Technical Field

This present invention relates to the field of medical diagnostics and detection, particularly of viral pathogens. More particularly, it relates to diagnostics using avian antibodies to detect the presence of dengue viruses in biological samples.

Background of the invention

Dengue fever (DF) is a vector-borne disease that causes acute febrile illness. It is very common in tropical and subtropical areas mainly in Asia, where the vectors Aedes aegypti and Aedes albopictus thrived. Worldwide, the prevalence of the disease has grown 30-fold, affecting more than 100 countries (Coffey et al. 2009). In 2007, the worldwide incidence is about 50 million with 2.5 billion of the population being at-risk for the infection (WHO 2009). In the Philippines, the incidence ranged from 4,000 to 23,000 from reported cases from 2001 to 2008 (NSCB 2011). Without treatment, the case fatality rate could be as high as 20% (WHO 2009).

The natural history of dengue involves 5-7 days of incubation followed by a week of fever (Nishiura and Halstead 2007). The fatal complication of dengue fever is dengue hemorrhagic fever (DHF), which usually occurs just when the fever subsides, and it is characterized by vascular permeability, hypovolemia, and severe shock (Gubler 2002). About 2-3% of afflicted develops complications.

Dengue is caused by four related dengue viruses, dengue virus 1 to 4 (Guzman and Kouri 2002). Of the four strains, a second infection involving strain 2 is more commonly associated with the development of DHF.

For the most part, mortality in dengue is due to misdiagnosis as treatment is readily available and simple. This could be due to because the differential diagnosis for acute febrile illnesses are protean and the diagnostic tools cost are costly, These kits are quite expensive mainly because the antibodies used are derived from mammalian sera. A new source of antibody, the chicken egg yolk, offers a cheaper alternative in order to make kits more accessible to patients and health workers. This will likely lower further fatalities associated with DHF.

There are presently several means to test for dengue fever. The most commonly employed diagnostics are real-time polymerase chain reaction (PCR) to detect viral nucleic acids and serological testing for host IgM to test for host response. Other tests include viral culture, viral antigen detection by immunoassays or fluorescence assays (Guzman et al. 2010), and mass spectrometry (US 13124362 A);. The present tests have serious limitations that may compromise their utilization in some communities. Dengue cultures are expensive, and the results can only be verified after a few days. Nucleic acid detection requires an expensive polymerase chain reaction machine. Other immunoassays use expensive mammalian-based antibodies, which is a major usage limitation in many afflicted communities. Further, immunoassays targeting the expression of host immunoglobulins often lead to false- positive diagnosis. Thus, albeit each having a different design, almost all diagnostics are either expensive or have required an expensive equipment or a specialized laboratory (Guzman et al. 2010).

As expected, the most specific means to diagnosing dengue is by detecting the virus itself. There are several targets for detection ranging from the various components of the virus, such as non-structural protein 1 (NS l), to the different differentially expressed biomolecules in the affected host/patients, such as the cytokine receptor family member ST2 (US200810316; US 13124362A; Guzman et al. 2010).

