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Summer Research Fellowship Programme of India's Science Academies

Laboratory detection of dengue NS-1 antigen in human serum/plasma & expression and purification of MBP-ZIKV EDIII

M. Pranathi

Apeejay Stya University, Sohna-Palwal Road, Haryana 122103

Dr. Navin Chandra Khanna

International Centre for Genetic Engineering and Biotechnology, Aruna Asif Ali Marg, Jawaharlal Nehru University, New Delhi 110067

Abstract

Both Zika fever and Dengue fever are caused by viruses transmitted by a mosquito-borne disease, primarily by Aedes aegypti. Both viruses belong to the same family, Flaviviridae. Zika fever is similar to Dengue in its initial symptoms but has other effects causing microcephaly. Dengue is caused by a positive-sense single-stranded RNA virus. There are four distinct but antigenically related serotypes-- DENV-1, DENV-2, DENV-3, and DENV-4. Its genome is about 11,000 nucleotide bases which code for 3 structural proteins and 7 Non-structural proteins (NS). Clinically, the Dengue disease manifests as either mild Dengue fever (DF) or potentially fatal Dengue haemorrhagic Fever (DHF) and Dengue shock syndrome (DSS). The NS-1 is involved in membrane-associated viral replication. It is present in the serum of infected persons directly at the onset of clinical symptoms in primary dengue infection which allows rapid detection within 1 to 2 days following infection and up to 9 days after the onset of symptoms. Earlier diagnosis of Dengue reduces the risk of complication such as DHF and DSS. The micro well ELISA kit test, which is designed for the in-vitro qualitative detection of Dengue NS-1 Antigen in human serum/ plasma and is used as a screening test for testing of collected blood samples suspected for DENGUE. This kit detects all four serotypes of Dengue. The principle involved is the sandwich complex formed in the well wherein dengue NS-1(from a sample) is trapped/ sandwiched between Antibody and enzyme conjugate (monoclonal anti-dengue NS-1 antibodies). In addition to this, also performed the expression and purification of recombinant MBP-ZIKV-EDIII protein that could be used as a potential diagnostic intermediate for detection of ZIKV specific antibodies in samples.

Keywords: dengue virus, zika virus, flaviviridae, structural proteins, non-structural proteins

Abbreviations

Abbreviations
Ab Antibody/antibodies 
 AgAntigen/antigens 
 ADE Antibody-Dependent Enhancement
APS Ammonium Persulfate 
 BCA Bicinchoninic Acid
 BSA Bovine Serum Albumin
 EDTA Ethylenediaminetetraacetic acid
 NSNon Structural Proteins 
 DF Dengue Fever
 DHF Dengue Haemorrhagic Fever
 DENVDengue Virus 
 DSSDengue Shock Syndrome 
EDIII Envelope Domain III 
 ELISAEnzyme Linked Immunosorbent Assay 
 HRPHorsr Radish Peroxidase 
 IPTGIsopropyl β- d-1-thiogalactopyranoside 
 KDaKilo Dalton 
 LLitre 
 LMW Low Molecular Weight Marker
M Molar 
 μgMicro-gram 
 mgMilli-gram 
 mlMilli-Litre 
 MBPMaltose Binding Protein 
 MWCO Molecular weight Cut Off
 ODOptical Density 
 PBS Phosphate Buffer Saline
 PMS Precision plus Protein Marker Standards
 PMSF Phenyl Methyl Sulfonyl Fluoride
 PVDF Polyvinylidene difloride
 RER Rough Endoplasmic Reticulum
 rpmRevolutions per minute 
SDS Sodium Dodecyl Sulfate 
 TEMED Tetramethylethylenediamine
 TMB3,3′,5,5′-Tetramethylbenzidine 
 WHOWorld Health Organization 
 WR Working Reagent
 ZIKV Zika Virus

INTRODUCTION

Background/Rationale

The World Health Organization (WHO) considers dengue to be one of the fastest spreading arboviral diseases. A mosquito-borne disease, dengue affect almost 400 million people worldwide. India is home to nearly a third of the global population at risk of dengue, a viral disease caused by four antigenically and genetically distinct dengue viruses(1). Primary infection with dengue virus results in a self-limiting disease characterized by mild to high fever lasting 3 to 7 days. Secondary infection is more serious and can result in Dengue haemorrhagic fever (DHF) and Dengue shock syndrome (DSS). In severe cases, plasma seeps out of capillaries, liquid pools around organs, massive internal bleeding ensues, and the brain, kidneys, and liver begin to fail.

