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Production and application of monoclonal antibodies against dengue for early detection of dengue virus infection on blood smear preparation*

Rabu, 23 Februari 2011 2 komentar
Umniyati SR1,  Sutaryo1,   Wahyono D2,Artama WT3
1Faculty of Medicine, Gadjah Mada University
2Faculty of Pharmacy, Gadjah Mada University
3Faculty of Veterinary Medicine, Gadjah Mada University
 
*This paper was presented in BIT Life Sciences’ 1st Annual World Congress of Virus and Infections 2010 (WCVI-2010) on July 31-August 3, 2010, in Busan, South Korea.     
Abstract
   Background: Commonly used diagnosis methods for confirming dengue infection involve  virus isolation, detction of virus antigen, or RNA in plasma, or serum or tissues, and the presence of dengue virus specific antibodies in serum and other body fluids. Monoclonal antibodies (mAb) are used extensively in basic biomedical research, in diagnosis of disease. Producing mAb requires immunizing an animal, usually a mouse; obtaining immune cells from its spleen; and fusing the cells with a cancer cell (such as cells from a Myeloma) to make them immortal, which means that they will grow and divide indefinitely. A tumor of the fused cells is called a hybridoma, and these cells secrete mAb.
Objective. The laboratory studies are aiming at producing, characterizing, optimizing and applying the monoclonal antibody against dengue for early detection of dengue virus infection on blood smear preparation based on immunocytochemical streptavidin–biotin- peroxidase complex (ISBPC) assay.
Methods.  Monoclonal antibody against dengue was produced by fusing of immunized splenocytes against dengue-3 strain H-87 antigen with NS-1 Myeloma cells. Intraperitoneal injection of 106-107 hybrid cells into Balb/C mice were carried out to obtain high level of antibody.  Determination of the antibody Class and subclass was carried out based on antigen mediated ELISA. The presence of dengue antigen on blood smear preparation was carried out based on ISBPC assay.
Results. Monoclonal antibody against Dengue-3 was produced by Dengue Team of Gadjah Mada University through three times of fusions.  It has been produced 50 cc of antibody against Dengue secreted by DSSC7 clones generated from the third fusion in ascites of Balb/c mice. The monoclonal antibody belongs to IgG Class, IgG1 subclass. The monoclonal antibody DSSCC7 is successfully applied for confirming the presence of dengue antigen in the cytoplasm of monocytes and lymphocytes of blood smear preparation from dengue fever and dengue haemorrhagic fever patients on the second and the third day of fever based on ISBPC assay.
Conclusion.  It has been produced monoclonal antibody against dengue by Dengue Team of Gadjah Mada University. Monoclonal antibody in ascitic fluid induced by DSSC7 hybridoma generated from the third fusion belongs to IgG Class, IgG1 subclass.  The monoclonal antibody is successfully applied as primary antibody for early detection of dengue infection on blood smear preparation based on ISBP assay.
Key words: monoclonal antibody, dengue, antigen, blood smear, ISBP

INTRODUCTION
Dengue Hemorrhagic Fever (DHF) caused by dengue virus and transmitted by Aedes mosquitoes, especially Aedes aegypti is still a serious health problem in Indonesia. Progression of the disease is very fast, therefore, if it does not immediately get appropriate treatment, is often fatal. Until now, diagnosis of DHF in Indonesia is still mainly based on clinical diagnosis without followed by virological diagnosis. Virological confirmation could be done early, because viremia occurred two days before and during fever1, however a simple diagnostic tool for this purpose until now is not yet available. Actually there have been a fast and simple diagnostic tool for dengue infection that only takes 5 minutes, that is a Dengue Fever Rapid Test Kit based on immunochromatography method (ICT) but this method only detects IgM and IgG, whereas IgM just appeared after 3-5 days fever. In this period, DHF patients usually recovered gradually or becoming shock and even death, so the kit is more appropriate for the purposes of research or surveillance than for treatment of patients. Diagnostic kit for early detection of dengue infection can be performed with monoclonal antibodies to detect dengue antigen circulating in the blood, usually because viremia has occurred two days before the fever and reached its peak at the time of fever. For this purpose specific monoclonal antibody against dengue virus is required as a diagnostic reagent.
After Kohler and Milstein discovering a technique of hibridoma in 1975 and received the Nobel award in 1984 for his services in finding a monoclonal antibody technique, the technique is extended in various areas of biomedical and multidolar businesses in the world. With the help of these new techniques makes possible the desired antibody is produced in unlimited quantities. Their use is no longer limited to basic research, but monoclonal antibodies have a new revolution in the industrial field, for certain purposes in medicine, such as diagnostic reagents and therapy of a disease with specific target2.
Monoclonal antibodies have great benefits due to its specificity and the ability to recognize just one epitope. Monoclonal antibodies can be produced by making immortalized from cells that produce antibodies. When the antibody-producing cells fused with tumor cells belonging to cell line, the hybrid cells will have two properties that the parent can live continually in culture and produce specific antibodies in accordance with expected purposes, and these hybrid cells can be propagated indefinitely (hybridoma).2,3,4
Objective
The laboratory studies are aiming at producing, characterizing, optimizing and applying the monoclonal antibody against dengue for early detection of dengue virus infection on blood smear preparation based on immunocytochemical streptavidin–biotin- peroxidase complex (ISBPC) assay.