The invention targets the envelope (E) protein of the virus, 60 kD glycoprotein, as a model marker. It is involved in binding and fusion of the viral particle to the host cell. Modis et al (2003) and Rey (2003) determined the crystal structure and the 3D structure of the viral envelope protein, respectively (Figure 1). Their discovery of the structure of the dengue virus greatly facilitated understanding of the pathogenesis of dengue virus. The structure of the E protein is described by Carlosa (1998) as unusual in that the protein does not extend outward the virion surface but instead lie flat on the membrane. At physiological pH, the envelope protein forms a dimer. However, exposure to low pH induces conformational change, which results to the formation of the homotrimer. This event also triggers membrane fusion of the virus to the host cell. Monocyte macrophages are the principal target cells for dengue viruses. Virus attachment is temperature independent whereas penetration proceeds only at 37°C (Seema and Jain 2005). Two N-linked glycosylation sites (Asp 153 and Asp 67) are present in the envelope protein. Asp 153 is conserved among flaviviruses while Asp 67 is unique to dengue viruses. In mosquito cells, the use of glycosylation sites is serotype specific (Rey 2003). Studies cited by Seema and Jain (2005) demonstrated that mutations on the glycosylation sites greatly affect neurovirulence and virus-mediated membrane fusion. These N-linked glycosylation sites are important for the recognition of the C-type lectin DC-SIGN, expressed by dendritic cells on the skin, which aids in the efficient entry of the virus into cells (Seema and Jain 2005; Rey 2003). Analysis of the nucleic acid sequences of 17 several flavivirus E genes showed perfect conservation of 12 cysteine residues, which forms six disulfide bridges (Henchal and Putnak 1990). The location of these disulfide bridges are said to be correlated to the structural properties of different epitopes present on the envelope protein. Three domains compose the E protein, namely, domain I, domain II, and domain III (Figure 1). Domain I (DI) contains predominately type-specific non-neutralizing epitopes. It is theorized to be the molecular hinge region involved in low-pH- triggered conformational change. Domain II (Oil) is the dimerization domain. It makes important contacts with itself in the homodimer and is involved in virus-mediated membrane fusion. Domain III (Dili) has an immunoglobulin like structure. It comprises amino acids 300 - 395 within a 500 amino acid envelope protein (Sim, A.C.N et al. 2007). It is highly stable, folds independently and lies exposed and accessible on the virion surface (Babu, J.P. et al. 2008). Dili contains the host-cell binding anti-receptor and most of the epitopes located on this domain are multiple type- and subtype-specific that only elicit virus neutralizing monoclonal antibodies (Crill and Roehrig 2001). Mapping of neutralizing epitopes showed that epitopes within Dili elicited the strongest monoclonal antibody blockers of virus absorption (Crill and Roehrig 2001 ; Wahala, W.M.P.B et al. 2009; Sukupolvi-Petty et al. 2007). Dill reactive antibodies were found to be present in primary and secondary dengue virus immune human sera. These antibodies recognize mainly type specific epitope after a primary infection and a cross-reactive epitope after a secondary infection (Wahala, W.M.P.B et al. 2009). Cross-reaction studies by Falconar (2008) and Crill and Chang (2004) reveals that DII contains most of the major flavivirus group and sub-group cross-reactive epitopes, which are sensitive to reduction and denaturation and are formed from discontinuous amino acid sequences.

Avian immunoglobulin, otherwise known as immunoglobulin Y (IgY), is gradually receiving more attention from the scientific community as an alternative source of polyclonal antibodies mainly because of the less invasive manner in harvesting the antibody and the high yield that results after isolation and purification. Immunoglobulin Y is often compared to mammal-derived IgG since IgG is one of the antibodies primarily used in diagnostics and therapeutics. Following the idea of refinement and reduction in the use of laboratory animals, IgY presents several advantages over IgG. First, the cost of breeding chickens is relatively much cheaper than breeding rabbits or goats. Also, more antibodies are generated from one egg compared to bleeding a rabbit. A single egg can generate up to 75% antibodies after purification process. Bleeding of animal is also omitted making this approach more appealing and easier to perform. The phylogenetic distance between birds and mammals also adds to the advantage of IgY over IgG since: a) a higher probability of producing antibodies of greater specificity to mammalian antigens can be produced, b) humoral immune response is ensured, c) non-specific binding is reduced because IgY does not bind bacterial or mammalian Fc receptors and does not cross-react with mammalian IgG, and d) the need for cross-species immunoabsorption is eliminated (Cova 2005; Leslie and Clem 1969; Malmarugan et al. 2005; Raj et al. 2004): Lastly, IgY is relatively stable. A study by Larsson and colleagues (1993) shows that purified chicken IgY can be stored for up to 10 years at 4°C, for 6 months at room temperature, and for 1 month at 37°C without significant loss of antibody activity (Bizhanov et al. 2004; Leslie and Clem 1969; Malmarugan et al. 2005; Raj et al. 2004).

IgY is characteristic of other egg-laying animals, such as birds and reptiles. Examples of avian IgY sources include the ducks, quails and the ostriches (Adachi et al. 2008; Magor et al. 1992). In reptiles, IgY has been found in lizards (Wei et al. 2009), snakes (Deza et al. 2007; Gabon-Deza et al. 2012), turtles (Xu et al. 2009). Such IgY should generally possess similar properties cited above in terms of advantages compared with mammalian antibodies. In addition, the serum antibodies of such animals could also be used for generate specific antibodies against various non-self antigens, and may substitute for the egg immunoglobulins.

