Evaluation of polyclonal antibodies raised in rabbit against Dengue NS1 antigen

doi:10.21203/rs.3.rs-4635752/v1

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Evaluation of polyclonal antibodies raised in rabbit against Dengue NS1 antigen

https://doi.org/10.21203/rs.3.rs-4635752/v1

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The vector-borne disease, dengue is caused by the dengue virus (DENV) and the illness spread by Aedes mosquitoes like AedesaegyptiandAedesalbopictus.In the absence of antiviral drugs and vaccines, dengue prevention is centred on mosquito vector control. Current methods of dengue detection suffer from limitations. The non-structural protein-1 (NS1) antigen in secretory form is being employed as a diagnostic marker to identify dengue infection andAedes vector infections. This method involves the use of commercial dengue NS1 ELISA kits. In the present study, we have evaluated Rabbit Polyclonal Antibodies raised against the Dengue NS1 antigen (RPAD). The RPAD were tested against commercial recombinant dengue virus-2 NS1 antigen (rDNS1Ag) by standardized ELISA and Western Blotting. Further, the sensitivity of RPAD was checked against the NS1 antigens of different serotypes of DENV. The specificity of RPAD towards the NS1 antigens of other flavivirus was also tested.We found that the RPAD is specific and sensitive for detecting rDNS1Ag of all serotypes and it can be explored to detect DENV infection in the mosquito vectors collected from the field during the dengue outbreaks.

Biological sciences/Biological techniques

Biological sciences/Immunology

Health sciences/Medical research

Aedes aegypti

Dengue virus

NS1 antigen

polyclonal antibody

vector-borne disease

Dengue virus (DENV) infections are among the most serious illnesses in the world spread bymosquitoes¹. In tropical Asia, it is currently aprimary factor ofmorbidity andin the absence of adequate treatment,mortality in children and it is frequently occurring in the Western Hemisphere.Dengue is an ongoing problem in 112 nations worldwide².Globally, millionsof dengue fever and 100 million cases of dengue hemorrhagic feverare documented eachyear.

The dengue virus (DENV), is a member of the family of Flavivirus. Its positive-sense single-strand ribonucleic acid (10.7 kb) codes for three structural proteins and seven nonstructural proteins³. Thehighly conservedNS1 protein is found in all flaviviruses.Viral replication requires it through a mechanism that could involve interactions with NS4A and NS4B.Infected cells initially express NS1 as a monomer and continually secrete NS1. The serum concentration of NS1 can exceed 50µg/ml, and the concentration is positivelycorrelated to the severity of the condition^4,5. Because of these characteristics of NS1, a variety of commercially available rapid dengue diagnostic procedures as well as the Enzyme Linked Immunosorbent Assay (ELISA) is developed.The commercial dengue NS1 ELISA kits are found to be highly sensitive and specific for the detection of recombinant dengue NS1 antigen^6,7.

The whole virus of dengue, itsproducts such as secreted NS1 protein, or measurement of virus-specific immunoglobulins (IgM and IgG) of the host-to-virus infection have all been targeted for diagnosis of DENV infection⁸.However, several challenges have been noted in the laboratory diagnosis of dengue⁹. Polyclonal antibodies have been explored for a variety of general research applications and diagnostic procedures as they exhibit high affinity for the target antigen and tolerance for slight changes in the target protein(https://www.creative-diagnostics.com/polyclonal-vs-monoclonal-antibodies.htm). Compared to monoclonal antibodies, polyclonal antibodies have the benefit of being manufactured more quicklyin a matter of months. In addition to being stable over a wide range of pH and salt concentrations, they often have minimal production costs and low technical skill requirements. The fact that they can be used as therapeutic immunoglobulins is more significant. Furthermore, because polyclonal antibodies are produced by numerous B-cell clones, each of which produces an antibody against a distinct epitope, they have higher specificity than monoclonal antibodies¹⁰. Therefore, in the present study, we raised the polyclonal antibody against the dengue NS1 antigen and explored its sensitivity and specificity towards the detection of recombinant dengue NS1 antigen (rDNS1Ag).