No drugs are available in the market till date for the treatment of dengue disease. In 2015, the first-ever vaccine licensed for dengue, Sanofi's Dengvaxia vaccine, which was approved in more than 10 countries, created havoc. However, it has some limitations and reported that it offers only limited protection in the age group 9-45 who had previous exposure to dengue. In children, under age 9, it enhances the disease. It is believed that second dengue infection can be much deadlier than the first because of Antibody-Dependent Enhancement (ADE) theory, in which any of the four dengue serotypes during infection, has the capacity to generate cross reactivity but not cross protective immune response. The cross reactive antibody response, generated by one serotype, can enhance infection of other three serotype viruses. Live attenuated vaccines, contains a large load of cross reactive epitopes, a major risk factor which may lead to enhancement phenomenon.

Similarly, Zika Fever which is caused by a Zika virus belongs to the same family of viruses, Flaviviridae as Dengue Virus. They are so similar in their initial symptoms that Zika virus had once believed to be one of the new serotype's of Dengue Virus, however, has other effects causing microcephaly.

The detection of these diseases is at utmost importance. The early diagnosis of DSS is particularly important, as patients may die within 12 to 24 hours if appropriate treatment is not administered. Primary Dengue virus infection is characterized by elevations in specific NS-1 antigen levels, present in the serum of infected persons, 0 to 9 days after the onset of symptoms; generally persists up to 15 days. Earlier diagnosis of Dengue reduces the risk of complication such as DHF or DSS, especially in countries where dengue is endemic. In Zika virus, the distinct ED III site could be used as a potential diagnostic intermediate for detection of ZIKV specific antibodies by expressing and purification of recombinant MBP-ZIKV-ED III (fusion protein).

Objectives of the Research

Overall objective

a) Laboratory Detection of Dengue NS-1 Antigen in human serum/plasma.

b) Expression and Purification of MBP-ZIKV-EDIII. The purified protein can be further detected and analyzed using western Blotting.

Outline- photo.jpg
    Outline of Research Objectives

    A)LABORATORY DETECTION OF DENGUE NS-1 ANTIGEN IN HUMAN SERUM/PLASMA

    LITERATURE REVIEW

    Dengue Virus replication

    Dengue virus outer surface is covered with envelope proteins surrounding lipid bilayer envelope. Inside the envelope, there is a capsid shell that contains viruses RNA genome. When the virus enters the host cell, due to lowered pH, the conformation changing of the envelope protein takes place. The virus is released into the cytoplasm which releases the viral RNA, which travels to Rough Endoplasmic Reticulum (RER). As it is a positive-sense strand, it can directly translate into proteins.

    The dengue virus genome is about 11,000 nucleotide bases which code for 3 structural proteins-- Capsid protein(C), Pre membrane protein(PrM), Envelope protein(E). The non-structural proteins(NS) are NS-1, NS-2A, NS-2B, NS-3, NS-4A, NS-4B and NS-5. 

    NS-1 is involved in membrane-associated viral replication. In the infected cell, NS1 is bound to the ER vesicles membrane on the lumen side and helps anchoring the viral replication complex. NS1 is present in the serum of infected persons directly at the onset of clinical symptoms in primary dengue infection and produces a strong humoral response which allows rapid detection within 1 to 2 days following infection and up to 9 days following symptom onset. Earlier diagnosis of Dengue reduces risk of complication such as DHF or DSS.

    NS-1 ELISA with Dengue Virus II supernatant

    It is a kit designed for in vitro qualitative detection of Dengue NS-1 Ag in human serum or plasma and is used as a screening test for testing of collected blood samples suspected for DENGUE. This kit detects all four serotypes of Dengue.

    Principle involved: A sandwich complex is formed in the well wherein dengue NS-1(from the sample) is trapped/sandwiched between Ab and enzyme conjugate. ​

    elisa.jpg
      Principle involved to detect Dengue NS-1 Ag

      1) Microwells coated with Anti-dengue NS1 antibodies with high reactivity for Dengue NS-1 Ag

      2) Samples are added in the well

      3) Followed by the addition of enzyme conjugate (monoclonal anti-dengue NS1 antibodies linked to Horseradish Peroxidase (HRPO))

      The dengue NS-1 (from serum samples) is trapped between the Ab and Ab HRPO conjugate.

