Claims:WE CLAIMS,
1. A method for detecting dengue, malaria and chikungunya infection in a human subject, the method comprising:
(a) providing at least one dengue, malaria and chikungunya specific antibody;
(b) testing at least one dengue, malaria and chikungunya specific antibody with a biological sample obtained from the human subject, the biological sample suspected of containing at least one binding partner capable of binding to at least one of the dengue, malaria and chikungunya specific antibody; and
(c) detecting the presence of a complex that forms between at least one of the dengue, malaria and chikungunya specific antibody and at least one binding partner, which presence indicate as infection in the subject.
2. The method of claim 1 wherein the binding partner to dengue antibody is the NS1 antigen causing dengue.
3. The method of claim 1 wherein the binding partner to malaria antibody is the Msp1 and Msp3 antigens causing malaria.
4. The method of claim 1 wherein the binding partner to chikungunya antibody is the chkE2 antigen causing chikungunya.
5. The method according to preceding claims, wherein dengue antibody is constructed against wing domain of NS1 antigen set forth as Seq. ID. No. 4.
6. The method according to preceding claims, wherein malaria antibody is constructed against fusion of Msp1 and Msp 3 antigen set forth as Seq. ID. No. 5.
7. The method according to preceding claims, wherein chikungunya antibody is constructed against ChkE2 antigen set forth as Seq. ID. No. 6.
8. The method according to claim 1, wherein, the antibody is labelled with a gold colloid of size in the range of 30 to 50 nm.
9. The method according to claim 9, wherein, the biological sample is whole blood, plasma or serum.
10. The method according to any one of the preceding claims, the method further comprising:
(a) providing a solid support having a first end and a second end;
(b) immobilising the colloidal gold labelled dengue, malaria and chikungunya antibody on the solid support;
(c) loading the sample on the solid support; and
(d) loading a buffer on the solid support, wherein, the presence of the complex formed on the solid support is detected by the labelled moiety of the molecule.
11. The method according to claim 11, the buffer is assay buffer having composition of distilled water, phosphate buffer, sodium chloride and detergent.
12. The kit for detecting a dengue, malaria and chikungunya infection in a human subject, the kit comprising:
(a) a solid support with dengue, malaria and chikungunya specific labelled-antibodies immobilized.
(b) an assay buffer.
13. The kit according to claim 13, wherein, solid support is a nitrocellulose membrane, ranging from 0.4 micron to 1.2 micron
14. The kit according to claim 13, wherein, the antibodies are labelled with colloidal gold particles of spherical shape/urchin shape/diamond shape.
15. The kit according to claim 13, wherein, biological sample obtained from human subject is loaded on solid support directly.

Dated this 22nd day of April, 2019
Mohsin R. Arabiani
(Agent of the applicant)
, Description:FIELD OF INVENTION
The present invention relates to the field of diagnostic assays.
BACKGROUND OF INVENTION
Diseases such as Dengue, Chikungunya, Malaria caused by pathogenic agents pose a significant public health issues due to its severity, high lethality, inter-human contagiousness, and lack of effective treatment. All three diseases show similar symptom of high-grade fever followed by severe body pain. Thus, it becomes difficult to distinguish the type of disease on the first day of the symptom.
The treatment for malaria, dengue and chikungunya is symptomatic and therefore, doctors wait for the symptoms to become evident in the body.
Several approaches have been applied for laboratory diagnosis of these viral infection(s). These methods include detection of the virus (by cell culture, immunofluorescence), detection of virus antigen (by enzyme-linked immunosorbent assay [ELISA]), detection of virus antibody (by hemagglutination inhibition [HI], complement fixation test [CF], neutralization tests, ELISA), and detection of virus nucleic acid (by real-time reverse transcription-polymerase chain reaction [RT-PCR]). In these state-of-art technologies, the tests for sensitive detection of vector borne diseases include an additional step for pre-enrichment of biological sample to obtain blood plasma/ blood serum. Such step(s) can take one-to-three days, before performing the detection test(s). Since time is a very crucial factor, there was a need to develop the detection test that can provide fast results. Presently, detection kits available in market provide accurate detection of disease in one day. However, the major drawback is that the kits available are not affordable for patients from all economic backgrounds or doctors with small practice. Each commercially available kit provide detection of one disease at a time which is also expensive for the patient. These kits also suffer from low specificity and sometimes sensitivity issues.
There are few detection kits commercially available in market for detection of these vector borne diseases. Most of these kits employ multiplex assay technique based on lateral flow immunoassay.
In WO2016022071, by MP Biomedical Asia Pacific, a multiplex-assay based diagnostic detection method is described for dengue. The method detects dengue specific antibody and antigen. However, the immunoassay described herein is based on lateral flow assay. Similarly, in WO2014151865, by Ran Biotechnologies, simultaneous detection of multiple biologicals is described which could be achieved by aggregating particle, labelling particle etc. These tests do not provide rapid detection, as it requires non-handy ingredients. In WO2015042593, by Assaypro, describes immunoassay using labelled fluorescent probes that provide multiplexing capability. Furthermore, in WO2016057749, by Theranos, method for detecting presence of virus by performing assay to detect antibodies through viral nucleic acid.
In US9086410, by Medmira, describes a rapid diagnostic device which uses principles of vertical/downward flow assay. However, this patent doesn’t elaborate disease specific detection device.
Antigen-based rapid diagnostic tests (RDTs) have an important role since these RDTs are estimated to be simple and the procedure could be performed on the field. These tests use finger-stick or venous blood, the test takes a total of 30 minutes, and a laboratory is not needed. However, commercially available rapid diagnostic tests (RDT) kits, at present, can detect only one disease at a time and hence one by one test takes more time for proper diagnosis.
Thus, there was a need for the development of a detection method that is easy to employ, fast, accurate and capable of detecting a broad spectrum of microorganisms.
Present invention provides a kit for rapid, accurate and simultaneous detection and facilitate differential diagnosis of common symptoms such as fever of Dengue, Malaria and Chikungunya in a single test based on multiplexed detection.

