Of infections of the nervous




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ASSAY OF POLIO VIRUS ANTIBODIES BY NEUTRALISATION TEST IN VITRO BY USING CELL CULTURE TECHNIQUE
Antibodies to polio viruses develop after vaccination with polio vaccine or after infection with wild virus. Antibodies to polio and other enteroviruses may be present in normal population and hence demonstration of four fold rise in the titre of antibody is absolutely necessary for the sero- diagnosis of enteroviral infections. For the test, two serum samples, one taken just after the onset of the disease and the second, after a gap of 2 weeks or more is required.

In the test proper, increasing dilutions of patient's serum is mixed with 100 LD50. Of the 3 types of polio viruses and incubated in water bath for 2 hrs. The mixture is then inoculated to preformed monolayers of either Vero or Hep 2 cells in flat bottom 96 well tissue culture plates and incubated for 7 days. Readings are taken at 3rd and 7th day and depending on the CPE seen in different dilutions, titre of the serum sample is calculated by using Reed and Muench method.


MATERIALS REQUIRED :


  1. 96 well flat bottom tissue culture plates

  2. Micro pipettes, 200 and 1000 Ul

  3. Microbiological safety cabinet (Laminar flow)

  4. CO2 incubator.

  5. Test tubes or penicillin vials

  6. MEM with added antibiotics and containing 2% FCS

  7. Water bath 56 C and 37 C


PROCEDURE :

Each serum sample has to be tested separately for antibodies against 3 polio viruses. Before starting the procedure a plan should be drawn for the distribution of samples and controls in the microtitre plates.



  1. Inactivate the serum samples at 56 C for min.

  2. Dilute the serum samples from 1:16 to 1:2048 in test tubes or penicillin vials using MEM with 2% FCS.

  3. Dilute the 3 types of polio viruses, P1, P2 and P3 in MEM to contain 100 TCID 50 per

    1. ml using MEM with 2% FCS.

  1. Mix equal serum quantities of serum dilution and virus dilution in test tubes and incubate in

water bath at 37 C for 2 hrs.

  1. Simultaneously prepare three 10 fold dilutions of challenge virus dose starting from the

dilution containing 100 TCID 50. Mix equal quantities of these dilutions and MEM with 2% FCS and incubate in a similar manner.

  1. At the end of incubation, dispense 100 ul of serum virus mixture and virus MEM mixture to

wells of the micro titre plate, starting from lower dilution of the serum to the higher. Include 2 wells for cell control and to this add 100 UL of MEM. Each serum dilution and virus dilution should be inoculated to at least 4 wells.

  1. Incubate the plates in a CO2 incubator and take the reading at the end of 3rd and 7th day. Look

for the CPE in wells in which virus dilutions were added and calculate the actual TCID50

employed in the test. This should be around 100 TCID50 per 0.1 ml. Note down the highest serum dilution showing complete neutralisation of CPE. Calculate the 50% endpoint dilution by using Reed -Muench formula. If there is initial neutralisation at day 3 followed by breakthrough at day 7, it indicates partial neutralisation due to antigenic relation with other enteroviruses.


ENZYME LINKED IMMUNOSORBENT ASSAY
Preparation of solutions and reagents for ELISA

  1. 0.05M carbonate buffer pH 9.6.

  1. Dissolve 0.53 g Na2CO3 (anhydrous) in distilled water and make final volume to 100 ml.

  2. Dissolve 0.42 g NaHCO3 in distilled water and make final volume to 100 ml.

To 32 ml of solution A, slowly B till pH is 9.6 (approximately 68 ml).


  1. Phosphate citrate buffer, pH 5.0.

  1. Dissolve 11.9 g of Na2HPO4, 2H2O in distilled water and make up to 1 litre (Solution A).

  2. Dissolve 7.0 g of citric acid in distilled water and make up to 1 litre (Solution B).

Mix 55 ml of Solution A with 45 ml Solution B.

Adjust pH to 5.0 with either a or b if necessary.


  1. 0.01 M Phosphate buffer saline (PBS), pH 7.2.

Dissolve 8.0 g of NaCl, 0.2g of KC1, o.2g of KH2PO4 and 1.5 g of Na2HPO4. 2H2O in distilled water and bring the final volume to 1000ml. Adjust pH to 7.2 with 0.1 N NaOH or 0.1N HC1 if necessary. Add 0.5 ml/L Tween 20 (Polyoxyethylene sorbitan monolaurate) to 1X PBS to make PBST.