2.1. Ethics statement

All the animal experimental protocols were approved by the Institutional Animal Ethics Committee of ICMR-Vector Control Research Centre (approval # ICMR-VCRC/IAEC/2021-A2. Dated 2502.2021) and all methods are reported in accordance with ARRIVE guidelines.

2.2. Raising polyclonal antibodies against recombinant dengue NS1 antigen in Rabbits:

For raising the polyclonal antibodies in Rabbits at the Animal house facilities of ICMR-VCRC, the Institutional Animal Ethics Committee (ICMR-VCRC/IAEC/2021-A/2 Dated 25.02.2021)approval was obtained. Raising the polyclonal antibodies was done by using 10 µg and 20 µg recombinant Dengue virus 2 NS1 antigen (DG R&D Systems, Inc. Canada, Catalog No. 9439) mixed in Freund’s incomplete adjuvant and injected subcutaneously into the rabbits, following aseptic procedures. The booster dose was given on the 7th and 21st day after the primary dose. Seven days after the first and second booster (14th and 28th day), the blood was collected from the rabbits and screened for the development of antibodies against the NS1 antigen by ELISA. One milli litre of blood was collected from the ear vein of each animal at 7 days intervals once the rabbit tested positive.

2.3. Developing RPAD-ELISA for detection of NS1 antigen:

For developing RPAD-ELISA, the ELISA plate (Costar, Corning) was coated with different concentrations of recombinant dengue NS1 antigen (DENV-1 to 4 and JE, YFV, WNV; The Native antigen company, Oxfordshire, UK. SKU: REC31778 to 1781). The plate was incubated overnight at 4º C for effective binding of rDNS1Ag. The next day, the ELISA plate was washed three times with phosphate-buffered saline (PBS) and the wells were blocked with buffer (2% BSA;200µl/ well) and the plate was incubated at 37° C for 2 hrs.After washing the plate three times with PBS-Tween (PBS-T), anti-dengue NS1 polyclonal antibody (serum dilutions made with 1% BSA; 50 µl/well) was added to each well and further incubated for 1hr at 37º C. After washing with PBS-T followed by PBS, anti-rabbit HRP (50 µl 1:5000 dilutions in 1% BSA) was added to individualwells and incubated (1hr at 37°C), After washing with PBS-T followed by PBS, 50µl of TMB substrate was added to each well. After colour development, 30µl of stop solution (1N H2SO4) was added immediately to stop the reaction. The absorbance was measured at 452 nm in an ELISA reader.

2.4. Detection of Dengue NS1 antigen using RPADby Western Blotting:

To check the specificity of the polyclonal antibody Western blot was performed at the Department of Biotechnology, Pondicherry University, Pondicherry. Recombinant DENV2-NS1 antigens of different concentrations separated on SDS-PAGE (12%) were transferred to the nitrocellulose membrane.The membrane was kept for overnight incubation in 2% BSA at 4° C. Next day, after washing the membrane with PBS, polyclonal antibody raised in the rabbits against the rDNS1Ag was added to the membrane (1:1000 dilutions) and incubated for 1 hr. at 37° C. After washing the membrane with PBS-tween, anti-rabbit-HRP (1:3000 dilutions) was added to the membrane and incubated for 1 hr. at 37° C. After washing the membrane with PBS-T followed by PBS, blot was developed using ECL solution in the dark.The signals captured on the X-ray were fixed and later developed by drying the X-ray films and the images were scanned.