SUMMARY OF INVENTION
Present invention relates to a method for detecting a dengue, malaria and chikungunya infection in a human subject, the method comprising:
(a) providing at least one dengue, malaria and chikungunya specific antibody;
(b) Testing at least one dengue, malaria and chikungunya specific antibody with a biological sample obtained from the human subject, the biological sample suspected of containing at least one binding partner capable of hybridizing to at least one of the dengue, malaria and chikungunya specific antibody; and
(c) detecting the presence of a complex that forms between at least one of the dengue, malaria and chikungunya specific antibody and the at least one binding partner, which indicates the presence of infection in the subject.
In one aspect of invention, the binding partner to dengue antibody is the NS1 antigen causing dengue, to malaria antibody is the fusion of Msp1 and Msp3 antigen causing malaria and to chikungunya antibody is the chkE2 antigen causing chikungunya. The antibody is labelled with gold particle of various forms.
In another aspect of present invention, the method comprises the steps of:
(a) providing a solid support having a first end and a second end;
(b) immobilising the colloidal gold labelled dengue, malaria and chikungunya antibody on the solid support;
(c) loading the biological sample on the solid support; and
(d) loading a buffer on the solid support, wherein, the presence of the complex formed on the solid support is detected by the labelled moiety of the molecule.
In yet another aspect of invention, it relates to the kit for detecting a dengue, malaria and chikungunya infection in a human subject, the kit comprising:
(a) a solid support with dengue, malaria and chikungunya specific labelled-antibodies immobilized.
(b) an assay buffer
(c) Cell separator
wherein, solid support is a nitrocellulose membrane and biological sample obtained from human subject is loaded on the solid support directly. The biological sample is selected amongst group of whole blood, blood serum, plasma.

BRIEF DESCRIPTION OF FIGURES
Figure 1 is overall process of detection in pictorial form wherein the various spots on the device interpret different infections
Figure 2 is interpretation of colours for detection of infection. Image A is finished test performed with the all positive recombinant antigen diluted in negative serum sample and Image B is the all positive recombinant antigen diluted in PBS.
Figure 3 is process flow diagram of antigen production.
Figure 4 is process flow diagram of antigen purification.
Figure 5 is process flow diagram of antibody production. It also relates to immunization and bleeding schedule of the antigen and the antibody respectively.
Figure 6 is process flow diagram of antibody purification.
Figure 7 is process flow diagram of antibody gold conjugate.
Figure 8 is flow diagram of device preparation.
Figure 9 is flow diagram of device testing.
Figure 10 is Flowchart for puri?cation of PfMSPFu24 Drug Substance.