ANTIGEN PREPARATION OF HERPES SIMPLEX VIRUS


  1. 0.5ml of a stock of Herpes simplex virus (HSV-1) is added to a Roux bottle with a monolayer of Vero cell line after discarding the growth medium.

  2. Adsorb the virus for 30 mins at 37 C and add 100 ml of maintenance medium. Incubate in a CO2 incubator at 37 C.

  3. Observe for evidence of CPE (24-48 h).

  4. Remove the supernatant medium and harvest the cells with 10-20 ml of sterile PBS, when >90% of the cells show CPE.

  5. Centrifuge for 5 mins at 1000 rpm and wash the cells thrice with sterile PBS.

  6. Resuspend the cells in a minimal amount of PBS (ex:1ml).

  7. Sonicate the suspension at 60 cycles/second continuously for 5 mins and pulsed sonication for 3 mins.

  8. Centrifuge the sonicate for 10 mins at 1000 rpm.

  9. The supernatant is used as the antigen after estimating the protein concentration.

  10. Control Vero cell antigen is prepared in a similar manner with mock infected Vero cells. (Sterile medium to be used instead of the virus suspension).

NOTE : The protein values of the HSV and Vero cell antigens should be matched before it is used in the test.



MEASLES VIRUS ANTIGEN PREPARATION


  1. 1 vial of commercial Measles vaccine reconstituted in 0.5 ml of cell culture medium and

made up to 5 ml with same medium and inoculated into 1 Roux bottle with a monolayer of Vero cell line.

  1. Adsorption is carried at 37 C for 1 h

  2. Maintenance medium is added.

  3. Cytopathic effect is first seen on third day and it gradually increases over next four days. On the seventh day extensive CPE can be seen

  4. The fluid is then frozen and thawed twice and kept as seed stock.


PREPARATION OF ANTIGEN


  1. Virus is grown in Roux bottle and fluid harvested aseptically.

  2. Spin at 1000 rpm for 30 min.

  3. To supernatant add sodium chloride (4gms/100ml) and PEG precipitate at a concentration of 10%. (10gms/100ml); (add Nacl first allow it to dissolve and then add PEG slowly)

  4. Stir for half an hour in ice bath and add 0.1% sodium azide while stirring.

  5. Keep for two hours or overnight in refrigerator.

  6. Spin at 10000 x g for 30 min.

  7. Discard supernatant.

  8. Resuspend pellet in 1/100th of the original volume with normal saline.

  9. Dialyse against sterile normal saline containing 1% sodium azide for 24h with three changes. (Dialysis bags are pretreated by boiling in 1% sodium carbonate solution in distilled water containing lmM EDTA).

  10. Collect the dialysed antigen centrifuge and collect supernatant 5MM EDTA + 200MM NaHCO3 in 100 ml D.W. x 20 min. and estimate the protein before using it in the test.

  11. Control Vero antigen is prepared in a similar manner with fluid collected from mock infected Vero cells.


EXTRACTION OF JAPANESE ENCEPHALITIS VIRUS ANTIGEN WITH SUCROSE AND ACETONE

1. The infected suckling mouse brain is homogenised with four volumes of chilled 8.5%

aqueous solution of sucrose in homogeniser (Waring blender) (ex. If the total weight of the mouse brain is 2g then 8ml of sucrose solution should be added)

2. The homogenate is added drop wise with brisk mechanical stirring to 20 volumes of

chilled acetone. The mixture is shaken vigorously and allowed to stand for 15 min.

The milky supernatant acetone is decanted, the sediment at this stage being a pink gummy mass which is tightly adherent to the bottom of the centrifuge bottle.

3. A volume of fresh acetone equal to that originally used is added to each bottle and the

preparation are allowed to stand in an ice bath for at least one hour to dehydrate the

sediment; after sufficient time the sediment is readily reduced to a fine suspension by use of a thick glass rod. The bottles are then centrifuged as before. The supernatant fluid is

aspirated out and the sediments from all bottles are pooled into one bottle with the help of small quantity of fresh acetone.