2.5. Purification of polyclonal antibody (IgG) from a rabbit immunized with rDNS1Ag:

RPAD was subjected to purification of IgG as per the manufacturer’s instructions (Thermo Scientific NAb Protein A/G Spin Kit). Briefly, the column was equilibrated with the binding buffer followed by the addition of a serum sample (250 µl). After incubation at 10 min., the column was kept over the spin column in a new collection tube and centrifuged for 1 min. After neutralizing the spin column, 400 µl of elution buffer was added to the column, mixed gently, and centrifuged for 1 min. This step was repeated two more times to collect three fractions. Later, the relative absorbance of each fraction was measured at 280 nm to check the purified antibody. The purified RPAD (IgG) was stored in aliquots at -30°C.

2.6. Comparison of RPAD with Commercial DNS1 Antibody

To test the minimum detection limit of rDNS1Ag, RPAD was compared with the commercial anti-Dengue virus NS1 protein (biotin) antibody (anti-DNS1Ab; Sigma Aldrich, Cat. No. SAB2702396) by Sandwich ELISA. Three different concentrations i.e. 1 ng, 10 ng and 100 ng RPAD were coated on the ELISA plate. After blocking (2% BSA), rDNS1Ag (10 ng, 100 ng and 250 ng) was added to the antigen-coated wells and the plates were further incubated at 37°C for 1 hr. Later, RPAD and anti-DNS1Ab were added (1:5000 and 1:2500 respectively). Finally, the wells with RPAD were detected with anti-rabbit-HRP and the wells with anti-dengue NS1 protein antibody were detected with Streptavidin-HRP (1:5000 each). The reaction was developed with TMB substrate and the absorbance was measured at 452 nm.

3.1. ELISA for detection of recombinant dengue NS1 antigen using RPAD:

For the detection of rDNS1Ag, polyclonal antibodies raised in the rabbits were tested. It was observed that Animal-1 exhibited higher absorbance (optical density; OD at 452 nm) against rDNS1Ag as compared to Animal-2. At dilution 1:10, Animal-1 displayed an absorbance value above 6 which is beyond the detection limit of the ELISA reader. Further, the absorbance values gradually decreased from dilution 1:25000 onwards(Fig. 1).

3.2. Western Blot analysis of dengue NS1 antigen using RPAD.

After confirming the sensitivity of the RPAD, the polyclonal antibody was tested by using Western Blotting. It was observed that the rDNS1Ag showed a protein band at 51 kDa.

3.3. SDS-PAGE analysis of RPAD

After purifying the polyclonal antibody i.e., IgG, the samples were run on 12% SDS-PAGE.IgG antibody has two light and two heavy chains. It was observed that the purified samples showed two distinct protein bands at 25 kDa and 50 kDa corresponding to the IgG.

3.4. RPAD is sensitive for detection of dengue NS1 antigen

The polyclonal antibody, RPAD showed high sensitivitytowards NS1 antigen of dengue virus serotype-2 by our initial experiments. The sensitivity of RPAD for detection of NS1 antigens of all the serotypes of DENV was carried out by ELISA. It was observed that Animal-1 exhibited higher absorbance (OD ≥ 1) for Dengue serotypes 1, 2 and 3. However, the OD for dengue serotype 4 was < 0.5. In the case of Animal-2, none of the serotypes displayed an OD of ≥ 1 (Fig. 2).

3.5. RPAD is specific forthe detection of dengue NS1 antigen only

To test the specificity of RPAD, ELISA for the detection of recombinant NS1 antigens of flaviviruses was tested.It was observed that none of the animals exhibited absorbance against the NS1 antigens of flaviviruses such as JE, TBE, WNV and YFV, indicating that RPAD is specific towards dengue NS1 only (Fig. 3).

3.6. Comparison of RPAD with Commercial DNS1 Antibody

The RPAD was compared with commercial anti-DNS1 antibody (DNS1Ab) using ELISA. The result showedthat,at 100 ng RPAD coating, RPAD (secondary antibody)displayed almost similar antibody responses to that of anti-DNS1Ab. However, at 1 ng RPAD coating, RPAD displayed higher antibody responses as compared to anti-DNS1Ab (Fig. 4). It is interesting to note that these observations were at 1:5000 dilutions of RPAD which is two-fold higher than the anti-DNS1Ab (i.e., 1: 2500).