BRIEF DESCRIPTION OF INVENTION
Definition
Throughout the description, the following terms, unless otherwise indicated, shall be understood to have following meanings:
the terms "antibody" or "antibodies" include the entire antibody and antibody fragments containing functional portions thereof. The term "antibody" includes any monospecific or bispecific compound comprised of a sufficient portion of the light chain variable region and/or the heavy chain variable region to effect binding to the epitope to which the whole antibody has binding specificity. The fragments can include the variable region of at least one heavy or light chain immunoglobulin polypeptide, and include, but are not limited to, Fab fragments, F(ab') 2 fragments, and Fc fragments.
The term “biological sample” refers to any samples which have been obtained from a human subject and which might contain antibodies or antigen. In a preferred embodiment, said biological sample is chosen from whole blood, serum, plasma, urine, seminal fluid, cerebrospinal fluid and saliva. A biological sample may also be modified prior to use, such as by centrifugation, dilution, and the like. Accordingly, a biological sample may refer to a homogenate, lysate or extract prepared from a whole organism or a subset of its tissues, cells or component parts, or a fraction or portion thereof.
The term “Chikungunya” refers to a febrile disease that caused by a Togavirus (species Chikungunya virus of the genus Alphavirus) transmitted especially by female Aedes mosquitoes. This is a small, black and white, highly domesticated tropical mosquito that prefers to lay its eggs in artificial containers found in and around homes that may hold water such as buckets, flower vases and other water containers. Following the bite of an infective female mosquito, the virus undergoes an intrinsic incubation period of 3 to 12 days (average 4 to 7 days) after which the person may experience acute onset of fever accompanied by other non-specific signs and symptoms. During this viraemic period (which may be between 2 to 7 days) the virus circulates in the blood of infected humans. The genome of Chikungunya virus is 11.8 kbs and consists two open reading frames (ORF), one contains four non-structural proteins (nsp1-4) and the other contains five structural proteins (capsid, E3, E2, 6he conclK and E1). The virus contains three structural proteins, glycosylated E1 and E2 are embedded in the viral envelope and a non-glycosylated capsid protein E3 associates with E2 during budding and formation of mature virions. Infection with other alphaviruses and CHIKV has revealed that the neutralizing antibody response is primarily directed against E2 and to a lesser extent to E1. In the case of CHIKV, E2, E3 glycoprotein, capsid and nsP3 proteins are targets of the anti-CHIKV antibody response. Both E1 and E2 have been major targets for the development of recombinant subunit vaccine and IgM or IgG based diagnostic assays.
The term “Dengue” refers to an acute infectious disease caused by a flavivirus (species Dengue virus of the genus Flavivirus), transmitted by female Aedes mosquitoes. Dengue virus infection causes a spectrum of illness in humans depending on the infecting virus, the host's age and immunological conditions. It may result in asymptomatic illness or ranges from an undifferentiated flu-like illness (Viral syndrome) to dengue fever (DF), to dengue haemorrhagic fever (DHF), and the severe and fatal dengue shock syndrome (DSS). Dengue virus has four serotypes (DENV1–4), all of which are responsible for the spectrum of disease ranging from benign dengue fever to severe DHF/DSS. The DENV genome contains three structural proteins (capsid, pre-membrane and envelope) and seven non-structural proteins (NS1, NS2a, NS2b, NS3, NS4a, NS4b and NS5). Amongst them, NS1 is a 46–50 kDa glycoprotein expressed in infected mammalian cells in membrane-associated (mNS1) and secreted (sNS1) forms [5]. DENV NS1 is known to be a major target of humoral immunity in DENV infection.
The term ‘immunogenic component” refer to any component derived from cells or otherwise that has been infected with the dengue virus or any antigen or antibody against such antigen etc.
The term “Malaria” refers to a human disease that is caused by sporozoan parasites (genus Plasmodium) in the red blood cells, is transmitted by the bite of Anopheles mosquitoes. Plasmodium MSP1 is a leading candidate for the malaria vaccine, especially a blood-stage vaccine, as it is abundantly expressed in the intraerythrocytic cycle (ring, trophozoite, and schizont stages). Plasmodium MSP1 is known as a useful marker to differentially diagnose falciparum and vivax malaria (Cho et. al. Int J Biol Sci. 2016 May 25;12(7):824-35). MSP1 is proteolytically cleaved into a 42-kDa C-terminal polypeptide at the time of invasion into red blood cells. It is further processed to 33-kDa and 19-kDa fragments. The 19-kDa C-terminal fragment remains on the surface of the merozoite and is highly immunogenic. MSP-Fu24 has conserved regions of the two leading malaria vaccine candidates, Plasmodium falciparum merozoite surface protein 1 (C-terminal 19-kDa region [PfMSP-119]) and merozoite surface protein 3 (11-kDa conserved region [PfMSP-311]).
The term “Multiplex Technology” refers to a multiplex immunoassay which is a type of assay used in research to simultaneously measure multiple analytes (dozens or more) in a single run/cycle of the assay. It is distinguished from procedures that measure one analyte at a time.

The present invention provides a multiplex technology based rapid, accurate, sensitive and specific test kit for detection of malaria, dengue and chikungunya antigen in single test.
More particularly present invention relates to a kit which is a device for detection of all three vector borne diseases namely Malaria, Chikungunya and Dengue together, wherein, separate kits for each individual disease is not required. Due to the sensitivity of this detection kit, it can detect the disease on first day of symptom.
The inventors have used recombinant proteins to develop antibody against antigen of respective virus, wherein, this antibody is used in the kit for detection. Since it is antigen-based detection technique, the kit can provide accurate results and does not show false positive results.
Due to use of antigen-detection technique, the conclusive test can be conducted by present invention detection kit on the first day of symptom. The technique provides added advantage of rapid in terms of time.
In one embodiment, the detection is colour based, thus interpretation of results is easier and can be done by layman upon reading the manual.
In preferred embodiment, the instrument shall be portable as seen in figure 2. This simplicity feature provides added advantage of easement of use of RDT on field.

Rapid, sensitive, multiplex-technology based infection detection kit
The present invention relates to a rapid, accurate, sensitive and specific test kit for detecting malaria, dengue and chikungunya infection in a human subject.
Inventors prepared NS1 antigen for dengue, fusion of truncated MSP-1 and truncated MSP-3 antigen for malaria and chkE2 antigen for chikungunya using protol described further in the description. The antibody was prepared against the antigen for the purpose of detection of infection.
In present invention, there is provided a method for detecting a dengue, malaria and chikungunya in a human subject. The method comprising: (a) providing antibody against NS1, Msp1 and chkE2 specific antibody; (b) testing the antibody with a biological sample obtained from the human subject, wherein the biological sample suspected of containing at least one binding partner capable of hybridising to the antibody; (c)detecting the presence of a complex that forms between the at least one of the antibody and the at least one binding partner, which presence indicates a infection in the human subject.
Binding partner is meant to include any molecule or component that can bind to a specific antibody. Any such binding method may be used that is known to a person skilled in protein characterization techniques. The present invention use colloidal gold as binding partner. The process of conjugation of antibody with colloidal gold is described further in description.
The present invention further relates to a kit wherein the colloidal gold lebelled antibodies for dengue, malaria and chikungunya are immobilized on a solid surface.
Preferably, the solid support is selected from the group comprising of a bead, a disc, a magnetic particle or a fiber optic sensor, a microtitre plate, glass slide or biological microchip or a membrane including nitrocellulose membranes, polytetrafluorethylene membrane filters, cellulose acetate membrane filters and cellulose nitrate membrane filters with filter paper carriers. Preferably, the solid support is a nitrocellulose membrane.