4. After most of the final supernatant acetone has been removed by aspiration the bottle is

closed with a rubber stopper fitted with glass tubing, which is connected to a vacuum pump for drying. The drying is continued for one hour with the bottle kept immersed in an ice bath. The pump is protected from contamination by a trap containing a large quantity of non-absorbent cotton.

5.To the dry powder a volume of 0.9% NaCl is added which is equal to 2/5th of the total

Volume of homogenate originally used (Ex. If the homogenate is 10ml, 4ml of 0.9% NaCl is added for rehydration). The bottle with antigen is left overnight in ice water in the cold room. The next morning it is centrifuged at 10000 rpm for one hour. The supernatant fluid is antigen. For gel diffusion test uncentrifuged antigen is used.


Note : Tris buffer (0.1M) pH 8 to 9 can be used rehydration of sucrose acetone antigen instead of 0.9% NaCl.
Procedure for biotinylation of monoclonal antibody
Reagents :

  1. Monoclonal antibodies : Ascitic fluids from BALB/c mice inoculated with desired clones have to be precipitated using the sodium sulphate technique. After dialysis the protein concentration is measured (absorbance at 280nm) and adjusted to 1mg in 0.1M sodium bicarbonate solution (stock solution).

  2. Biotin-N-hydroxy succinamide (BRL Bethesda): 1mg of Biotin-n-hydroxy succinamide is dissolved in 1ml of dimethylsulfoxide (Sigma or Merck). Prepare it fresh on the day of use. DO NOT STORE SOLUTION PREPARED EARLIER.

3) Good quality dialysis bags (25000 mol.wt cutoff)
PROCEDURE

To 1000 ug of monoclonal antibody in 1ml of sodium bicarbonate solution, add 200 ul of stock biotin solution drop-wise (1mg/ml DMSO, it can be 100, 200 & 400 accordingly check before use). After mixing well incubate at room temp. for 4 hours with periodic mixing. At the end of 4 hours dialyse the mixture against sterile normal saline (500 ml x 3 changes) for 18 hours at 4 C to remove unbound biotin. After dialysis measure the exact volume. If the volume is 1 ml then the protein concentration is 500 ug/ml. Distribute 50 ul volumes into 20 Eppendorf tubes (bullets). Add 50 ul of sterile glycerol to each bullet (cryopreservative). Each bullet now contains 50 ug of biotinylated monoclonal antibody. Store at -70 C. Estimate optimal concentration of this diluent for this antibody. DO NOT USE PLAIN PBS SINCE HIGH BACKGROUND IS OBTAINED. The conjugate used is STREPTAVIDIN PEROXIDASE (BRC Bethesda) at a dilution of 1:1000 (50 ul/well in MAC-ELISA). Incubation with biotinylated monoclonal antibody is for 2 hours at 37 C in a water bath (float the plates).


DETECTION OF IgG ANTIBODIES TO HSV-1 BY ELISA


  1. Coat ELISA plates with HSV and Vero antigens at a predetermined concentration in carbonate

buffer pH 9.6, 100 ul/well.

2. Incubate overnight at 4 C.

3. Discard and wash the plates thrice with PBST.

4. Quench the plates with 1% skimmed milk powder in PBST, 150 ul/well.

5. Incubate for 2hrs at 37 C.

6. Discard, wash plates thrice with PBST.

7. Add 100 ul/well sera, CSF appropriately diluted in PBST. Include positive, negative

controls and blanks.

8. Incubate for 2 hrs at 37 C.

9. Discard, wash thrice with PBST.

10. 100 ul/well of goat/rabbit anti-human IgG peroxidase conjugate (Bangalore Genei)

(dilution to be used: 1:2000 in PBST with 1% normal goat serum)

11. Incubate for 2 hrs at 37 C.

12. Discard, wash thrice with PBST.

13. Add 100 ul/well of a solution containing OPD (4mg/10ml of PCB, pH 5) with 10 ul

of H2O2.

14. Incubate in the DARK at room temperature for 20 minutes

15. Stop the reaction with 50 ul/well of 4N H2SO4.

16. Read the plate at 492 nm on an ELISA reader.

17. A sample is considered positive if the difference in OD values is >0.1 between the

HSV antigen and Vero antigen at that particular dilution. Antigen is considered

significant at that dilution.