      4) Upon addition of TMB substrate buffer, a blue color develops

      5) Addition of stop solution terminated enzyme-substrate reaction and changes to yellow color

      The intensity of the color is proportional to the concentration of dengue NS-1 in sample

      METHODOLOGY

      PREPARATION

      Well Plan
       12 
       A 25F25F 
       B 50F50F 
       C100F  100F
       D 1000F1000F 
       E 10,000F10,000F 
      F +ve control +ve control 
      G  -ve control-ve control 
      H  1x PBS1x PBS 

      For one well,

      25F=> 2µlL(DENV-2 supernatant) + 48µL(1x PBS) = 50µL/well

      50F=> 1µL(DENV-2 supernatant) + 49µL(1x PBS) = 50µL/well

      Serial Dilutions,

      100F=> 1.2µL(DENV-2 supernatant) + 120µL(1x PBS)==> Add 50µL/well

      For 1000F, take 12µL from 100F dilution and add 120µL of 1x PBS and mix it well. Add 50µL in each well.

      For10,000F, take 12µL from 1000F dilution and add 120µL of 1x PBS and mix it well. Add 500µL in each well.

      a) Enzyme Conjugate (100µL/well)

      Dilute conjugate 1:50 in diluent= 40µL+1.96ml(diluent) i.e. 100µL/well

      b) Working wash buffer (200µL/well)

      Check the buffer concentration for the presence of salt crystals. If crystals are present in solution, resolubilize by warming at 37ºC until all crystals dissolve= 800µL+19.2ml(H2O)

      c) Working substrate solution (150µL/well)

      Mix TMB substrate and TMB diluent in 1:1 ratio to prepare a working substrate

      1.2ml of A+1.2ml of B= 2.4ml

      PROCEDURE

      1) Add 50µL of 25F, 50F, 100F, 1000F, 10,000F diluents in each well.

      2) +ve and -ve controls are present in the kit. Add 50µL/well each.

      3) Add 1x PBS (50µL/well) as a control.

      4) Add enzyme conjugate (100µL/well) and mix well.

      Enzyme conjugate consists of monoclonal anti-dengue NS-1 antibodies.

      5) Incubate at 37°C for 1 hour and 30 minutes.

      6) Take out the plate from the incubator after the incubation time is over and wash the wells 6 times with working wash solution. Add 200µL/well for each wash.

      7) Add working susbtrate solution (150µL/well) in each well.

      TMB substrate chromogenic substrate used in staining procedures.

      8) Incubate at room temperature(20º-30°C) for 30 minutes in dark.

      9) Add 100µL of stop solution in each well.

      Stop solution is used to terminate enzyme substrate reaction after attaining desired color intensity.

      10) Read absorbance at 450nm with 30 minutes in ELISA READER (Spectra Max pro 6.1).

      RESULTS AND DISCUSSION

      Sheet1
      1  2 
      A(25F) 3.9269 3.8298
      B(50F) 3.9197 3.8878 Average 
      C(100F) 3.9616 3.8236 100F 3.8926
      D(1000F) 1.1957 0.6875 1000F 0.9416
      E(10,000F) 0.206 0.1292 10000F 0.3352
      F(+ve control) 2.9472 2.4004
      G(-ve control) 0.1502 0.07240000000000001
      H(1x PBS) 0.0743 0.0469
      http://assets.authorcafe.com.s3.amazonaws.com/10037/assets/docxassets/NS1_graph_5d52be41b32e3/xl/media/image1.png
        This graph represents serial dilutions against OD at absorbance 450nm. The linear equation shows the presence of Dengue (DENV II) NS-1 Ag's

        CONCLUSION

        a) The optical density for 25F,50F is saturated.

        b) For, 100F, 1000F, 10000F, have a linear graph which indicates the presence of Dengue NS-1 antigens.

        LIMITATIONS

        If there are more number of NS-1 Antigens in given human serum/plasma, then there be a chance of showing some false-negative results through the Direct method. This limitation can be overcome by using the Indirect method (a two-step method).

        B) EXPRESSION AND PURIFICATION OF r MBP-ZIKA-ED III

        LITERATURE REVIEW

        Maltose binding protein (MBP) is a common protein expression tag. It is one of the most well-known and accomplished means of tagging recombinant proteins expressed in microbes. A fusion of a target protein to MBP permits its one-step purification using amylose resin. Additionally, in E. coli, MBP is known to have significantly enhanced the solubility of many proteins it has been fused to. The pMAL-c5x vector allows to create a fusion between an expressed target protein and MBP, and purify the fusion protein in a single step.