IgY has been utilized in the detection of both bacterial and viral infections. A study by Sunwoo, et.al (2006) demonstrated that IgY can detect the presence of Escherichia coli 0157:H7 where IgY was used as the capture antibody. As low as 40 CFU/ml of the bacteria can be detected using this method. Kim, et.al (2005) developed a sandwich ELISA using HRP-labeled IgY for the detection of Listeria spp. Sensitivity and specificity of the developed ELISA was analyzed and showed that no cross-reactivity with other bacteria tested occurred, including Salmonella enteritidis and E.coli 0157:H7. The detection limit of the ELISA using IgY was compared with ELISA using a pair of monoclonal antibodies. IgY detection limit was 10 times lower than MAb pair. A sandwich ELISA was also developed for the detection of enterotoxigenic Escherichia coli (ETEC) 88+ antigen. This test by Kim, et.al (1999) is quantitative rather than qualitative. Nevertheless, it showed that the assay using IgY antibody is sensitive and specific and can provide a good estimate of the fimbrial protein or the number of K88+ ETEC iii the sample. Malmarugan, et.al. (2005) were able to produce and purify anti-infectious bursal disease (IBD)-IgY while Lee, et.al (2002) used indirect ELISA in detecting the presence of Salmonella enteritidis and Salmonella typhimurium.

Some patented use of IgY include the detection of human telomerase reverse transcriptase (WO2006US24750), of the pili of Escherichia coli (KR199781987) and of the flagella of Salmonella typhi (KR20006341 1). The IgY product were intended for both diagnostic and therapeutic purposes. No prior art on the use of IgY in dengue was obtained.

Thus, our IgY-based dengue diagnostic has several advantages over existing prior art. First, it is accurate as it detects the viral antigen themselves which is crucial in the early diagnosis. Second, the test result is immediately obtained as immunoassays can be done within hours after collecting the samples from the patients. Third, it is less expensive than the more commonly used mammalian antibody because specific IgY in an egg is more than 100 more than the amount collected from a mouse, without killing the hen and without more inoculation with expensive antigens. Further, there is no need for complex equipment or a specialized lab, compared with PCR or viral culture. Fourth, the test is easily reproducible, as batches of IgY are stable and can be kept in bulk, and small peptides will be injected (making the antibodies pseudo-monoclonal). Lastly and most important, the test can be done in the point-of-care setting because immunoassays can be designed, as in the case of lateral flow assays, or sugar-complexed ELISA kits. In summary, IgY offers a cheaper and likely more sensitive means in the diagnosis of pathogens, such as the dengue virus. Summary of the Invention

Thus, the objective of the invention is to provide an alternative means to diagnose dengue fever, which is accurate, rapid, inexpensive, easily reproducible, and can be done in the point-of-care. The invention comprise immunoassays that will utilized chicken antibodies, which are abundant in the egg, and has been consistently found to perform well in various detection assays. Summary of Figures and Drawings

Figure 1 shows a representation of the structure of the envelope (E) protein of the dengue virus (as shown in Rey, F.A. 2003. Dengue Virus Envelope Glycoprotein Structure: A New Insight Into Its Interactions During Viral Entry. PNAS. 100:6899- 6901.

Figure 2 shows a schema for IgY extraction and purification by Ko and Anh

(1978) with modifications.

Figure 3 shows a schema for IgY extraction and purification by Shin and colleagues (2003) with modifications.

Figure 4 demonstrates the chromatographic profile of extracted IgY composed of non-denaturing (Panel A) and denaturing (Panel B) of representative IgY isolates.

Lane designations in Panel A: Lane 1, Standards; Lane 2, baseline isolate; Lane 3, first booster isolate; Lane 4, 2 weeks after immunization; and Lane 5, Kaleidoscope molecular weight markers. Lane designations in Panel B: Lane 1, IgY standard; Lane

2, baseline isolate; Lane 3, primary immunization isolate; Lane 4, first booster isolate; Lane 5, second booster isolate; Lane 6, 2 weeks after immunization; and Lane 7,

Kaleidoscope molecular weight markers.

Figures 5 is a graph showing peptide with SEQ ID #1 -antipeptide 1 activity at different concentrations demonstrating a concentration dependent increase in the absorbance reading reflective of activity.

Figure 6 is a graph showing peptide with SEQ ID #2-antipeptide 2 activity at different concentrations demonstrating a lesser concentration dependent increase in the absorbance reading reflective of activity.

Figure 7 details the binding activity of antipeptides for SEQ ID #1 and SEQ ID

#2 against whole dengue viruses showing the overall binding activity of antipeptide 2 is lower compared with antipeptide 1. Panel A-D correspons to serotype 1-4, respectively.

Figure 8 details the viability of binding of the antipeptides until 10 weeks. Absorbance reading of antipeptide 1, antipeptide 2 and negative control (from baseline to 2 weeks after last immunization) against dengue virion serotypes 1 -4. Panel A-D correspons to serotype 1-4, respectively.

