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  Table of Contents    
ORIGINAL ARTICLE  
Year : 2013  |  Volume : 56  |  Issue : 3  |  Page : 269-271
Development and evaluation of flavi-immunoglobulinM capture enzyme-linked immunosorbent assay


Department of Virology, King Institute of Preventive Medicine and Research, Guindy, Chennai, Tamil Nadu, India

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Date of Web Publication24-Oct-2013
 

   Abstract 

In this study, we report the evaluation of In-house flavi virus immunoglobulin M (IgM) capture enzyme-linked immunosorbent assay (ELISA), which can be used as a screening test to determine the infecting flavivirus serotype over the current serological methods. A panel of 88 sera (inclusive of well characterized dengue, Japanese Encephalitis (JE) and West Nile virus (WNV) positive and negative samples tested and confirmed by commercial kit) was used for evaluation of the kit. The sensitivity and specificity of the In-house capture assay versus the commercial kit for the sero-diagnosis of dengue was 100% and 87% respectively, for JE IgM, it was found to be 90% and 100% respectively, and for West Nile it was 87.5% and 90.9%. Based on the study, we concluded that this flavivirus-serotyping ELISA provides rapid results and may be used as an accurate alternate to other serological tests for the specific diagnosis of flavivirus infections.

Keywords: Dengue, In-house flavi immunoglobulinM capture enzyme-linked immunosorbent assay, Japanese encephalitis, West Nile

How to cite this article:
Palani G, Padmanabhan PP, Ramesh K, Asadullah KS, Sambasivam M, Arunagiri K, Krishnasamy K. Development and evaluation of flavi-immunoglobulinM capture enzyme-linked immunosorbent assay . Indian J Pathol Microbiol 2013;56:269-71

How to cite this URL:
Palani G, Padmanabhan PP, Ramesh K, Asadullah KS, Sambasivam M, Arunagiri K, Krishnasamy K. Development and evaluation of flavi-immunoglobulinM capture enzyme-linked immunosorbent assay . Indian J Pathol Microbiol [serial online] 2013 [cited 2020 Jan 25];56:269-71. Available from: http://www.ijpmonline.org/text.asp?2013/56/3/269/120391



   Introduction Top


Viruses of the genus flavivirus (family-flaviviridae) constitute some of the most important emerging viruses known to man. They are transmitted by mosquitoes or ticks and are thus encompassed by the ecological description 'arboviruses' (arthropod-borne viruses). Derived from a common ancestor 10-20,000 years ago, they are evolving rapidly to fill new ecological niches. [1]

Flaviviruses are positive-stranded RNA viruses with a genome approximately 11 kb in size . More than 70 flaviviruses have been identified; approximately half of which can cause diseases in humans, but only a few are of medical importance. The viruses are categorized according to their vector, geographical area of occurrence, or the clinical symptoms with which they most commonly present. The three clinical syndromes caused by flavi viruses are fever-arthralgia-rash, viral hemorrhagic fever with or without hepatitis, and central nervous system disease. There are no antiviral drugs against flaviviruses, and vaccines exist only for a few types. [2]

The most important group medically, causing human and animal disease is the mosquito-borne group, which includes 21 viral species. Of these, the dengue fever (DF), yellow fever (YF), Japanese encephalitis (JE) and West Nile (WN) viruses have been implicated in causing human disease globally. [3]

Arboviruses spread by competent mosquito vectors across great distances and pose substantial risk to other regions in which the disease is currently non-endemic. [4] The immunoglobulin M (IgM) capture enzyme-linked immunosorbent assay (ELISA) is the preferred test used for the diagnosis of flavivirus infection as the IgM antibodies appear first in the serum and are markers for early diagnosis. The diagnosis of virus infections in humans pose difficulty in geographic areas where multiple flaviviruses are circulating due to cross reactions. Because of 'original antigenic sin', the highest antibody titer may be due to a previous flavivirus infection rather than to the etiologic agent. Serological diagnosis of DEN, JE and WN infections is extremely difficult in dengue virus hyper endemic areas. [5]

The aim of the present study was to compare the performance of the In-house flavivirus IgM capture ELISA with standard commercial ELISA kits. The In-house ELISA may be used as a preliminary assay to screen for flavivirus antibodies before confirming the etiology by specific serological and molecular methods.