MATERIAL AND METHODS
A. Production of Monoclonal antibody
Monoclonal antibodies were produced through a series of activities as follows: (1) preparation of antigen; (2) immunization; (3) fusion; (4) selection and cloning; (5) preservation (6) production of monoclonal antibodies in vitro and in vivo.

B. Characterization of Monoclonal Antibody
            Determination of isotype and cross reactivity. Determination of the antibody Class and subclass was carried out based on antigen mediated ELISA (enzyme linked immunosorbent assay). The specificity of monoclonal antibody DSSC7 was determined by Western blotting method5 and inhibition ELISA6, using DENV-1, DENV-2, and DENV-3, DENV-4, Japanese encephalitis (JE) and Chikungunya antigen. The DEN, JE and CHIK viral antigen were obtained from Namru-2 Jakarta.

C. Detection of dengue virus infection of blood smears preparation based on ISBPC assay 

Preparation of blood smears

Blood was obtained from the finger of dengue and dengue haemorrhagic fever patients. The area to be punctured must be clean and dry. In making the thick film, three small drops of blood were placed near one end of the slide and spread by stirring with the corner of another clean slide. The glass slide should touch only the drop of blood, not the skin, to avoid introducing artifacts into the specimen. The resulting film should be about the size of a dime; if too thick, the film crack and may not stick. The thick film should dry for an hour and was hemolyzed with aquadest. After a few minutes of drying it was fixed with cold absolute methanol (-200C) for ten minutes.


Figure 1. Thick and thin blood smear before staining based on immunocytochemical assay
Immunocytochemical Streptavidin biotin-peroxidase-complex assay
The principle of immunocytochemical Streptavidin biotin-peroxidase-complex (ISBPC) assay was performed as follows (Figure 2).

Figure 2. Diagram of immunocytochemical Streptavidin biotin-peroxidase-complex (ISBPC)
The endogenous peroxidase activity was destroyed by treating a specimen with hydrogen peroxide solution. The non- specific background is eliminated by incubating the specimen with non immune serum.The primary antibody to specific antigen is incubated to target antigens. This is followed by addition of biotinilated second antibody which serves as the linker between the primary antibody and peroxidase-streptavidin conjugate. Streptavidin peroxidase is then added to bind to the biotin residues on the linking antibody. Diaminobenzidin tetrachloride (DAB) was adding according to manufactures protocols. Between steps, the slides were washed in PBS for 5 minutes.Finally, the slides were washed in running water, counterstained in Mayer hematoxylin, dehydrated, and mounted with DPX.
The specificity of the ISBPC procedure was validated by negative controls that ensure that the labeling method accurately identifies the antibody bound to the tissue and by positive controls that show that the antibody is binding to an appropriate structure. Negative result was detected as blue color in the lymphocytes and monocytes. Positive result was detected as discrete brownish granular in the cytoplasm of lymphocytes and monocytes. The presence of dengue antigen on thin and thick blood smears were detected in Dengue Fever and Dengue Haemorrhagic Fever  patients using binocular microscope at magnification of 20x, 200x, 400x, and 1000x based on ISBPC assay.