Description of the Invention

The present invention involves constructing an immunoassay using dengue- specific IgY. The embodiment is preferably an enzyme-linked immunosorbent assay (ELISA) test in which the biological sample is applied for a while to the solid substrate so the dengue protein can adhere to it. After blocking the dengue-specific IgY is applied to the container holding the substrate and will be incubated with the adhered sample. After further incubation, a secondary antibody conjugated to an enzyme is applied, and subsequently, a substrate is applied to be catalyzed by the conjugated enzyme. The presence and degree of colorometric change will be compared to control to indicate the positivity/negativity of the test.

The assay is optimized to work in the following conditions: a primary antibody specific to epitopes PI or P2 (see below) with the minimum concentration of 0.125 mg/ml and 1 mg/ml, respectively, with maximum recommended concentration up to 2 mg/ml. The epitopes can be extended to any specific dengue epitopes that is suspected to be of practical application for a test.

The following description teaches the means and justifies the parameters for the invention:

Immunization and egg collection

The antibody are produced in chicken hens injected at day 0, day 14 and day 28, with a peptide from designed from a surface epitope present on the domain III of the dengue virus envelope glycoprotein with sequence 352- ITVNPIVTEKDSPV E- 368. From the studies done by Roehrig, JT et al (1990) and Amexis and Young (2006), this amino acid sequence was able to produce high anti-dengue 2 titers and elicit virus neutralizing antibodies when used as an immunogen on BALB/c mice. N- terminal acetylation and C-terminal amidation was added as modification by the manufacturer to ensure better stability and to mimic the Dengue virus glycoprotein which can increase the biological activity of the peptide. Two forms of this peptide were used in this study. Peptide 1 (PI) was purchased conjugated to BSA at the N- terminal using glutaraldehyde as linking reagent, while Peptide 2 (P2) has cysteine residues on both N and C terminals (Table 1). Though both proteins can discriminate dengue, PI seems to be better than P2. For the immunization, the protocol for polymerization was adapted from the work of Frisco, H. et al (2010) with modification. Briefly, one milligram of peptide 2 was dissolved in 20 μΐ of carbonate buffer (l .OM, pH 9.6) and incubated overnight at 4°C. Nine hundred eighty microliters of PBS was added the following day to make 1 ml solution. For the BSA - conjugated peptide (PI), a stock solution of 1 mg/ml was prepared using PBS. To prepare the immunogen, 600 μΐ of the peptide stock solution (1 mg/ml) was emulsified with an equal volume of Freund's Complete Adjuvant. A total of one milliliter of the emulsified solution was injected into four different sites (250 μΐ per site) at the pectoral muscle of each chicken. Intramuscular administration was selected since it results to higher antibody levels with high specificity (Dias da Silva and Tambourgi 2009). Booster doses were administered at 2 and 4 weeks after the primary immunization. The booster doses contain the same component as the primary immunization except for Freund's Incomplete Adjuvant. The negative control group was injected with PBS for all immunizations.

Table 1. Amino acid Sequence of Peptides Used in the study

Isolation of IgY

Extraction of IgY was initially carried out (for the first set of hens) using the method described by Ko and Ahn (2007) with modification (Figure 2). Briefly, the yolk was diluted with 9 volumes of acidified (pH 2.5) cold distilled water, homogenized and centrifuged at 2,800 x g for 40 minutes at 4°C. After centrifiigation, the supernatant was collected and 0.1% (w/v) λ-carrageenan was added to remove residual lipoproteins in the supernatant. The mixture was centrifuged again at 2,800 x g for 30 minutes at 4°C. The supernatant was filtered using Whatman no. 1 and 40% (w/v) ammonium sulfate was added gradually to the filtrate. The pellet was collected through centrifugation at room temperature and dissolved in 0.01M Phosphate Buffered Saline (PBS) pH 7.4. The isolated IgY was stored at -20°C until use.

A similar but simpler method by Shin et al (2003) was also performed using the eggs from the second set of hens (Figure 3). In this method an equal volume of distilled water was added to the yolk contents followed by an equal volume of 0.15% (w/v) λ-carrageenan. The mixture was then set to incubate for 30 minutes at room temperature. After centrifugation at 10,000 x g at 4°C for 30 minutes, the supernatant was collected and filtered using Whatman filter paper no. l . To the filtrate, 19% (w/v) of sodium sulfate was gradually added and the solution was centrifuged for 30 minutes at room temperature to collect the precipitate. The precipitate was dissolved in 0.01M PBS pH 7.4 and was passed through Econo-Pac 10DG desalting column (Biorad) to remove excess salt which can interfere with the succeeding assays such as electrophoresis and ELISA. The desalted solution was then lyophilized to increase shelf-life.