        The EDIII of ZIKV was fused with E.coli bacteria for the expression of MBP-DIII (MBP-DIII) fusion protein.

        pMAL-c5x-1.png
          pMAL-c5x vector

          METHODOLOGY & RESULTS

          PROCEDURE

          1) Take LB Agar mixture of 4.1g in 100ml H2O

          2) Autoclave it for 20 minutes.

          3) Add (stock-100mg/ml) Ampicillin (100μL) and mix it well.

          4) Pour it into Petri dishes and allow to solidify for half-hour.

          5) Using tip streak the plates with MBP-ZIKV-EDIII clone glycerol stock and put it in the incubator at 37°C overnight. 

          MEDIA PREPARATION FOR PRIMARY AND SECONDARY CULTURE

          For Primary Culture

          2.5g of LB broth in 100ml of H2O and autoclave it.

          For Secondary Culture

          Take a 1L flask and add: 1) Yeast extract - 5g

          2) NaCl - 5g

          3) Tryptone - 10g

          4) Glucose (Dextrose) - 2g

          Mix it well. Cover with a foil and autoclave it.

          PRIMARY INOCULATION

          1) Add 100μL of Ampicillin (stock-100mg/ml) to 100ml of LB broth (of primary culture).

          2) Using tip pick a single colony from petri plate and inoculate the culture.

          3) Culture overnight @37°C at 120rpm

          SECONDARY INOCULATION

          1) Add 1ml of ampicillin (stock-100mg/ml) to 1L of secondary culture.

          2) Inoculate 10ml of primary culture to secondary culture.

          3) Grow until OD (Optical Density) reaches 0.6 (OD-600nm)

          Absorption @ 600nm

          Blank - 0

          1 hr 10 mins - 0.196

          +20min - 0.392

          - 0.476

          +20min - 0.784

          4) Stop the bacterial replication when OD reaches 0.5 and Induce the culture with 1ml of 1M IPTG

          IPTG a molecular mimic of allolactose, a lactose metabolite that triggers transcription of the lac operon, therefore, used to induce recombinant protein expression in E.coli.

          5) Also, collect 500μL of uninduced culture in two eppendorf tubes and centrifuge it @ 10,000rpm for 5 min, collect the pellet and store @-20°C

          6) Grow the culture for 4 hours @37°C, 120rpm

          7) Pellet the culture at 6000rpm for 5 mins.

          8) Wash the pellet with 0.9% NaCl and spin at 6000rpm for other 5 mins. Store the pellet @-80°C until use.

          INDUCTION CHECK ON SDS-PAGE
          Preparation of Resolving and Stacking Gels

          RESOLVING GEL
          2x 
          Milli Q 3.2ml 
           Glycerol200μL 
           Tris pH 8.8 2.6ml
           30% acrylamide 4.0ml
           10% SDS 100μL
           10% APS 100μL
          TEMED  4μL
          STACKING GEL
          2x 
           Milli Q 3.0ml
           Glycerol 100μL
           Tris pH 6.81250μL 
           30% acrylamide650μL 
           10% SDS50μL 
           10% APS 25μL
           TEMED 5μL

          APS - 10% 10g in 100ml => 0.1g in 1ml

          SDS - 10g in 100ml => 4g in 40ml

          NOTE: 10% APS and TEMED should be added only when the solution is ready to use.

          1) Set thick and thin glasses in such a way that it doesn't get leaked out.

          2) Using pipette add resolving gel solution without any air bubbles in it and add isopropanol/water to remove any air bubbles in it and to level the resolving gel solution. Leave it for 20 mins or until polymerized.

          3) Remove water and add stacking gel solution and then fix the combs in it.

          4) Plates can be stored at - 20°C and do not remove combs if not gel gets dried.

          PREPARATION OF SAMPLE BUFFER (2x)

          SAMPLE BUFFER
           Tris HCl pH 6.8120mM 
          SDS 4% 
           β-mercaptoethanol10% 
           Bromophenol bluepinch 
          Glycerol  20% 
          H2O little  
          Sample 

          NOTE

          (i) β-mercaptoethanol should be added before use.

          (ii) The sample protein and sample buffer should have the same volume.

          Making samples

          Add the same amount of sample proteins and 2x sample buffer. Boil it for 10-12 mins and centrifuge it for 5 mins at 10,000 rpm. And then load it in the wells.