RESULT AND DISCUSSION
A. Production of Monoclonal antibody
1. Preparation of antigen
Antigen were prepared as follows: monolayer of C6/36 cells were grown to 90% confluence in 75-cm2 flasks, then inoculated with dengue virus , and incubated for 1 hour at 28°C in an atmosphere of 5% CO2. Flasks were supplemented with 15 mL of maintenance medium (minimal essential medium, 2% fetal bovine serum [FBS], 1x non-essential amino acids, 100 U/mL of penicillin, and 100 µg/mL of streptomycin) and maintained at 28°C in an atmosphere of 5% CO2. Infection was monitored daily by an inverted microscope and cell supernatants were harvested at seven or eight days post-infection. Maintenance medium was changed after 2 to 4 days (depending on the virus) and the culture supernatants and infected cells were harvested when cytopathic effect was apparent throughout the monolayer. The culture supernatants were clarified by centrifugation for 10 minutes at 1000 rpm at 4°C, and stored in aliquots at -80°C until use. The infected monolayer were washed with phosphate buffered saline (PBS) and lysed in 2 ml of a hypotonic buffer containing 1% TX-100. Intact nuclei were removed by brief centrifugation at 14,000 rpm in a micro centrifuge and the lysate supernatants (referred to as "lysates") were aliquoted and stored at -80°C until use. Virus stocks were stored as individual 1mL aliquots in 20% FBS at -80°C.
            Antigen purification. All infective medium was centrifuged at 3000 rpm for 30 '. Supernatant was taken and then directly purified by continuous gradient sucrose ultracentrifugation techniques.  Dengue-3 antigens subsequently were inactivated with formalin 0.2%. Furthermore, antigen levels were counted according to Bradford with bovine serum albumin (BSA) as a standard using spectrophotometer at a wavelength of 595 nm. The quantitative analysis of the dengue antigens vary from 450 µg, 620 µg, 1000 µg/ml. Antigen was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Protein was monitored by Coomassie blue and silver staining and the use of pre-stained molecular weight markers, respectively. The antigen used in this study comprised NS3 protein (68.9 kDa), E protein (57.9 kDa), and NS1 protein (48kDa).
           
2. Immunization
Immunizations are intended to stimulate a number of clones of lymphocytes. The clones of stimulated lymphocytes will proliferate and differentiate into plasma cells that capable of producing antibodies. Adjuvant was used in immunization of experimental animals to enhance the immune response. Adjuvant may affect imunogen through structural changes or electrostatic thereby increasing the immunogenicity and influence the immune system through the  imunogen depot thus slowing the release of antigen from the injection site, so that stimulation of the immune system will still be there. Four days until two days before fusion mice were immunized once daily with dengue 3 antigens via the lateral vein of tails2, 3, 4, 6. Blood sampling of mice to determine the immune response of mice against dengue-3 virus was performed by ELISA 10 days after immunization II, III, IV and V.
Serum dilution. Each serum sample and positive control serum (hyper immune mouse ascitic fluid (HMAF) anti-dengue-3 from Namru-2 Jakarta) diluted with PBS to a series of concentration: (1:100), (1:200), (1:400), (1:800), (1:1600) (1:3200), (1:6400), (1:12800). When a sufficient antibody titer is reached in serum, immunized mice are euthanized and the spleen removed to use as a source of cells for fusion with myeloma cells.
3. Fusion
Fusing antibody-producing spleen cells, which have a limited life span, with cells derived from an immortal tumor of lymphocytes (myeloma) results in a hybridoma that is capable of unlimited growth. Myeloma cells are immortalized cells that are cultured with 8-azaguanine to ensure their sensitivity to the hypoxanthine-aminopterin-thymidine (HAT) selection medium used after cell fusion. The HAT medium allows only the fused cells to survive in culture2, 4, and 6.
            Fusion of Myeloma Cells with Immune Spleen Cells.  Single spleen cells from the immunized mouse are fused with the previously prepared myeloma cells. Fusion is accomplished by co-centrifuging freshly harvested spleen cells and myeloma cells in polyethylene glycol, a substance that causes cell membranes to fuse. Only fused cells will grow in the special selection medium. The cells are then distributed to 96 well plates containing feeder cells derived from saline peritoneal washes of mice. Feeder cells are believed to supply growth factors that promote growth of the hybridoma cells2, 4, 6.