Assessment of IgY purity

To determine the presence and purity of the IgY in the isolates, Native PAGE was performed. The presence of a protein band similar to the electrophoretic profile of the IgY standard confirms the presence of IgY for all weekly isolates (Figure 4). However, several other protein bands were also observed indicating that the isolates are not yet pure. This is also one explanation why the protein concentration of the IgY isolates exceeds the amount reported by Akita and Nakai (1992) and other researchers. Figure 4 shows the electrophoretic profile of IgY isolates that were subjected to desalting and lyophilization. A 1 mg/ml solution was prepared from the powdered sample after lyophilization and diluted 1 :2 with the sample buffer. Fifteen microliters of the diluted sample was loaded to each well. This profile shows that at low concentrations of the isolates, we can greatly reduce the presence of other proteins while still having reasonable amounts of IgY. However, the IgY present in these bands is still a mixture of specific and non-specific IgY. Assessment of stability of IgY samples

To determine the stability of the IgY isolates, representative samples from the group PI of the first set of hens were subjected to indirect ELISA. Different peptide sequences available at the Department of Biochemistry and Molecular Biology were used as capture antigen to test the IgY activity and specificity. Absorbance readings for DEN, PI and P2 were above the cut-off value by 9.43%, 106.6% and 54.72%, respectively, indicating a positive Peptide-Antipeptide interaction. While no peptide- antipeptide interaction was observed for HBV-1 and HBV-2. The results also reveal high stability for the protein which is consistent with the findings of Larson et al (1993) that IgY preparations can be stored up to 10 years at 4°C, for 6 months at room temperature and 1 month at 37°C.

Assessment of antibody activity towards the antigens

Indirect ELISA was performed to assess the peptide-antipeptide activity at different concentrations. Tables 2 and 3 shows the summary of the checkerboard titration performed for Peptide 1 (PI) versus Anti-peptide 1 (API) and Peptide 2 (P2) versus Anti-peptide 2 (AP2), respectively. The highlighted values indicate absorbance readings higher than the computed cut-off value. The computed cut-off value for Table 2 is 0.104 while Table 3 has a cut-off value of 0.095. The relative activity is also shown in Figures 5 and 6, respectively.

Table 2. Summary of the Absorbance Readings (450nm) for Checkerboard Titration Performed for Peptidel (PI) and Anti-Peptide 1 (API)

*Cut-off value is computed at 99.0% confidence interval: 0.104 Table 3. Summary of the Absorbance Readings (450 nm) for Checkerboard Titration Performed for Peptlde2 (P2) and Anti-Peptide 2 (AP2)

*Cut-off value is computed at 99.0% confidence interval: 0.095

Comparison of the anti-peptide activity of API and AP2 against their respective peptides shows considerable difference. While API can detect peptide concentrations up to 0.625 μg/ml, AP2 is limited only to the highest concentration which is 10 μ^ηιΐ. This difference may be attributed to the modification incorporated to Peptide 2. Cysteine residues were added on both the N and C terminals of the peptide fragment to induce polymerization thereby increasing the immunogenicity. However, this technique produces a wide range of oxidized peptidic species ranging from the cyclic monomer and dimer to high molecular weight linear peptides (Andreu et al. 1994; Guzman et al. 2007). Chemical control and scrupulous attention to experimental details such pH, temperature, time and concentration should be regarded when using this technique (Andreu et al. 1994). In this study, Peptide 2 fragments may have formed several different looped/cyclic structures or aggregates which hindered their accessibility when used as capture antigen for indirect ELISA thus lowering the signal produced. This finding is similar to the work of Frisco et al (2010) using unconjugated polymerized peptide. The HPLC profile of Frisco et al (2010) shows a mixture of polymerized peptides: cyclic and linear which may have occurred during the 24-hour oxidation period. Since indirect ELISA requires coating the antigen, in this case the peptide, onto the 96-well plate, aggregation or formation of looped structures may have caused high-density binding of antigen which promoted steric inhibition and prevented antibody binding (Crowther 1995). Assessment of affinity to all strains of dengue

When tested against Dengue virions, both Antipeptide 1 (API) and Antipeptide 2 (AP2) showed increasing binding activity as the viral concentration increases for all Serotypes (Figures 7 and 8). Although the binding activity of Anti- peptide 2 (AP2) is not as high as the binding activity Anti-peptide 1 (API), this data illustrates that both API and AP2 are able to recognize and react with the intact Dengue envelope glycoprotein.