The kit further comprising: (a) a solid support having a first end and a second end; (b) immobilising the dengue, malaria and chikungunya antibodies on the solid support, the anti-human antibodies are immobilised separately and spaced apart from each other along the solid support; (b) loading the sample on the first end of the solid support,; and (c) loading a buffer on the second end of the solid support adjacent or on the antigen such that the buffer solution, wherein the presence of the complex formed on the solid support is detected by the labelled moiety.
Preferably, the kit comprises antibodies for dengue, malaria and chukungunya immobilised on the solid support separately and spaced apart from each other. More particularly the chikungunya on top spot, malaria on left spot and dengue on right spot of the solid support.

The process workflow of present invention summarized as follows:
Step 1: Antigen production (as shown in Figure 3)
Step 2: Antigen purification (as shown in Figure 4)
Step 3: Antibody production (as shown in Figure 5)
Step 4: Antibody purification (as shown in Figure 6)
Step 5: Preparation of Antibody Gold Conjugate (as shown in Figure 7)
Step 6: Preparation of Immuno Filtration Flow – Through Devices (as shown in Figure8)
Step 7: Device Testing (as shown in Figure 9)

Example 1: Detection kit preparation protocol
The requirements of kit are:
1. Antibodies for Dengue, Malaria and Chikungunya
2. Flow Through cassettes assembled with Nitrocellulose membrane
3. Chemicals and bio materials for coating of antibodies and gold conjugation
4. Consumables like droppers, pipettes, tips, & cell separator etc.

The procedure for the infection detection test using the kit is:
1. Coating of the devices
2. Preparation of colloidal gold conjugate
3. Preparation of the Assay buffer
4. Testing protocol
5. Result Interpretation

Step 1 Coating of the devices
Dengue Stock: 5 mg/ml to 10 mg/ml, preferably, 8.25 mg/ml
Malaria Stock: 5 mg/ml to 10 mg/ml, preferably, 7.28 mg/ml
Chikungunya Stock: 10 mg/ml to 15 mg/ml, preferably, 11.45 mg/ml

1. Preparation of Carbonate Buffer. pH 9.0. 50.0 ml
1. Take a glass container of volume 100 ml.
2. Add 30.0 ml glass distilled water.
3. Add 0.079 gm Sodium carbonate
4. Add 0.1463 gm Sodium bicarbonate
5. Add 0.4 gm Sodium Chloride.
6. Make up volume to 50.0 ml and measure the pH of the buffer.
7. pH of the buffer should be 9.0

2. Preparation of working solution of coating antibodies
1. Take the carbonate buffer prepared above and dilute the antibody solution to 3.0 mg/ml as below
Working solution = (Required Quantity X Required Volume)
Stock concentration
• Dengue : 3.0 mg/ml X 1.0 ml
8.25 mg/ml
= 0.363 ml of antibody solution should be make up to 1.0 ml using carbonate buffer
• Malaria : 3.0 mg/ml X 1.0 ml
7.28. mg/ml
= 0.412 ml of antibody solution should be make up to 1.0 ml using carbonate buffer
• Chikungunya : 3.0 mg/ml X 1.0 ml
11.45 mg/ml
= 0.262 ml of antibody solution should be make up to 1.0 ml using carbonate buffer
Goat Anti Mouse IgG should be taken as 2.0 mg/ml solution for the control spot.

1.3 Coating of the devices.
Take the devices assembled with the 0.6 micron nitro cellulose membrane. The dual absorbent pad of 2 X 2 CM2 are kept just below the nitro cellulose membrane.
The various spots on the device interpret different infections (as shown in figure 1).
Top Spot : Chikungunya
Left Spot : Malaria
Right Spot : Dengue
Centre Spot : Goat/rabbit Anti Mouse IgG for control.

Make the spot of 0.5 µl of each antibodies of all above concentration in the device. Allow the device to dry at 37ºC for 30 minutes and put it into dessicator or aluminum pouch with activated silica bags. The devices should not be exposed to humidity. The coated devices further tested with the colloidal gold conjugates.

Step 2 Preparation of Colloidal Gold Conjugate
2.1 Preparation of Raw Colloidal Gold
1. Take a conical flask of volume 250 ml.
2. Add 100 ml of glass distilled water into it.
3. Add 1.0 ml of 1% gold chloride hydrate solution.
4. Put the flask on hot plate for heating with stirring.
5. Add 2.0 ml 1 % Tri Sodium Citrate Dihydrate solution at the boiling temperature.
6. Keep heating for 20 minutes and cool the solution to room temperature.

The observed optical density of the colloidal gold is 1.012 at maximum absorbance of 520 nm. The size of the colloidal gold particle ranged from 30 to 50 nm.

2.2 Preparation of Colloidal Gold Conjugate
In case of Malaria, Dengue and Chikungunya antibodies, the optimum coupling concentration was found to be 4.0 µg/ml. Therefore 4.0 µg/ml was selected and proceeded further for gold conjugation to all three antibodies.