4. Selection and cloning
 At this step new, small clusters of hybridoma cells from the 96 well plates can be grown in tissue culture followed by selection for antigen binding. Cloning by “limiting dilution” at this time ensures that a majority of wells each contain at most a single clone. Considerable judgment is necessary at this stage to select hybridomas capable of expansion versus total loss of the cell fusion product due to under population or inadequate in vitro growth at high dilution. The selection growth medium contains the inhibitor aminopterin, which blocks synthetic pathways by which nucleotides are made. Therefore, the cells must use a bypass pathway to synthesize nucleic acids, a pathway that is defective in the myeloma cell line to which the normal antibody-producing cells are fused. Because neither the myeloma nor the antibody-producing cell will grow on its own, only hybrid cells grow.

5. Production of Monoclonal antibody
Hybrids were selected for growth in HAT medium, under these conditions, unfused myeloma cells dye because they can not use the salvage pathway and the B cells can not survive for more than 1 to 2 weeks because they are not immortalized, so that only hybrids will grow. Hybrid cells producing antibodies were grown in flask containing complete RPMI medium. The hybrid cells were inoculated intra peritoneal into pristane-treated BALB/c mice. After 2–3 weeks, the ascitic fluid produced by each mouse was collected with a syringe or by puncturing the abdomen, and then stored for further steps, while the hybrids were stored in liquid nitrogen. It has been produced 50 cc of antibody against DENV secreted by DSSC7 hybridoma in ascites of two Balb/c mice.  It has also been produced 238.5 mL and 19.5 mL of antibodies against DENV secreted by DSSE10 and WDSSB5 hybridomas respectively.
2. Characterization of monoclonal antibody
            Determination of isotype. The class of antibody was selected for by the screening tests used during the establishment of the hybridoma. For example, since the majority of desired monoclonal antibodies will be IgG, if the anti-mouse whole IgG label in the test is replaced with an anti-mouse IgG label, only IgG antibodies wil be selected. Similarly, if only It was used a commercially available testing kit isotypic specific reagent (Sigma ISO-2) based on antigen mediated Elisa to perform isotype analysis during this study. In this study monoclonal antibody secreted by hybridoma DSSC7 belongs to IgG Class, IgG1 Subclass, and antibody secreted by hybridoma DSS2F1 belongs to IgM Class, whereas antibody secreted by hybridoma DSSE10 belongs to IgM and IgG1 Subclass, and antibody secreted by hybridoma DSSG7 belongs to IgM and IgG2a. After several times recloning, the antibody secreted by DSSE10 clone belongs to IgG1 Subclass.
            Specificity. An important early characterization test of any panel of antibodies is the analysis of whether they react with the same, close or totally different epitopes. Monoclonal antibody secreted by a single hybridoma (DSSC7) which generated from the third fusion recognized DENV complex specific epitope based on Western blotting analyses. The result exhibited that Mab DSSC7 recognized DENV antigen (DENV1, DENV-2, DENV-3, DENV-4) at molecular weight of about 48,000 Da (48 kDa). According to Henchal and Putnak7 non structural (NS-1) DENV protein has molecular weight of 48,000 Dalton. The NS-1 protein could be found in the cell, at plasma membrane or it is secreted out of the cell during the infection. Dengue virus belongs to Flaviviruses. They are enveloped, single-stranded, positive-sense RNA viruses. The genomic RNA is about 11 kb long and contains 10 genes encoding three structural proteins (capsid [C], envelope [E], and membrane [M]), and seven nonstructural proteins (NS1, NS2a, NS2b, NS3, NS4a, NS4b, and NS5)8. The polycistronic coding region is flanked by non-coding regions at its 5′ and 3′ ends. The nonstructural protein, NS1, is a highly conserved glycoprotein, but its biological activity has not been established. During in vitro9. In solution, secreted NS1 protein behaves as a hexamer; it circulates and accumulates in the sera of dengue virus-infected patients throughout the clinical phase of the disease10. A recent study demonstrated that soluble NS1 protein binds to endothelial cells and, following recognition by anti-NS1 antibodies, could contribute to plasma leakage during severe dengue virus infection .The detection of secreted NS1 protein represents a new approach to the diagnosis of acute dengue infection11,12,13. infection, the flavivirus NS1 protein is expressed as an intracellular membrane-associated form essential for viral replication
             The result showed that there was no cross reactivity between the mAb DSSC7 toward CHIK antigen. The mAb DSSC7 and DSS4E10 also showed high immunoreactivity toward DENV-1, DENV-2, DENV-3, DENV-4 and no cross-reactivity toward Japanese encephalitis and chikungunya antigen based on indirect ELISA, whereas mAb secreted by DSS2F1 hybridoma showed immunoreactivity toward DENV-1, DENV-2, DENV-3, DENV-4, and cross-reactivity toward Chikungunya antigen based on Western blotting and inhibition ELISA.