   Materials and Methods Top


This study was performed at the Department of Virology, King Institute of Preventive Medicine and Research, Guindy, Chennai. The standard strains - Dengue 2 (Group Antigen) New Guinea strain, JE Nakayama strain, WNV (NY385-99) were procured (NIV). We had also procured control antisera against the three viral strains for analysis (from NIV). The individual viruses were inoculated into the mid ventral brain of 24-48 hours old Swiss albino suckling mice and antigen was extracted by sucrose-acetone method [6] and concentrated by Marian method. [7] Antigen was purified by Ammonium sulphate precipitation and dialysis. The protein level was estimated by Bradford's method with the standard controls. The purified antigen was titrated by HA test and spectrophotometry. [8]

In-house prepared mouse brain antigen was diluted in BAPS to yield concentrations of 20 μg, 40 μg, 60 μg, 80 μg per 100 μl (concentration confirmed by a spectrophotometer) and was used for coating ELISA plates. Effective antigen concentration to detect IgM (diluted up to 1 in 2000) was determined. The antigen quantity standardized for In-house ELISA is as follows, dengue-20 μg, JE-40 μg and West Nile-80 μg. [9]

Samples received from Government General Hospital, Institute of Child Health, other Government hospitals and private institutions all over Chennai, which were positive for DEN, JE, and WNV were used for evaluating the In-house method. [10]

Serum samples were tested for dengue and JE using Panbio Diagnostics, Brisbane, Australia (catalogue number EDEN01M) and diagnosis of West Nile fever was performed by IgM capture ELISA by Focus Diagnostics.

Requirements

Anti-human IgM (DAKO Code No - A0425) diluted at 1:2000, patients serum, In-house prepared mouse brain Ag (Den2/WN/JE), Anti flavi Group Monoclonal Antibody (MBS602538, 1:100), carbonate-bicarbonate buffer (pH 5.6), Tween 20 (Hi-media RM156), NUNC-maxisorp immune modules (the plates used in in-house ELISA and the commercial kit were of same binding capacity), blocking buffer (1% skimmed milk powder), phosphate buffered saline (PBS), serum samples and wash buffer with 0.1% Tween 20.

Procedure

Flavi IgM ELISA is an IgM-capture assay format wherein patient and control samples were diluted to 1:100. Micro titer plates (96 well) were coated with anti human IgM antibody and incubated overnight at 4°C. On day 2, the plates were washed and blocked with 1% skimmed milk powder (prepared in PBS) and incubated for 2 hrs at 37°C. Low positive controls for each of the three viruses were prepared by double dilution of well characterized positive sera and by performing ELISA (run in duplicate). E value was calculated and a graph constructed with E ratio on x-axis and OD units on the y-axis. The dilution against the E ratio of 1.2-2 was chosen and taken as low positive control. 0.1 ml portions of the In-house standardized control sera were added to micro titer wells and incubated at 37°C for 1 hour . [11] Meanwhile, the In-house prepared individual antigens (DEN, JE, and WN) in different dilutions, which were added to the group specific anti-flavi antibody and incubated at room temperature for 1 hr. After washing, the pre-incubated In-house individual antigens (DEN, JE, and WN) and the anti flavi antibody were added into the respective wells and incubated for 2 hrs at 37°C. The presence of IgM antibody was detected by adding 100 μl of horse radish peroxidase-labeled conjugate complex (DAKO-P0215) and incubating for 1hour. The chromogen TMB was added and the final reaction product was measured in an ELISA reader at a wavelength of 450 nm. [12] The cut off was calculated based on the formula:

Cut off OD = Sample OD/Low positive OD

Index values of <0.9 were considered negative, values from 0.9 to 1.1 were considered equivocal and values of >1.1 were considered positive for IgM antibody against the three viruses [Figure 1].
Figure 1: Flow Chart

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   Results Top


The well characterized 88 serum samples were tested by the In-house IgM ELISA as described above and the results were compared with those previously obtained for the serum samples by commercial IgM ELISA kits.

Of the dengue patient sera, 30 (68%) gave results consistent with the original results. The sensitivity and specificity of the In-house capture assay against the commercial kit for the sero- diagnosis of dengue was 100% and 87% respectively. For JE, it was found to be 90% and 100% respectively. The evaluation of the WN results was found to have an overall low specificity and sensitivity, possibly due to the less number of samples tested. The evaluation results of ELISA kits used are shown in [Table 1].
Table 1: Performance of in-house IgM ELISA test

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Table 2: Evaluation of antigen concentration by hemagglutination and spectrophotometry

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   Discussion Top


The number of flaviviruses infecting humans in India is potentially more than in any other area of the world and is also associated with high mortality and morbidity. [12] The infecting flavivirus serotype may be determined by various serological methods, such as the HAI test; it requires two blood samples, an acute serum sample and a convalescent serum sample to demonstrate a rise in titer.