          PREPARATION OF RUNNING BUFFER

          RUNNING BUFFER
          10x 
           Glycine578g 
           Tris base120.8g 
           SDS40g 
           H2O4L 

          For 1x, 100ml buffer + 900ml H2O

          Running SDS-PAGE Gel Electrophoresis

          1) Take the SDS apparatus and remove the combs and fix plates properly in the stand.

          2) Pour running buffer into the apparatus and load the samples into the well.

          WELL PLAN
           1Marker (M)5μL 
           2Un-induced (U)15μL 
           3 Induced (I)15μL 
           4 Dye (D)15μL 
           5 Un-induced (U)3μL 
           6 Induced (I)3μL 
           7 Un-induced (U) 10μL
           8 Induced (I) 10μL

          3) Close the lid and set the voltage to 80V until the sample pass stacking gel. Later increase it to 130V and run the gel until the dye leaves the gel.

          4) Remove the gel and add it in Coomassie blue and leave it overnight with continuous shaking for better staining.

          5) Prepare destaining solution with Methanol - 40% and Glacial acetic acid -10%.

          6) Destatin the gel and scan the gel in geldoc instrument.

          1st_4.jpg
            IPTG Induction check of MBP-ZIKV-EDIII. The gel was kept in Coomassie blue and added in a destain solution. UI represents uninduced with IPTG and I represents Induced which shows the band at 52Kd which indicates presence of MBP-ZIKV-EDIII. 

            PURIFICATION OF MBP-ZIKV-ED III

            BUFFER PREPARATION

            1) Column buffer (for 1L)

            COLUMN BUFFER (for 1L)
             20mM Tris20ml (from 1M stock) 
             200mM NaCl40ml (from 5M stock) 
             1mM EDTA2ml (0.5M stock) 
             H2O 900ml

            Adjust pH=7.4 with HCl (1N) and make final volume to 1L. Filter (0.45μL) and use.

            2) Lysis Buffer (for 200ml)

            Add 2.0ml of 100mM PMSF into 200ml of column buffer.

            PMSF- is a serine protease inhibitor commonly used in the preparation of cell lysates.

            3) Elution Buffer (for 100ml)

            Add 10mM maltose monohydrate (360.31mg) into 100ml of column buffer. Filter and use.

            It is used to wash away unbound proteins at first and at a higher concentration it released the desired protein from the ligand.

            PURIFICATION PROTOCOL

            1) 2.0g biomass from 2L culture

            2) Take 40ml of lysis buffer (pre-chilled lysis buffer) and suspend the pellet.

            3) Freeze/ Thaw 2-cycle in liquid Nitrogen/ 37°C H2O.

            4) Sonicate the lysate for 5min (until the lysate turns transparent) (10 sec--on & 30 sec off cycle) for 15 mins.

            5) Spin the lysate @1500rpm for 1 hour at 4°C and collect the supernatant (protein), not the pellet (cell debris).

            6) Filter the sup with 0.45μ filter, to remove debris.

            7) Take 10ml of amylose NEB resin and wash with 5cv of H2O in the column tube. (5cv => 5x => 5*10= 50ml H2O)

            8) Wash it with column buffer in column tube only.

            9) Equilibrate the resin with column (5cv)

            10) Keep 40ml of lysate to bind with resin for 1 hour at rotation (on gentle rotation).

            11) Pack the column and pass through the lysate for 3-5 times.

            12) Collect the flow throw in a falcon tube.

            13) Wash the column with column buffer. Collect the wash.

            14) Elute with elution buffer and collect 1ml aliquots in eppendrof tubes and check the status of protein it with Bradford's reagent (1x).

            => (1x/10x) *10 => 1ml + 9ml (H2O)

            15) Add 1x Bradford reagent, 1ml each in separate 15-17 eppendorf tubes.

            16) Now add 5μL each from collected elution fractions to Bradford's reagent and mix to see the blue color development.

            17) Continue this process until all proteins are eluted from the resin.

            NOTE: The color change indicates the presence of protein.

            18) They can be stored @-80°C.

            Presentation1-1.jpg
              This figure represents the column eluted fractions of MBP-ZIKV-EDIII protein from the resin, F-1 & F-2 shows no color change which indicates no presence of protein. F-3 to F-15 shows color change to blue which indicates the presence of protein.

              Important Points

              1) In Lysis buffer,

              EDTA and PMSF are the protease inhibitors, which inhibits the function of the proteases present in the E.coli.

              The step of freeze/thaw 2-cycle in liquid Nitrogen/37°C H2O helps in the breakdown of the cell wall and cell membrane of E.coli.