Anti-peptide 1(AP1) and Anti-peptide 2 (AP2) were isolated and purified from chicken egg yolks immunized with synthetic peptides which were supposed to be analogues of epitopes found exclusively on the envelope glycoprotein of Dengue Serotype 2. The results of the binding affinity assay showed otherwise. Sequence alignment of the Domain III envelope glycoprotein of the four serotypes showed very similar amino acid sequences. Differences in the amino acid sequence seem to be negligible since amino acid changes have the same polarity or size which does not affect the overall three-dimensional structure of the epitope on the virus. As an example the shift from Valine (Serotype 2) to Alanine (Serotype 1) on amino acid 353 or the shift from isoleucine (Serotype 2) to valine (Serotype 3) on amino acid 357 confer little effect on the three-dimensional structure of the epitope. This is further supported using the 3D models downloaded from NCBI Molecular Model Database. The amino acids 358-362 which are located on the exposed portion of the loop have little or no difference in their properties across the four serotypes. In fact, three out of the five amino acids are identical for serotypes 1 to 3. Surprisingly, Anti-peptide 1 and Anti-peptide 2 also gave acceptable binding activity for serotype 4. This result may have been due to the difference in the binding capacity of mammalian and avian antibodies. Schade et al. (1996) noted differences in the binding capacity of chicken and rabbit antibody when tested against cholecystokinin octapeptide. He noted that avian antibody was more directed to short sequences and that longer sequences are less well recognized which he attributed to the structural organization of IgY. Beltramello et al (2010) generated 70 monoclonal antibodies (mAbs) against the Dengue virus envelope glycoprotein. Of the 70 Dengue virus reactive mAbs, 13 were mapped to domain III of the envelope glycoprotein. Only five of the 13 mAbs were serotype specific whereas the rest were cross-reactive to two, three or all four serotypes. Regardless of being serotype specific or cross-reactive, antibodies generated against the domain III of Dengue virus envelope glycoprotein are potent in neutralizing Dengue virus infections. According Beltramello et al (2010) cross- reactive mAbs were able to neutralize infection in the same range concentration as observed for serotype-specific mAbs. This finding may be beneficial for this study since the epitope sequence chosen may be used as source for the production of neutralizing antibodies which can be used for immunotherapeutic or immunodiagnostic purposes.

Figure 8 shows the change in specific activity of IgY during the immunization period. A sharp increase in activity can be observed on week 2 (first week of primary immunization) for both Anti-peptide 1 and Anti-peptide2 when reacted with the Dengue virion (serotype 1 to 4). The activity was maintained until 2-weeks after the last immunization.

Assessment of binding specificity

Anti-peptide 1 (API) and Anti-peptide 2 (AP2) were tested against several other peptide sequences available in the Department of Biochemistry and Molecular Biology to the check binding specificity. Table 4 summarizes the Peptide-Antipeptide binding specificity assay. The computed cut-off value is 0.53. Although E2 which is the Negative Control group gave positive response based on the cut-off value for DEN, PI and P2 peptides, an absorbance reading with 0.02 difference can be regarded as insignificant. Anti-peptide 1 (E3) and Anti-peptide 2 (E4) also gave positive response for DEN, PI and P2 which is expected.

Table 4: Peptide-Antipeptide Binding Activity of IgY Isolates expressed as Absorbance in 450

Alternative Embodiments

The invention can further be extended by using other avian sources, such as ducks, turkeys, quails and other birds; and reptilian sources, such as lizard, snakes, alligators, crocodiles and turtles.

The IgY antibodies may exist in suspension, solution, lyophilized matter, or conjugated to various means for test reporting, storage, preservation or for other beneficial purposes.

The IgY antibodies can further be purified by methods, such as affinity chromatography or other means to enhance its activity or storage.

Moreover, the immunoassay methods cited above can be modified, such as using sandwich-type immunoassay, multiplex-type and radial immunoassays.

The detection method may also vary, including, but not limited to, flourescence, radiation, and heat-sensing.

The use of the antibody may utilize other means, such as lateral flow assays, Western blots, immunochemistry and even, in vivo detection assays.

The antibody produces can be used for the diagnosis and prognosis of dengue. It can also be used as a monitoring means to assess responses to treatment. The methods and the products can be used for research and educational purposes to study the dengue virus, and the pathogenesis of dengue fever.

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