DETECTION OF IgG ANTIBODIES TO MEASLES VIRUS BY ELISA


  1. Coat ELISA plates (100 ul /well) with measles virus antigen and Vero antigen at a

predetermined concentration in carbonate buffer pH 9.6

  1. Incubate overnight at 4C.

  2. Discard contents of the wells and wash thrice with PBST.

  3. Quench with 1% skimmed milk powder in PBST (150 ul/well). Incubate at 37C for 2 hrs.

  4. Discard and wash thrice with PBST.

  5. Add the required dilutions of CSF/Serum samples (100 ul/well) and incubate at 37 C for 2 hrs. Include positive , negative controls and blanks.

  6. Discard and wash thrice with PBST.

  7. Add 100 ul/well of rabbit anti-human IgG peroxidase conjugate (Bangalore Genei) diluted 1:2000 in PBST with 1% normal goat serum and incubate for 2 hrs. at 37 C.

  8. Discard and wash thrice with PBST.

  9. Add 100 ul/well of a solution containing OPD (4mg/10ml of PCB,pH5) and 10 ul of H2O2. Incubate at RT for 20 mins in the dark.

  10. Stop the reaction with 50 ul/well of 4N H2SO4.

  11. Read the results at 492 nm on an ELISA reader.

  12. A sample is considered positive if the difference in OD values is > 0.1 between the measles virus antigen and Vero antigen at any given dilution.


DETECTION OF IgM ANTIBODIES TO JEV BY AN IGM ANTIBODY CAPTURE ELISA (MACELISA)


  1. Coat ELISA plates with 100 ul/well of a predetermined concentration of rabbit anti-human IgM antibodies (DAKOPATTS) in carbonate buffer pH 9.6.

  2. Incubate at 4 C overnight.

  3. Discard and wash with PBST thrice.

  4. Quench with 1% skimmed milk powder in PBST (150 ul/well). Incubate at 37C for 2 hrs

5. Discard and wash thrice with PBST.

  1. Add 100 ul/well of CSF (diluted 1:100 in PBST) in duplicates. Incubate for 4hr at 37 C.

  2. Discard and wash with PBST thrice.

  3. Add 100 ul/well of JEV mouse brain antigen (50HA units/100 ul diluted in PBS). Incubate at 4 C overnight.

  4. Discard and wash with PBST thrice.

  5. Add 100ul/well of biotinylated NIV cross-reactive monoclonal antibody to JEV (25ul/10 ml of PBST containing 1% skimmed milk powder and 1% normal goat serum). Incubate at 37 C for 2 hr.

  6. Discard and wash with PBST thrice.

  7. Add 100 ul/well of streptavidin peroxidase conjugate (DAKOPATTS) diluted 1:5000 in PBST containing 1% normal growth serum. Incubate for 15 mins at RT.

  8. Add 100 ul/well of a solution containing OPD (4mg/10ml of PCB,pH5) and 10 ul of

H2O2. Incubate at RT for 20 mins in the dark.

14. Stop the reaction with 50 ul/well of 4N H2SO4.

15. Read the results at 492 nm on an ELISA reader.


  1. Appropriate high positive control, low positive control, negative control for CSF and serum as well as blanks should be included in every assay.

  2. The results are expressed as MACELISA units which is calculated using the following

formula :
ELISA units = (OD of test sample - OD of negative control)

--------------------------------------------------------- x 100



(OD of weak positive - OD of negative control)
Interpretation of results is as follows :

  1. Any test sample with > 100 units is considered positive for IgM antibodies to JEV.

  2. A sample with ELISA units between 30-99 is considered probably positive for IgM antibodies to JEV but requires further testing with Dengue and West Nile virus antigens as well as normal mouse brain antigen to exclude false positive reactions.

  3. A sample with ELISA units < 30 is considered negative IgM antibodies to JEV.


IMMUNOASSAYS FOR THE DETECTION OF RETROVIRAL ANTIBODIES
Western blot analysis for HTLV-1 proteins


  1. Preparation of Western blot strips : Use solubilized virus obtained from MT2 or HUT102 cells which has been purified and concentrated 1000 fold by sucrose gradient centrifugation .