Conjugation Process
Take a glass container of volume 500 ml and label it as Colloidal gold.
1. Add 150 ml of Colloidal gold into the container.
2. Adjust pH of colloidal gold to 8.0 using 1.0 % sodium carbonate.
3. Take 3 different glass container of volume 100 ml and add 5.0 ml of Phosphate buffer in each container
4. Label each container as Dengue, Malaria and Chikungunya.
5. Add 200 µg of Dengue, Malaria and chikungunya antibodies in respective containers.
6. Add 50 ml of pH adjusted colloidal gold in each container and keep stirring for 60 minutes.
7. Add 0.5 ml of 10.0 % w/v BSA solution in each colloidal gold conjugate solution and stir for another 30 minutes.
8. Centrifuge the colloidal gold conjugate at 7000 rpm for 30 minutes and remove the supernatant.
9. Collect the pellete and suspend into 10 mM phosphate buffer of pH 7.4 containing 1 % sucrose & 1 % BSA as stabilizers and 0.1 % sodium azide as preservative.
10. Filter the gold conjugate from 0.45 micron nitro cellulose membrane and adjust the OD to 5.0 using the same phosphate buffer.
11. Label each gold conjugate as Dengue, Malaria and Chikungunya gold conjugate respectively.
The prepared gold conjugate were further tested with the antibody coated cassettes.

2.3 Preparation of Gold Conjugate Cocktail Solution
1. Take a glass container of quantity 50 ml
2. Add 5.0 ml of Malaria gold conjugate of 5.0 OD
3. Add 5.0 ml Dengue gold conjugate of 5.0 OD
4. Add 5.0 ml of Chikungunya gold conjugate of 5.0 OD
5. Mix all the gold conjugate on roller mixer for 20 min and then store in 2 – 8 ºC.

Step 3 : Preparation of Assay Buffer. Quantity 100 ml
1. Take a glass container of capacity 250 ml
2. Add 70.0 ml of glass distilled water
3. Add 1.212 gm of potassium phosphate dibasic
4. Add 0.414 gm of potassium phosphate monobasic
5. Add 0.875 gm of sodium chloride
6. Add 0.1 ml of triton X – 100
7. Check the pH of the buffer. It should be 7.4

Step 4 : Testing protocol
Preparation of antigen dilution to use as a sample.
Prepare the 1.0 µg/ml of the stock concentration of all the antigen i.e. Dengue, Malaria and Chikungunya.

Testing protocol
1. Bring all the reagents and devices to room temperature
2. Add 2 drops (100 µl) of assay buffer
3. Add 2 drops (100 µl) of antigen sample
4. Add 2 drops (100 µl) of assay buffer
5. Add 2 drops (100 µl) of colloidal gold conjugate
6. Add 4 drops (200 µl) of assay buffer

Step 5 : Result Interpretation (as shown in figure 2)
If the red color spot is observed at the top corner of the device, it indicates that the sample is infected by the Chikungunya antigen.
If the red color spot is observed at the left corner of the device, it indicates that the sample is infected by the Malaria antigen.
If the red color spot is observed at the right corner of the device, it indicates that the sample is infected by the Dengue antigen.
The red color spot at the center must be present that indicates the proper testing of the device. If the control spot at the center does not appear it indicates that the test is not performed properly or the reagents are not proper. It serves as the positive control.
Image A is finished test performed with the all positive recombinant antigen diluted in negative serum sample and Image B is the all positive recombinant antigen diluted in PBS.

Example 2: Process protocol for Dengue NS1 antigen preparation
In present invention, inventors have cloned a truncated version of the NS1 protein (483bp) (shown in SEQ ID NO 1). NS1 antigen was gene-synthesized and cloned in pET28b vector. It was then transformed in BL21 DE3 cell for induction check and protein purification. The molecular weight of the recombinant NS1 protein is 18 KDa. The dengue antibody was constructed against wing domain of NS1 antigen set forth as Seq. ID. No. 1 or Seq. ID. No. 4. Following protocol was followed for production of truncated version of the NS1 protein.

1. Preparation of Luria Broth: it was prepared using Casein, enzymatic hydrolysate 10.000, Yeast extract 5.000, Sodium chloride 10.000 at final pH (at 25°C) 7.5±0.2.
Heated if necessary, to dissolve the medium completely. Sterilize by autoclaving at 15 lbs pressure (121°C) for 15 minutes. Dispensed as desired.
Kanamycin (100 mg/mL) was prepared by 5mL of Water for injection IP (Temp <15 °C) in a 15mL capacity falcon tube and add 0.5 gms of kanamycin. Allow it to dissolve by mixing. Make up the volume to 5 ml with water(<15ºC). Stored at -20°C.

2. Reagent preparation for generation of antigen.
Kanamycin (100 mg/mL)
1 M IPTG
Lysozyme stock 4mg/ml
Lysis Buffer 100 ml: pH-8.0 Add 20mM Tris-Cl, 300mM NaCl, 10mM imidazole; Adjust pH 8.0 with HCl. Make up volume 100 mL. Check conductivity and pH of the buffer.
Filter using DHS glass assembly using 0.2um filter and store in DHS glass wares.

