA in 0.2 M borate buffer was used as blocking agent as compared to 5% SMP in 0.2 M Borate buffer. This has similarity with an earlier report [3] wherein the BSA-blocked latex beads caused non-specific reactions. Since BSA is a serum protein, in certain circumstances it could cause non-specific signals [14]. One of the major disadvantages of EDAC is that it causes non specific cross linking resulting in clump formation which persisted even after vigorous pipetting and vortexing. Therefore, a brief sonication of the beads prior to storage was done for uniformly dispersing the latex beads and thus reduced the difficulty in interpreting the results. The ideal reaction condition was the one in which 5 μl beads at 2.5% concentration and 10 μl serum were reacted for a period of 5 min. When the reaction time was increased, there was difficulty in interpreting the result due to settling of the particles and drying of the mixture leading to false positive interpretations. The immuno-agglutination assay does not reach an end point and hence reaction time analysis is an important factor to consider when optimizing an assay. Optimizing concentration of beads is also an important factor in optimizing the latex
Fig. 1. LAT by covalent coupling. (a) Positive control. (b) Negative control.
the various blocking agents tried, 5% SMP in 0.2 M borate buffer (pH 8.5) at room temperature gave the least number of false positive reactions. The specificity of the test increased when blocking time was increased from 30 min to 2 h at room temperature. The best visual agglutination was observed when 5 μl beads and 10 μl serum was reacted (Fig. 1). The reaction time up to 5 min was found to be optimum, with longer time leading to settling and drying of the mixture. Carboxylated latex beads of 0.9 μm mean particle size (10% solids) (Sigma-Aldrich) were tried to improve the sensitivity of the test. Since the beads were colorless, the visibility of agglutination reaction was poor. Hence, it was concluded that carboxylate modified 1.0 μ sized blue dyed microspheres (Polyscience, Inc.) are better for visualization of the reaction. 3.5. Suitability of LAT for field testing Based on the results of the study, format IV was found to be the most ideal one and it was used to screen the field samples (Fig. 2). A total of 207 pig serum samples were tested using format IV and the seropositivity for JE was found to be 41.5% (86/207). 3.6. Diagnostic efficacy of LAT Diagnostic sensitivity, specificity, efficiency, positive predictive value and negative predictive value of LAT in comparison with in-house rNS1 protein based indirect IgG ELISA were 80.2%, 95.2%, 88.3%, 94.2% and 83.5%, respectively. 3.7. Storage life of the latex beads The storage life of the beads was evaluated by keeping the beads at 4 °C and checking at periodic intervals. There was no appreciable change in sensitivity and specificity when the beads were reacted after 60 days interval. 4. Discussion In present study, rNS1 based latex agglutination test utilizing
Fig. 2. Screening of field samples using LAT. 1,2,4–8: Negative samples. 3: Positive sample. 75
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Outreach Program on Zoonotic Diseases.
agglutination assay. At low particle concentration, the naturally occurring proteins found in serum samples can interfere with immunoassays while at high particle concentration there is decreased formation of immunocomplexes due to imbalance between antigen and antibody concentrations or due to the increased steric hindrance or non-specific agglutination [1]. The relative diagnostic sensitivity and specificity of carboxyl beads based latex agglutination test for sero-diagnosis of JE in pigs was 80.2% and 95.2%, respectively. The sensitivity of LAT in comparison to ELISA was low for which we do not have any plausible explanation but the other advantages of LAT like it give results in 5 min, compared to 3 h of ELISA and non requirement of any equipment, make this assay a suitable screening test for use in field conditions. Further, it is warranted to validate this assay including the production procedure at wider scale before recommending its direct use in field conditions.
References [1] Garcia VS, Gonzalez VDG, Marcipar IS, Vega JR Gugliotta LM. Optimisation and standardisation of an immunoagglutination assay for the diagnosis of Trypanosoma cruzi infection based on latex- (recombinant antigen) complexes. Trop Med Int Health 2014;19(1):37–46. [2] Gonzalez VD, Garcia VS, Vega JR, Marcipar IS, Meira GR, Gugliotta LM. Immunodiagnosis of Chagas disease: synthesis of three latex–protein complexes containing different antigens of Trypanosoma cruzi. Colloids Surfaces B Biointerfaces 2010;77(1):12–7. [3] Horie M, Ogawa H, Yamada K, Hara A, Bui VN, Awad SS, et al. A latex agglutination test using a recombinant nucleoprotein for detection of antibodies against avian influenza virus. J Virol Methods 2009;161(2):259–64. [4] Inzana TJ. Simplified procedure for preparation of sensitized latex particles to detect capsular polysaccharides: application to typing and diagnosis of Actinobacillus pleuropneumoniae. J Clin Microbiol 1995;33(9). 2297-230. [5] Yu Z, Jin M, Xu X, Zhang R, Zhou H, Hu Q, et al. Development of a specific latex agglutination test based on a recombinant hemagglutinin protein to detect antibodies to H5 avian influenza viruses. Avian Dis 2006;50(2):264–8. [6] Grace MR, Dhanze H, Pantwane P, Sivakumar M, Gulati BR, Kumar A. Latex agglutination test for rapid on-site serodiagnosis of Japanese encephalitis in pigs using recombinant NS1 antigen. J Vector Borne Dis 2019;56:126–31. [7] Molina-Bolivar JA, Galisteo-Gonzalez F. Latex immunoagglutination assays. J Macromol Sci C Polym Rev 2005;45(1):59–98. [8] Dhanze H, Bhilegaonkar KN, Rawat S, Chethan Kumar HB, Kumar A, Gulati BR, et al. Development of recombinant nonstructural 1 protein based indirect enzyme linked immunosorbent assay for sero-surveillance of Japanese encephalitis in swine. J Virol Methods 2019https://doi.org/10.1016/j.jviromet.2019.113705. [9] Grace MR, Dhanze H, Pantawane PB, Sivakumar M, Kumar A. Sero-positivity of Japanese encephalitis virus in swine using virus neutralization test. J Vet Public Health 2016;14(1):9–12. [10] Veterinary Thrusfield. epidemiology. third ed. London, UK: Blackwell Science Ltd.; 2005. [11] Joullié MM, Lassen KM. Evolution of amide bond formation. Arkivoc 2010;8:189–250. [12] Bastos-Gonzalez D, Ortega-Vinuesa JL, De Fj L, Hidalgo-Alvarez R. Carboxylated latexes for covalent coupling antibodies. I. J Colloid Interface Sci 1995;176(1):232–9. [13] Jang LS, Keng HK. Modified fabrication process of protein chips using a short-chain self-assembled monolayer. Biomed Microdevices 2008;10(2):203–11. [14] Xiao Y, Isaacs SN. Enzyme-linked immunosorbent assay (ELISA) and blocking with bovine serum albumin (BSA)-not all BSAs are alike. J Immunol Methods 2012;384(1):148–51.
5. Conclusion Latex agglutination test developed in the present study can be used as rapid on-site screening assay for sero-surveillance of JE in pig population. Timely detection of JEV antibodies in pig population will aid in taking adequate preventive measures to control outbreak in humans. Since this test does not require any specific secondary antibody, theoretically it can also be used to screen human and equine sera samples for JEV specific antibodies. The novel approach adopted in present study for covalent coupling of the recombinant protein onto the latex beads may be adopted for the development of rapid on site diagnostics assays for other infectious diseases as well. Conflicts of interest None. Funding This study was funded by Indian Council of Agricultural Research-
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