              The protein of interest (i.e. MBP) is present in the cytosol of the cell.

              2) In Lysate,

              Protein of interest (i.e MBP= Maltose Binding Protein), maltose have the affinity to bind amylose resin.

              When elution buffer is added after wash, (elution buffer consists of maltose monohydrate) the amylose resin has the more affinity towards maltose monohydrate than our protein of interest (MBP).

              Hence, the pure form of protein is collected as 1ml aliquots.

              PURIFICATION CHECK ON SDS-PAGE

              Preparation of resolving gels solution and Stacking gel solution:

              RESOLVING GEL SOLUTION
               2x
               Solution A 4ml
               Solution B 4ml
               10% APS 40μL
               TEMED 4μL
              STACKING GEL SOLUTION
               2x
              Solution A 2ml 
              Solution B 2ml 
              10% APS 20μL 
              TEMED 4μL 
              Well Plan (Gel-1)
               1 LMW (L)
               2 Un-induced (U)
               3Induced- Lysate(LOAD) (I)
               4 Flow throw (FT)
               5 Wash (W)
               6 Resin (R)
              Well Plan (Gel-2)
               1LMW 
               2 Fraction-3
               3Fraction-5 
               4 F-7
               5 F-9
               6 F-11
               7 F-13
               8 F-15
               9 ------
               10 Control (C)

              18) Pool the selected fractions (from F-3 to F-15)

              19) These fractions contain pure protein and dialyze against column buffer.

              2nd_2.jpg
              SDS gel for analysis of MBP-ZIKV-EDIII. UI represents the uninduced with IPTG. I represents Induced (i.e. Lysate), it is the protein collected from the pellet. FT represents the Flow throw. W represents the wash collected after washing the column with column buffer. The was has no signs of protein. R is the amylose resin which is taken from the column, which has few traces of MBP-ZIKV-EDIII
                3rd gel.jpg
                SDS gel for purification analysis of MBP-ZIKV-EDIII. F-3 to F-15 indicates the pooled fractions of protein in which, F-5 fraction consits of higher concentration of the protein. 
                  Purification check of MBP-ZIKV-EDIII

                  DIALYSIS

                  a) Make column buffer for 3L

                  20mM Tris - 60ml (from 1M stock)

                  200mM NaCl - 120ml (from 5M stock)

                  1mM EDTA - 6ml (from 0.5M stock)

                  Adjust pH=7.4 with HC l(1N) and make final volume to 3L.

                  b) Take dialysis membrane (MWCO-12-14Kd; diameter-29mm; width-45mm) tie a knot at the bottom so that it doesn't leak and pour the pooled out fractions of protein into the membrane and close at the top with a knot.

                  c) Take 1L of prepared column buffer and place the membrane containing pooled fractions.

                  d) Keep it at 4°C on a stirrer for 4 hours.

                  e) 3 cycles of it by changing the column buffer

                  f) Collect the purified protein from the dialysis membrane, filter it with 0.45µ filter and Store it at 4°C.

                  BCA PROTEIN ASSAY KIT

                  It is performed to analyze the concentration of the purified protein (Detection and Quantitation of total protein). Water soluble complex exhibits a strong absorbance @562nm.

                  The protein concentration is generally determined and reported with reference to standards of a common protein such as Bovine Serum Albumin (BSA).

                  a) PREPARATION OF DILUTED ALBUMIN (BSA) STANDARDS

                  Preparation of Albumin Standards
                   Vial Volume of diluent (μL)Volume & source of BSA (μL)Final BSA concentration (μL/ml) 
                  A 700 100 of stock 250 
                   B400 400 of A dilution 125 
                   C450 300 of B dilution 50 
                   D 400400 of C dilution 25 
                   E400 100 of D dilution 5 
                   F 4000 0= Blank 
                  Note
                  Diluent = Column buffer

                  b) PREPARATION OF BCA WORKING REAGENT (WR)

                  Add 50 parts of BCA reagent A (12.5ml) + 1 part of BCA reagent B (0.25ml)

                  TEST TUBE PROCEDURE

                  For test tube procedure
                   SAMPLE-1 (1:10)SAMPLE-2 (1:20) 
                  sample : column buffer(1:10) sample : column buffer (1:20)  
                  20μl : 200μL 10μL: 200μL 

                  1) Pipette out 50μL of each standard fractions and the unknown samples into each eppendorf tubes.