3. Total cell pellet preparation for generation of antigen
Reconstitute the Pre-autoclaved Luria Broth (10 mL) with 10 ul of Kanamycin (100mg/mL) stock. Inoculate 0.04 mL glycerol stock in 100 ml Luria broth Kanamycin (100 µg/ml). Incubate at 37OC/174 rpm for Overnight (~ 16 hrs.).
Reconstitute the Pre-autoclaved Luria Broth (1000 mL) with 1 mL of Kanamycin (100 mg/mL) stock. Inoculate 10mL O/N grown culture (1%) seed in 1000ml Reconstituted Luria broth supplemented with Kanamycin (100 µg/ml). Incubate at 37OC/174 rpm till OD 600 reaches 0.4 – 0.6.
Induce with 1 mL of 1M IPTG to the culture to achieve final concentration 1mM. Incubate at 16OC/174 rpm for overnight. Centrifuge the 1L culture / centrifuge bottle at 6000 g for 30 mins. Store the pellet at -20° C.

4 Cell lysis of total cells for generation of antigen.
1000 ml cell pellet (?2.5gm wet weight) dissolved in 50mL lysis buffer properly (prevent frothing). To it 200mg lysozyme added and incubated at 37°C for 3hr (invert mixed in between). Centrifuged for 15 min at 10000 rpm at 40C.
When the lysate becomes clear, it is transferred from falcons (50mL) to oak-ridge tubes (30mL) and spun at 10,000 rpm, 4°C for 45 mins in cold centrifuge. pellet was stored.

5. Solubilization of protein from inclusion bodies

6. Ethanol precipitation was carried out to remove the urea which hampered affinity purification.

7. Purification of antigen by Ni-NTA for generation of antigen.
7.1. Protein binding/ charging
Ni-NTA agarose bead (stored in 20% EtOH) slurry taken out from the 4°C and allowed to thaw in room temperature (you will see 2 layers bottom one is Ni-NTA agarose bead & upper one is 20% EtOH). EtOH mixed with Ni-NTA slurry slowly. 1mL Ni-NTA bead added to the Ni-NTA column. Bead slurry was washed with 20 CV de-ionized, nuclease & protease free water. 80 ml cell lysate was taken. To it, 1 ml pre-equilibrated bead was added. It was batch cultured for 1 hr. Then it was loaded onto an empty gravity flow column. Back flow pressure was maintained by adding rest of the lysate continuously.
Above process repeated 3 times. Flow through was collected in an oak-ridge tube (250mL) and stored at 4°C.

7.2 Washing of protein charged Ni-NTA bead and Target protein elution
The bound proteins were washed with 20 ml of 20 mM imidazole containing lysis buffer. The column was further washed with 10ml of each 50 mM imidazole and 100 mM imidazole containing lysis buffer. The protein was eluted with 2 ml of each 500 mM imidazole. Collect samples from NiNTA Load, Flow through, Wash and Fraction for analysis by SDS PAGE Coomassie staining.

Example 3: Process protocol for Chikungunya antigen chkE2 preparation
In present invention, inventors have cloned a truncated version of the chkE2 protein (386bp) (as shown in SEQ ID NO 3). Chikungunya antigen encoding genes are synthesized by oligo contig overlapping and extension method. Hybridized and gap filled DNA were finally amplified by terminal primers. The molecular weight of the purified recombinant chkE2 protein is 14 kDa. The chikungunya antibody was constructed against ChkE2 antigen set forth as Seq. ID. No. 3 or Seq. ID. No. 6. Purified protein is used for antibody development.
In present invention, chikungunya detection was designed to be done by over expression of a Surface Glycoprotein named Alpha_E2_Glycop (Tamaki et.al., J Clin Microbiol. 2015 Feb; 53(2): 382–388). This is also reported that the full length E2 protein expression in E. coli cell is extremely poor. The presence of 17 Cys residues in the full length E2 protein may cause a hindrance to over-express. A particular deletion from both N and C terminal of the protein, removes 13 number of Cys residues so that truncated E2 protein is over expressed in E. coli cell. Over expressed E2 Truncated protein is purified from the cell. Purified protein was used for antibody development.

1. Preparation of Luria Broth by Casein, enzymatic hydrolysate 10.000, Yeast extract 5.000, Sodium chloride 10.000 and Final pH (at 25°C) 7.5±0.2.
Heated if necessary, to dissolve the medium completely. Sterilize by autoclaving at 15 lbs pressure (121°C) for 15 minutes. Dispensed as desired.
Preparation of Kanamycin (100 mg/mL): Take 5mL of Water for injection IP (Temp <15 °C) in a 15mL capacity falcon tube and add 0.5 gms of kanamycin. Allow it to dissolve by mixing. Make up the volume to 5 ml with water(<15ºC). Stored at -20°C.

2. Reagent preparation for generation of antigen.
Kanamycin (100 mg/mL)
1 M IPTG
Lysozyme stock 4mg/ml
Lysis Buffer 100 ml, pH-8.0: Add 20mM Tris-Cl, 300mM NaCl, 10mM imidazole, adjust pH 8.0 with HCl, Make up volume 100 mL. Check conductivity and pH of the buffer.
Filter using DHS glass assembly using 0.2um filter and store in DHS glass wares.