Hemagglutination assay was performed for dengue, JE, WN antigens, before and after concentration. [Table 2] clearly indicates the rise in titer of the antigens used for the standardization of ELISA after the concentration method was employed. Similarly, the antigens were analyzed in a UV spectrophotometer, wherein there was an increase in the antigen concentration.

The results obtained in the In-house ELISA is comparable to that of the standard ELISA kits used. The need for the development of screening test for commonly occurring flaviviruses is vital and if tested positive by the initial test, the positives can be reconfirmed by performing specific ELISA tests which can be time and cost effective. In this study, we have demonstrated that an IgM capture ELISA using a panel of antigens was able to differentiate infecting common flavivirus serotypes for the majority of serum samples tested.

There are some limitations in this study. Our In-house standardized ELISA test detects IgM antibodies only. We have not standardized the test for detection of other flaviviruses, nor utilized positive sera from other f positives to rule out cross reactivity.

False positive results were noted in the present study. This issue can be settled with better purification methods of mouse brain antigen. Furthermore, tissue culture purified antigen will be more specific and sensitive to rule out false positivity. We believe that the In-house flavivirus IgM ELISA greatly improves the ability to provide timely, accurate, and meaningful results in case of flavivirus infection, particularly for the diagnosis of dengue virus infections in this part of the country where the occurrence of the vector is also high. [13]

 
   References Top

1.Gould EA, Zanotto PM, Holmes EC, Saluzzo JF, Dodet B. The genetic evolution of flaviviruses. Factors in the emergence of arbovirus diseases. Elsevier; 1997. p. 51-63.  Back to cited text no. 1
    
2.Simpson DH, Cook G. Arboviruses. Manson's tropical diseases. London: Saunders; 1996. p. 615-65.  Back to cited text no. 2
    
3.Gubler DJ. Emerging vector-borne flavivirus diseases. Are vaccines the solution? Expert Rev Vaccines 2011;10:563-5.  Back to cited text no. 3
    
4.LaBeaud AD, Sutherland LJ, Muiruri S, Muchiri EM, Gray LR, Zimmerman PA, et al. Arbovirus prevalence in mosquitoes, Kenya. Emerg Infect Dis 2011;17:233-41.  Back to cited text no. 4
    
5.Loroño-Pino MA, Farfan-Ale JA, Blitvich BJ, Beebe JL, Jarman RG, Beaty BJ. Evaluation of an epitope-blocking enzyme-linked immunosorbent assay for the diagnosis of West Nile virus infections in humans. Clin Vaccine Immunol 2009;16:749-55.  Back to cited text no. 5
    
6.Schmidt NJ, Lennette EH. Appendix: Basic technics for virology. In: Horsfall FL, Tamm I Jr, editors. Viral and rickettsial infections of man, 4 th ed. Philadelphia: J. B. Lippincott Co.; 1965.  Back to cited text no. 6
    
7.Horzinek M. A simple method for concentration of arboviruses propagated in tissue culture. Am J Trop Med Hyg 1969;18:588-91.  Back to cited text no. 7
    
8.Clarke DH Casals J. Techniques for hemagglutination and hemagglutination-inhibition with arthropod-borne viruses. Am J Trop Med Hyg 1958;7:561-73.  Back to cited text no. 8
    
9.Greiner M, Sohr D, Gobel P. A modified ROC analysis for the selection of cut-off values and the definition of intermediate results of serodiagnostic tests. J Immunol Methods 1995;185:123-32.  Back to cited text no. 9
    
10.Kienzle N, Boyes L . Murray Valley encephalitis: Case report and review of neuroradiological features. Australas Radiol 2003;47:61-3.  Back to cited text no. 10
    
11.Manual on quality standards for HIV testing laboratories. National AIDS Control Organisation. Ministry of Health and Family Welfare: March 2007.  Back to cited text no. 11
    
12.Martin DA, Biggerstaff BJ, Allen B, Johnson AJ, Lanciotti RS, Roehrig JT. Use of immunoglobulin m cross-reactions in differential diagnosis of human FlaviFlaviflaviflaviviral encephalitis infections in the United States. Clin Diagn Lab Immunol 2002;9:544-9  Back to cited text no. 12
    
13.Schmidt NJ. Tissue culture technics for diagnostic virology. In: Lennette EH, Schmidt NJ editors. Diagnostic procedures for virus and rickettsial infections, 4 th ed. New York: Amer. Pub. Health Ass., Inc.,.1969  Back to cited text no. 13
    

Top
Correspondence Address:
Kaveri Krishnasamy
Department of Virology, King Institute of Preventive Medicine and Research, Guindy, Chennai - 600 032, Tamil Nadu
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0377-4929.120391

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    Figures

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    Tables

  [Table 1], [Table 2]

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