                  2) Add 1ml of prepared WR in each of 8 tubes.

                  3) Cover and incubate tubes for 30mins @60°C and mix well.

                  4) Cool all the tubes at room temperature.

                  5) With spectrophotometer, @562nm, zero the instrument on a cuvette filled only with water. Subsequently, measure the absorbance of all the samples within 10mins.

                  NOTE: Because BCA assay does not reach a true endpoint, color development will continue even after cooling to room temperature.

                  6) Subtract the average 562nm absorbance measurement of the Blank standard replicates from the 562nm absorbance measurement of all other individual standard and unknown sample replicates.

                  7) Prepare a standard curve by plotting the average Blank-corrected 562nm measurement for each BSA standard vs. its concentration in µg/mL. Use the standard curve to determine the protein concentration of each unknown sample.

                  Readings
                  Actual OD OD-blank(0.261) concentration
                  F 0.261 0 0 250 0.903
                  E 0.268 0.007 5 125 0.484
                  D 0.34 0.079 25 50 0.177
                  C 0.438 0.177 50 25 0.079
                  B 0.745 0.484 125 5 0.007
                  A 1.164 0.903 250 0 0
                  S-2 0.604 0.343
                  S-1 1.07 0.8090000000000001
                  Test Dilution OD OD-(-0.0039) OD-(-0.0039/0.0037) Final mg/ml Average Total Volume Total Protein
                  MBP- ZIKA EDIII 1:10 0.8090000000000001 0.8129000000000001 219.7027027027027 2197.027027027027 2.197027027027027 2.036081081081081 14ml  28.505135135134
                  1:20 0.343 0.3469 93.75675675675676 1875.135135135135 1.875135135135135
                  http://assets.authorcafe.com.s3.amazonaws.com/10037/assets/docxassets/NS1_graph_5d53e9b792a86/xl/media/image2.png
                    It is the standard graph in which blue line indicates the concentrations of MBP-ZIKV-EDIII against particular OD monitored by absorbance at 562nm

                    Therefore, the concentration of MBP-ZIKV-ED III is 2.0361mg/ml.

                    SDS-PAGE

                    To run the gel for particular concentrations of protein.

                    a) For 2μL

                    10μL ---- protein

                    10μL ---- Column buffer

                    20μL ---- 2x Dye

                    40μL ---- TOTAL

                    b) Protein-1 (20μL+20μL dye)= 40μL

                    c) Protein-2 (20μL+20μL dye)= 40μL

                    Boil a,b,c for 10-12 mins and centrifuge it for 2mins

                    d) For 0.5μg

                    Take 3μL from 2μg + 9μL of 1x dye = 12μL

                    WELL PLAN
                     1Protein-1 (P-1)  5μL
                     2Protein-2 (P- 2) 5μL
                     3LMW  5μL
                     4 0.5μg 4μL
                     5 2μg 4μL
                     60.5μg  4μL
                     72μg 4μL 
                     8P-1 5μL 
                     9P-2 5μL 
                    4th_1.jpg
                      SDS gel analysis of MBP-ZIKV-EDIII. P-1 & P-2 are the proteins for control. 0.5μg & 2μg represents the concentrations of the protein. 

                      To store the protein--------

                      Make aliquots of 1ml and 200μL each in cryovial tubes and directly freeze them in liquid Nitrogen (Cryopreservation) and store them at -80°C.

                      WESTERN BLOTTING

                      It is a technique to detect and analyze proteins.

                      1) Protein Electrophoresis

                      Run the gel for the samples- 0.5μg and 2μg concentrations

                      WELL PLAN
                       1PMS 7μL 
                       2 0.5μg4μL 
                       3 2μg4μL 
                       40.5μg 4μL 
                       52μg 4μL 

                      2) Transfer proteins to the membrane

                      • Membranes-- have high affinity to proteins. Ex: Nitrocellulose, Polyvinylidene difloride (PVDF).
                      • A transfer is done for further detection.
                      • The membrane is thicker than gel, therefore, when proteins bind their epitopes or binding sites easily accessible to Antibodies
                      • Transfer buffer --- 25ml of transfer buffer + 50ml of methanol and make it up to 250ml.
                      • Take gel and sponges and place it in transfer buffer for some time.
                      • Now place them in following order on the Western blot apparatus:

                      1. Sponge

                      2. Membrane (towards -ve electrode)

                      3. Gel (towards +ve electrode)

                      4. Sponge

                      • Adjust them accordingly without any air bubbles.
                      • Close the lid and set the voltage to 12V for 30 minutes.
                      • Now, protein gets transferred from gel to the membrane.