3. Total cell pellet preparation for generation of antibody
1. Reconstitute the pre-autoclaved Luria Broth (10 mL) with 10 ul of Kanamycin (100mg/ mL) stock. Inoculate 0.04 mL glycerol stock in 100 ml Luria broth Kanamycin (100 µg/ml). Incubate at 37OC/174 rpm for Overnight (~ 16 hrs). Reconstitute the pre-autoclaved Luria Broth (1000 mL) with 1 mL of Kanamycin (100 mg/mL) stock. Inoculated 10mL O/N grown culture (1 %) seed in 1000ml Reconstituted Luria broth supplemented with Kanamycin (100 µg/ml). Incubate at 37OC/174 rpm till OD 600 reaches 0.4 – 0.6. Induce with 1 mL of 1M IPTG to the culture to achieve final concentration 1mM. Incubate at 36OC/174 rpm for 4 hrs. Centrifuge the 1L culture / centrifuge bottle at 6000 g for 30 mins. Store the pellet at -20° C.

4. Cell lysis of total cells for generation of antigen.
1000 ml cell pellet (?2.5gm wet weight) dissolved in 50mL lysis buffer properly (prevent frothing). To it 200mg lysozyme added and incubated at 37°C for 3 hr (invert mixed in between). Centrifuged for 15 min at 10000 rpm at 40C. When the lysate becomes clear, it is transferred from falcons (50mL) to oak-ridge tubes (30mL) and spun at 10,000 rpm, 4°C for 45 mins in cold centrifuge. Approximately 50mL of supernatant collected in oak-ridge bottle (250mL) and stored in 4°C / ice and pellet discarded.

5 Purification of antigen by Ni-NTA for generation of antigen.
Protein binding/ charging: Ni-NTA agarose bead (stored in 20% EtOH) slurry taken out from the 4°C and allowed to thaw in room temperature (you will see 2 layers bottom one is Ni-NTA agarose bead & upper one is 20% EtOH). EtOH mixed with Ni-NTA slurry slowly. 1mL Ni-NTA bead added to the NiNTA column. Bead slurry was washed with 20 CV de-ionized, nuclease & protease free water. 50 ml cell lysate was taken. To it, 1 ml pre-equilibrated bead was added. It was batch cultured for 1 hr. Then it was loaded onto an empty gravity flow column. Back flow pressure was maintained by adding rest of the lysate continuously. This process was repeated 3 times. Flow through was collected in an oak-ridge tube (250mL) and stored at 4°C.
Washing of protein charged Ni-NTA bead and Target protein elution. The bound proteins were washed with 20 ml of 20 mM imidazole containing lysis buffer. The column was further washed with 10ml of each 50 mM imidazole and 100 mM imidazole containing lysis buffer. The protein was eluted with 500 ul of each 500 mM imidazole. Collect samples from NiNTA Load, Flow through, Wash and Fraction for analysis by SDS PAGE Coomassie staining.

Example 4: PfMSPFu24 [fusion protein of trucated MSP1 and MSP3] fermentation process
The codon optimized synthetic genes coding for PfMSPFu24 antigen of P. falciparum was assembled separately by de novo synthesis based on the known amino acid sequences of these genes. The total number of amino acids for PfMSPFu24 is 172 (as shown in SEQ ID NO 2). The Malaria antibody was constructed against PfMSPFu24 antigen set forth as by Seq. ID. No. 2 or Seq. ID. No. 5. Flowchart for puri?cation of PfMSPFu24 drug substance is as elaborated in figure 10.
PfMSPFu24 fermentation process was as follows:
(i) Development of Seed
One vial of master cell bank (MCB) of E.coli cells harboring PfMSPFu24 gene was revived in 2000 mL shake ?ask containing 400 mL complex media and incubated for 14±1 hours at 30± 1°C and 200 RPM. In process controls: Sterility of the media and stock solutions used was also checked by conventional methods and the media passed in sterility. Seed cultures were analysed for optical density at 600nm to check the process step consistency.
(ii) Production in fermentor
Upstream production of PfMSPFu24 protein was performed at 20L scale (working volume) in fermentor by inoculating the 13-15 hours grown seed. Each batch production in fermentor was performed following a well standardized method, in which different parameters like pH, temperature, dissolved oxygen, stirrer speed and air ?ow were closely monitored and maintained within a narrow differential range of the respective set points. Cells were grown in batch mode and culture was induced at ~10 OD with 1mM IPTG. In process controls: During fermentation, sampling was performed at regular intervals and analysed on the basis of the following criteria.

Purity of the cultures was checked at regular intervals to rule out any contamination to the culture. Purity was checked by Grams staining of the culture. P.falciparum Malaria vaccine containing PfF2/ Alhydrogeland PfMSPFu24+PfF2/ Alhydrogel

Downstream process for puri?cation: Steps involved in the puri?cation of the product The downstream puri?cation process for the drug substance of PfMSPFu24 involves the following steps:
i. Cell Harvesting and cell pellet washing
ii. Cell Disruption and clari?cation
iii. IMAC chromatography
iv. Cation exchange chromatography
v. Concentration
vi. Sterile ?ltration