  1. Polyacrylamide Gel Electrophoresis :

  1. Layer the 10% resolving gel and incubate for 15min. at RT with distilled water layered over it.

  2. After this removing the water layer 4% stacking gel on to the resolving gel and insert combs into the stacking gel. Incubate at RT for 15 min.

  3. Remove the comb and flush the wells with distilled water.

  4. Carefully remove the clamps and mount the gel plates on to the Electrophoresis unit and add tank buffer into both lower and upper tanks. Ensure that the upper tank has sufficient buffer to cover the wells in the gel.

  5. Denature the protein sample (HTLV-1 antigen) in sample buffer by boiling for 3 min. in a water bath. Load 25 to 50 ul of this denatured sample into each of the wells carefully avoiding spills and air bubbles using a micro pipette.

  6. Run the gel in the constant voltage mode at a setting of 60 volts until the dye front reaches the bottom of the gel plate.

  7. Remove the gels carefully and use for transferring of proteins to nitrocellulose membranes by Western Blotting.



  1. Western Blotting :

  1. Transfer Electrophoresed gel to tray containing Western Blot buffer (20% methanol in 1x tris-glycine buffer which has been de-aerated for 15 min.)

  2. Place a pre-wetted nitrocellulose sheet, which has been cut to the same dimensions as the gel on the gel ensuring that there are no air bubbles trapped in between.

  3. Keep filter paper pads below the gel and above the nitrocellulose paper to make a sandwich.

  4. Place this sandwich in the appropriate clip and insert into the Electro blotting tank containing buffer.

  5. Ensure that the anode is towards the nitrocellulose membrane side while the cathode is towards the gel.

  6. The electroblotting is carried out using constant current mode at 150 MA for 2 hrs.

  7. Remove the nitrocellulose membrane (always handle gently using a forceps) and place in a tray containing 1% milk powder in PBS. Incubate at 4 C overnight.

  8. The nitrocellulose membrane is removed from the tray and carefully cut into thin strips. Store in a zip lock bag at 4 C for upto 1 month.

  9. Place thin strips in a Western Blot immuno assay tray and wash with PBS using a rocking platform.

  10. Add 2.5 ml of 1:50 dilution of CSF or 1:100 dilution of serum prepared in PBS containing 1% skimmed milk powder.

  11. Incubate at room temperature on the rocking platform for 2 hrs. Wash thrice with PBST on the rocking platform.

  12. Add 2.5 ml of appropriate dilution of anti-human IgG peroxidase conjugate prepared in PBST containing 1% milk powder and incubate on the rocking platform at RT for 2 hrs.

  13. Wash thrice with PBST on the rocking platform.

  14. Add 2ml of substrate solution (Diaminobenzidine 4 mg /10 ml of PBS and 10 ul of H2O2) and rock on the platform for 1 min. and keep the tray in the dark for 10 min. at room temperature.

  15. Stop the reaction by adding distilled water to each of the wells.


ELISA procedure for detection of anti HTLV-1 antibodies


  1. Use cell culture purified and concentrated virus solubilized virus for ELISA. The optimal

concentration should be pre-determined in a checkerboard titration. Usually 5-10 ug/ml

of viral antigen gives best results.



  1. Coat plates with 100 ul of antigen diluted in 50 mM NaHCO3 buffer, pH 9.6 and incubate

at 4 C overnight.

  1. Discard contents and rinse thrice with PBS. Add 200 ul of 2.5% BSA solution in PBS

containing 1.25 % milk powder and 5% sucrose to each of the wells and incubate at 37

C for 2 hrs. Wash plates thrice with PBST.



  1. Add 100 ul of CSF/serum samples diluted five fold (1:5 to 1:625) in PBST with 2% milk powder to the wells in the ELISA plate.

  2. Incubate at 37 C for 2 hrs. Wash plates thrice with PBST.

  3. Add 100 ul of goat anti-human IgG peroxidase conjugate (Bangalore Genei) diluted 1:1000 in PBST containing 1% normal goat serum. Incubate for 2hrs. at RT. Wash thrice in PBST.

  4. Add 100 ul of substrate (4 mg % of OPD in PCB with 0.005% H2O2) and incubate in the dark for 20 mins. at RT.

  5. Stop the reaction with 50 ul of 4N H2SO4.

  6. Measure the OD values at 492nm in an ELISA Reader.
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