                      3) Blocking

                      To prevent non-specific binding of Antibodies.

                      Blocking agents-- BSA, skim milk

                      • Take 5% of skim milk (2.5g of skim milk in 50ml of wash buffer)
                      • Take a western blot box and place the membrane in 5% skim milk and keep it on a stirrer for minimum 1 hour.

                      Wash buffer => 1x PBS

                      For 1 blot => 10ml of primary Antibody

                      => 10ml of secondary Antibody

                      TOTAL => 20ml of 2.5% skim milk

                      50ml of 5% skim milk + 50ml of wash buffer= 100ml

                      4) Antibody Probing

                      Primary Antibody - DENT4.mAb (4μL) against MBP-ZIKV-ED III

                      a) Add 4μL of primary antibody(DENT4.mAb) in 10ml of 2.5% skim milk

                      b) Mix it well and add it in blot box.

                      c) Leave it for 1 hour on a rocking platform.

                      d) 3 times wash with wash buffer=> 5 minutes/wash.

                      Secondary Antibody- Goat ā.mouse IgG.HRP Conjugated

                      2° Ab : 2.5% skim milk = 1 : 10,000

                      => 1μL: 10ml

                      a) Add 1μL of secondary Ab in 10ml of 2.5% skim milk

                      b) Mix it well and it in blot box and leave it for 1 hour on a stirrer.

                      c) 3 times was with wash buffer => 5 minutes/wash.

                      Add TMB-Insoluble substrate (@room temperature) => 5ml in blot box

                      Scan it and store the membrane.

                      western blot.jpg
                        Western blot membrane to detect MBP-ZIKV-EDIII. The thick bands at 52Kd detects the presence of the protein at 0.5μg and 2μg.

                        CONCLUSION

                        Early detection of diseases increases the chances for successful treatment that can improve symptoms and slow down the progress of the disease. So, it is very essential for the diseases where drugs are not available, like Dengue Fever, Zika Fever, to get diagnosed at its earliest. The advancements in researches are helping in designing an ideal vaccine for dengue fever and some are already at its clinical stages. However, in the coming years, the vaccination for dengue will hopefully help in preventing this disease, which is now endemic.

                        ACKNOWLEDGEMENTS

                        First and foremost, I need to thank Dr. Navin Khanna, as without his help I could not have completed my summer training. His visionary approach and guidance were very valuable in bringing up my project completion. I am very fortunate for having been given an opportunity to be a part of his lab.

                        I would like to thank Science Academies’ Summer Research Fellowship Programme by Indian Academy of Sciences for giving this unique opportunity to work as a Summer Research Fellow.

                        I express my sincere thanks to Dr. Hemalatha mam and Dr. Raj Gokul sir, for guiding me heart fully and helping me in completing my project work.

                        I sincerely thank Dr. Viswanathan Sir, for always helping me in learning basics, and enhancing my curiosity to learn more.

                        I also thank Dr.Richa Ahuja mam, for always being my support system and boosting my confidence to work.

                        I extend my warm thanks to Jogi bhaiya and Jeevan bhaiya, who has always extended their hands for helping me out in needed situations.

                        Finally, I immensely thank my family for encouraging and trusting my abilities.

                        REFERENCES

                        1. Swaminathan S, Khanna N(2019). Dengue vaccine: Global and Indian scenarios. International Journal for Infectious Diseases.

                        2. Shanmugam RK,Ramasamy V,Shukla R,Arora U,Swaminathan S,Khanna N (2019). Pichia pastoris-expressed Zika virus envelope domain III on a virus-like particle platform: design, production and immunological evaluation. Pathogens and Disease, Volume 77, Issue 3, April 2019.

                        3. Anika.W, Wibberg.D, Jan-Philipp.S, Tobias. L, Jorn.K, Karl.F,et al (2016). Integration event induced changes in recombinant protein production in Pichia pastoris discovered by whole genome sequencing and derived vector optimization. Microbial Cell Factories.

                        3. Seema Yasmin, Madhusree Mukherjee. (2019, April). How the World's First Dengue Vaccination Drive Ended in Disaster. Scientific American Magazine.

                        https://www.scientificamerican.com/magazine/sa/2019/04-01/

                        Source

                        • Fig 2: https://www.youtube.com/watch?v=6Ue1Hd3dyaQ 
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