Example 5: Protocol for Antibody development against the recombinant Dengue antigen
The antibodies against antigen was first requirement of the diagnostic kit of present invention.
The protocol was for the production of polyclonal antibody in 2 New Zealand White Rabbits which included 7 immunizations and 3 Production bleeds per animal. Pre-immune sera of 5-10 ml per animal and a total of 30-40 ml of crude immune sera was produced per rabbit. The total procedure took 12 weeks to complete.
15-20 mg of purified Antibody was generated using the described protocol.
Reagent(s) preparation:
1. Preparation of reagent for hyper immune sera 13.7 gm Sodium Phosphate monobasic (mol. wt. 137.99), was dissolved in 100 ml purified water. 14.16 gm Sodium phosphate dibasic (mol. wt. 141.96), was dissolved in 100 ml purified water. The dibasic sodium phosphate solution was titrated with monobasic sodium phosphate solution to achieve the pH of the buffer to 7.2.
2. Preparation of 20 mM PBS: To 50 ml of purified water, 2 ml of 1 M phosphate buffer pH 7.2 and 0.9 gm NaCl was added to it. It was stirred to dissolve and the volume was made up to 100 ml with purified water.
3. Preparation of hyper immune sera: The Antigen was reconstituted in 20 mM PBS pH 7.2. The rabbit (New Zealand white rabbit, weighing approximately 1 kg) was immunized with 100µg of antigen in Freund’s complete adjuvant (FCA) at four sites. The immunization and the bleeding was done as per schedule. Details are as elaborated in Figure 5. Serum was separated and stored at -20o C in 1 ml aliquots. Sample (1ml) was removed for analysis of antibody titer by DOT blot.

Functional assay of the raised antibody by DOT ELISA
1. One µl of antigen was dotted at the centre of nitrocellulose membrane with the help of micro pipette tips at 12 projections of plastic comb at an antigen concentration of 100 µg/ml in 20mM phosphate buffer pH 7.2 (coating buffer) for Dot ELISA.
2. Non-specific sites were blocked with 5% defatted milk powder in PBST (20 mM PB pH 7.2, NaCl 0.9 %, Tween -20, 0.05 %). 50ml per a single blot, at 37o C for one hour on a rocker.
3. Rabbit serum (primary antibody) was serially diluted starting from 1:200 up to 12 dilutions in an ELISA PLATE such as 1:200,1:400,1:800, 1:1600, 1:3200, 1:6400, 1:12800, 1:25600, 1:51200, 1:102400,1:204800, 1:409600 with 5% skim milk in 20 mM PBST to ensure that different dilutions are not mixed.
4. The comb was washed thrice with PBS-T at 7 mins interval and was incubated with goat anti-rabbit immunoglobulin- HRP conjugate at 1:1000 dilutions in 5 % skim milk PBST for one hour at 37o C.
5. The colour was developed by adding 3, 3’di-amino benzidine (DAB) 1mg/ml, H2O2 substrate solution 1µl/ml (Approx. volume 50 ml for one strip). The reaction was stopped by washing the comb with distilled water.

Purification of the Raised antibody using protein A agarose
1. Buffers were prepared using following ingredients and stored at 4oC for 1-2 weeks
1. Protein A Loading Buffer: 1 M potassium phosphate, pH 9.0
2. Elution Buffer: 0.1 M citric acid, pH 3.0
3. 1.5 M Tris base, for neutralization of the eluate
2. Column sleeve was prepared. Plunger from syringe was removed and discard. A small amount of glass wool into the bottom of the syringe was pressed, enough to form a cushion about 1/2 - 1 cm thick. The stopcock was attached. Rinsed with 1-2 ml loading buffer. The glass wool cushion remains firmly in the bottom of the syringe was ensured.
3. The resin (Protein A agarose) was suspended in 2 ml loading buffer by inverting and rotating the bottle. Avoid Excessive shaking was avoided. The slurry was not vortexed.
4. The slurry was poured into the syringe. Allow the excess buffer to drain through, then the column was washed with 5 ml loading buffer. Do not allow the resin to run completely dry.
5. Based on the capacity of the resin for Ig from the species of the starting material the amount of Ig in the starting material, volume to load was calculated.
6. The serum was diluted with 3 volumes of loading buffer. The supernatant was diluted with 1 volume of loading buffer. The diluted material was applied to the column. The unbound fraction was collected in a test tube or beaker and save for possible reprocessing. The unbound proteins were washed through with 5 ml loading buffer per ml resin.
7. The Elution Buffer was applied to the column. Fractions equal to 1/2 column volume was collected. 10 ml of elution buffer was used per ml resin.
8. The column was equilibrated with 5 ml loading buffer per ml resin. pH of effluent was checked to ensure that column is equilibrated at the pH of the loading buffer.
9. The eluate fractions were assayed for protein by absorbance at 280 nm. Fractions were pooled which are positive for protein. Neutralized to pH 6 - 8 with 1.5 M Tris base.
10. If desired, the unbound fraction may be re-applied to the column to recover any Ig that did not bind on the first pass, which may occur if the amount of material loaded exceeds the column capacity.
11. The purified antibody was dialyzed into desired buffer, i.e. PBS, pH 7, at 4o C. The volume of the dialysis buffer should be at least 20 times the volume of the protein solution. At least 2 changes of dialysis buffer, for at least 2-4 hours each, should be done to ensure complete equilibration in the dialysis buffer.
12. If no more runs are to be performed, the column was washed with PBS containing 0.05 - 0.1% sodium azide. The column was sealed with a stopper or Parafilm and stored at 4o C.

The antibodies for chikungunya and malaria were prepared by the similar process.

Conclusion
The detection kit of present invention results in improved outcomes for patients by offering differential diagnosis or detecting simultaneous infections. Simultaneous detection also reduces the number of visits for differential diagnosis, reducing the time of correct treatment, and the cost per diagnosis.
Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications. The invention also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations of any two or more of said steps or features.