Dengue is caused by a flavivirus that has four unique serotypes, DENV-1, DENV-2, DENV-3, and DENV-4.Dengue hemorrhagic fever (HDF)/dengue shock syndrome (DSS), a more serious clinical variant of dengue infection was initially identified in the Philippines in 1954 before spreading to rest the of Southeast Asia¹.According to WHO estimates, every year 390 million dengue virus infections occur and among these, 96 million show clinical symptoms. Even though infection is a concern in more than 100 nations, 70% of the burden occurs in Asia.¹¹As of April 30, 2024, more than 7.6 million dengue cases have been reported to WHO this year, including 3.4 million confirmed cases, over 16,000 severe cases, and more than 3,000 deaths¹².India had the highest number of cases, with roughly 33 million evident and the occurrence of 100 million asymptomatic cases every year¹³.

One of the most common assays used for the diagnosis of DENV is ELISA⁵.However,the commercial kits are costly and the expiry date will be short¹⁴.During the acute stage of dengue infection, diagnosis using the virus isolation method by animal models or cell culture is time-consuming and requires skilful personnel to handle¹⁵. Early dengue infection at febrile stages can be detected by reverse transcription-polymerase chain reaction,but it is an expensive procedure¹⁶.Since in the infected patient’s sera,a high level of NS1 antigen issecreted, it is being widely used as a promising diagnostic marker. Dengue NS1 antigen has been used in several types of polyclonal or monoclonal antibody-based immunoassays^17–21.At the early stage of infection, NS1is detectable from zero days onwards and achieves peak by the fourth day. Later on, IgM starts appearing in the blood from the third day to the ninth day of infection but its presence does not affect detection of antigen⁵.

Polyclonal antibodies are a collection of immunoglobulin molecules released in response to a specific antigen; each antibody identifies a unique epitope. As a result, polyclonal antibodies are an assortment of different antibodies. Every kind of antibody in the combination is produced by a particular clone of plasma B cells. As a result, several plasma B cell clones are involved in the synthesis of polyclonal antibodies. The process of producing polyclonal antibodies also begins with immunization/natural infection. A specific antigen can immunize an animal. Numerous epitopes may be present in this antigen. Each of the epitopes on the injected antigen is targeted by a unique antibody that the immune system makes. Directly from the animal's serum, polyclonal antibodies can be obtained²². Polyclonal antibodies have been explored for a variety of general research applications and diagnostic procedures as they exhibit high affinity for the target antigen and tolerance for slight changes in the target protein(https://www.creative-diagnostics.com/polyclonal-vs-monoclonal-antibodies.htm).Considering these applications, the polyclonal antibodies generated against recombinant dengue NS1 antigen from rabbits were evaluated in the present study.

On comparing the animals for antibody responses, Animal 1 displayed higher OD at 500 ng rDNS1Ag concentration. The absorbance level gradually decreased from 6 at 1:10 to 2.5 at 1:10000 dilutions. A similar study with rabbit polyclonal and monoclonal antibodies tested by Young et al.,²³showed the optimum dilution of 1:1500 for the absorbance. Further, the RPAD was tested by Western blotting which showed it as highly specific for detection of rDNS1Ag. We also purified the IgG using a commercial polyclonal antibody purification kit. The SDS-PAGE analysis of protein fractions showed two distinct protein bands at 25 kDa and 50 kDa corresponding to the IgG.