C. Detection of dengue virus infection of blood smears preparation based on ISBPC assay

            The monoclonal antibody DSSC7 is successfully applied for confirming the presence of dengue viral NS1 protein in the cytoplasm of monocytes and lymphocytes of thin and thick blood smear preparation from dengue fever and dengue haemorrhagic fever patients on the first, second and the third day of fever based on ISBPC assay. 

 
                                          Positive sample                                 Positive Control

 
                                                                     Negative Control
Figure 3
Thick blood smear preparation of dengue haemorrhagic fever (DHF) patient (3rd day fever) showing dengue viral NS1 antigen (brown color) in the cytoplasm cells of leucocytes (monocytes) based on immunocytochemical streptavidin biotin peroxidase complex (ISBPC) assay using monoclonal antibody against DENV (DSSC7 1:10)


In this study thick blood smear preparations were made as positive control from febrile patients clinically suspected of having dengue fever and confirmed  with one step single tube multiplex RT-PCR, whereas healthy volunteers were included as negative control. Evaluation of the ISBPC assay for detecting dengue viral NS1 protein in the cytoplasm of monocytes and lymphocytes of thick blood smear preparation has been done using RT-PCR as a gold standard. The ISBP assay on thick blood smears using DSSC7 mAb gave a sensitivity of 94.29% with a specificity of 90%14.


Figure 4
Thin blood smear preparation of dengue haemorrhagic fever (DHF) patient (3rd day fever) showing dengue viral NS1 antigen (brown color) in the cytoplasm cell of monocyte based on immunocytochemical streptavidin biotin peroxidase complex (ISBPC) assay using monoclonal antibody against DENV (DSSC7 1:10)

            In reference laboratories, dengue disease is confirmed by virus isolation or genome detection during the acute phase and by serological methods during the early convalescent phase. The viral NS1 protein circulates in the sera of infected patients throughout the clinical phase of the disease. Novel diagnostic tests based on NS1 detection have been recently developed and marketed. The performance of two tests for detecting dengue NS1 protein during the clinical phase of dengue infection (an immunochromatographic test (ICT) from Bio-Rad and a two-step sandwich-format ELISA from Panbio) has been evaluated. The ICT test performed better than the ELISA test from Panbio. The sensitivity of the ICT was 81.5% (95% CI: 75.8% to 86.4%) after 15 minutes and 82.4% (95% CI: 76.8% to 87.2%) after 30 minutes. Both tests had a specificity of 100% (97.5% CI, one-sided test: 92.6% to 100.0%) 15.  It means that the ISBPC assay on thick blood smear preparation using mAb DSSC7 for detecting dengue viral NS-1 more sensitive than the ICT. Our findings support the use of diagnostic tools based on the NS1 antigen detection for the diagnosis of acute DENV infection. The ISBPPC assay the first simple rapid diagnostic test for DENV infection was highly sensitive and specific, and would therefore be a suitable first-line test in the field. This study confirms that diagnostic tests based on NS1 could be used in routine clinical practice in poorly equipped laboratories and that dengue diagnosis could therefore be confirmed without the need for testing in reference laboratories. This represents a crucial step towards the control of dengue disease in the human population.
CONCLUSION
            It has been produced monoclonal antibody against dengue by Dengue Team of Gadjah Mada University. Monoclonal antibody in ascitic fluid induced by DSSC7 hybridoma generated from the third fusion belongs to IgG Class, IgG1 subclass.  The monoclonal antibody is successfully applied as primary antibody for early detection of dengue infection on blood smear preparation based on ISBPC assay.
ACKNOWLEDGEMENT
The authors gratefully thank the Dean of Faculty of Medicine, and the Head of Department of Parasitology, Gadjah Mada University, for their support in the conduct of these studies. Thanks are also due to Muslimah, Purwanti, Suprihatin, Arsiah, Purwono, and Utami for their valuable assistance in the laboratory.

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