The sensitivity of RPAD for the detection of NS1 antigens of all the serotypes of DENV was carried out. It was observed that Animal-1 exhibited higher absorbance (OD ≥ 1) for Dengue serotypes 1, 2 and 3. In a similar study, with the polyclonal antibodies, as low as 1 ng per ml of NS1 protein of DENV-1 serotypes was detected in the dengue patients. However, the antibodies were not sensitive to detect NS1 of other serotypes of DENV as the assay was no less than 10 times lower for serotypes other than DENV-1⁵. Since the polyclonal antibodies were purified against the NS1 antigen of the DENV-1 serotype, they were less sensitive to detect the NS1 antigen of other serotypes. On the contrary, we observed that RPAD displayed lower absorbance values for serotype 4 only. Further, to test the specificity of RPAD, for detection of recombinant NS1 antigens of only rDS1Ag, NS1 antigens of flaviviruses were tested. It was observed that none of the animals exhibited absorbance against the NS1 antigens of flavivirus indicating that RPAD is specific towards dengue only. We also compared the RPAD with commercial anti-DNS1 antibodies for the detection of rDNS1Ag. It was observedthat RPAD (as a secondary antibody)displayed almost similar antibody responses to that of anti-DNS1Ab. However, at 1 ng RPAD coating, RPAD displayed higher antibody responses as compared to anti-DNS1Ab. It is interesting to note that these observations were at 1:5000 dilutions of RPAD which is two-fold higher than the anti-DNS1Ab (i.e., 1: 2500).

Overall, using the RPAD, an in-house assay to detect recombinant dengue NS1 antigen is developed. Our preliminary study suggests that RPAD-ELISA may be further explored todiagnose dengue virus infection in humans as well as mosquito samples after laboratory confirmation. However, laboratory experiments require facilities such as insecticide biosafety level laboratories, culture and maintenance of dengue viruses and approval of the biosafety committee.

Competing interests

The authors declare no competing interests.

Author Contribution

PRA: Conceptualization, Data curation, Formal analysis, Project administration, Visualization, Supervision.; PD: Resources; BL & EP: Methodology, Formal analysis, Investigation. Writing – original draft; AD: Validation, Formal analysis, Writing –review & editing; AK: Funding acquisition, Resources. All authors reviewed the manuscript.

Acknowledgement

The project was funded by the Indian Council of Medical Research-Vector Control Research Centre, Puducherry, through an intramural grant (IM-1903). We thank Dr. Arunkumar Dhayalan, Dept. of Biotechnology, Pondicherry University for the Western Blotting experiment. The Authors are thankful to Mr. S. Rajkumar for the technical support for maintaining the Animals for the study. We express gratitude to Dr. S. Poopathi, for the facilities of SDS-PAGE.

Data availability statement

The data supporting the conclusions of this article are included within the article. The datasets analysed during the present study are available from the corresponding author (PRA) upon reasonable request

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Evaluation of polyclonal antibodies raised in rabbit against Dengue NS1 antigen

Status:

Version 1

Abstract

Figures

1. INTRODUCTION

2. MATERIALS AND METHODS

2.1. Ethics statement

2.2. Raising polyclonal antibodies against recombinant dengue NS1 antigen in Rabbits:

2.3. Developing RPAD-ELISA for detection of NS1 antigen:

2.4. Detection of Dengue NS1 antigen using RPADby Western Blotting:

2.5. Purification of polyclonal antibody (IgG) from a rabbit immunized with rDNS1Ag:

2.6. Comparison of RPAD with Commercial DNS1 Antibody

3. RESULTS

3.1. ELISA for detection of recombinant dengue NS1 antigen using RPAD:

3.2. Western Blot analysis of dengue NS1 antigen using RPAD.

3.3. SDS-PAGE analysis of RPAD

3.4. RPAD is sensitive for detection of dengue NS1 antigen

3.5. RPAD is specific forthe detection of dengue NS1 antigen only

3.6. Comparison of RPAD with Commercial DNS1 Antibody

4. DISCUSSION

Declarations

Competing interests

Author Contribution

Acknowledgement

Data availability statement

References

Additional Declarations

Supplementary Files

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