Test Procedure for Dengue IgM antibodies estimation in the Serum Samples

PURPOSE: This SOP explains the test procedure for detection of IgM antibodies for

Dengue by IgM capture ELISA kits provided from NIV, Pune.

2. STAFF RESPONSIBLE AND AUTHORIZED TO PERFORM THE PROCEDURE: Laboratory technicians

3. PRINCIPLE OF PROCEDURE USED FOR EXAMINATION:

IgM antibodies in the patient’s blood are captured by Anti-human IgM (µ chain specific) that is coated on to the solid surface (wells). In the next step, dengue antigen is added which binds to captured IgM, if the IgM and antigen are homologous. Washing is carried out to remove unbound antigen. In the subsequent step, biotinylated flavivirus cross reactive monoclonal antibody is added followed by addition of Avidin-HRP and then chromogen. It is observed for development of colour. The reaction is stopped by 1N H2SO4 and the colour intensity is measured by spectrophotometer reading of Optical density(OD) at 450nm. OD readings are directly proportional to the amount of Dengue virus specific IgM antibodies in the sample.

4. SAMPLE – Serum sample

5. TYPE OF CONTAINER – Plain test tube or vacuettee or screw capped vial

6. MATERILS, EQUIPMENTS AND REAGENTS USED:

• IgM ELISA Kit- NIV, Pune Kit
• Pipettes – 5ul, 50 ul, 500ul
• Microtips
• Discarding container of freshly prepared 1%Sodium hypochlorite.
• Disposable gloves
• Autoclavable eppendrof tubes – 1.8 ml.
• Autoclavavable external threaded vials. -  2 ml.
• Plastic beaker.
• Aluminium foil.
• Rubber Balb
• Pasteur pipette.
• ELISA washer
• ELISA reader

PREPARATION OF REAGENTS:

1.  Antigen-Ready to use

2.  Wash buffer

·           Wash buffer is to be diluted 1:10.

·           For 1 strip i.e.: 8 samples, approximately 100ml of diluted wash buffer is required.

·           Add 10 ml of wash buffer concentrate in 90 ml of distilled water in a sterile glass flask.

·           Wash buffer is diluted fresh every time test is performed.

   3.  Antibody-Ready to use

   4.  Controls- Positive & Negative - Ready to use

   5.  Avidine HRP- Ready to use

   6.  TMB Substrate- Ready to use

   7.   1 N H2SO4- Ready to use

   8.   Dilution buffers—ready to use

7. PROCEDURE - IgM Capture ELISA test method

TEST PROCEDURE:

1. Dengue Kit, reagents and serum samples to be tested are brought at room temperature. They are kept at room temperature half an hour before the start of test.
2. Test protocol is prepared according to the samples and positive & negative controls supplied in the kit. Two internal quality control samples- (In house controls - one positive and one negative) are also included in each test run.
3. Clean the work surface with 1% sodium hypochlorite solution. Wear apron and gloves.
4. Samples are arranged in the rack as per the protocol.
5. Antigens, Biotinylated Monoclonal Antibody & Controls are as specified in kit literature provided with the kit.
6. Dilution of sample: Serum is diluted 1:100 with sample dilution buffer. 5µl Serum sample is added to 500µl sample diluent in sterile 1.8 ml eppendorf tube. Each sample vial is vortexed for uniform mixing.
7. Washing of the wells: Coated plates are washed with wash buffer for 3 times in automatic ELISA washer.(Refer SOP No 2.4, Page no 15-16 of Instruments SOP issue no 3)

1. If ELISA washer is not working then washing is performed manually with multichannel pipette. 300µl wash buffer is filled with multichannel pipette in all the wells, mixed & aspirated. This is discarded in the container of 1% Na hypochlorite. Same step is repeated for 4 times. Plate is tapped on blotting paper for drying. 
2. 50 µl of diluted samples is transferred to the appropriate wells of ELISA plates mentioned in the protocol. 50 µl Positive control and 50 µl Negative control which are ready to use are transferred to respective wells. 50 µl inhouse positive control and 50 µl inhouse negative control are transferred to respective wells. Use separate tip for each sample and controls. Discard the tips in the discarding container of 1% sodium hypochlorite.
3. Plate is kept in a humidified box and incubated at 37ºC for 1 hour.
4. At the end of incubation, plate is washed 5 times with wash buffer. Tap the plate after last wash on a blotting paper.
5. 50 µl of antigen is added to each well.
6. Plate is kept in a humidified box and incubated at 37ºC for 1 hour.
7. Plate is washed for 5 times with wash buffer as mentioned in step no.7.
8. 50 µl of Flavivirus cross reactive monoclonal antibody (biotinylated) is added in each well.
9. Plate is kept in a humidified box and incubated at 37ºC for 1 hour.
10. Plate is washed for 5 times with wash buffer as mentioned in step no.7.
11. 50 µl Avidin-HRP is added to each well.
12. Plate is kept in a humidified box and incubated at 37ºC for 30 minutes.
13. After incubation, Plate is washed for 5 times with wash buffer as mentioned in step no.7.
14. 100 µl TMB substrate is added in each well.
15. Incubate the plate for 10 minutes in dark at room temperature. 
16. 100 µl of 1N H2SO4 is added to stop the reaction.
17. OD values are taken at 450 nm within 10 minutes in ELISA reader.

(Refer SOP No 2.5, Page no 17-18 of Instruments SOP issue no 3)

18. O.D value print out is pasted in the protocol and each O.D value is entered in the Protocol & sample receiving register.
19. At the end of day’s work clean the work surface with 1% sodium hypochlorite solution.
20. Discarding container is autoclaved at the end of day.

8. PERFORMANCE SPECIFICATION - IgM capture ELISA is a qualitative test for IgM antibody estimation.

CALIBRATION PROCEDURE – Not applicable

10. QUALITY CONTROL PROCEDURE-

INTERNAL QUALITY CONTROL

1. 1 Positive and 1 negative serum sample from test run is stored in a screw capped vial. Each quality control serum sample is given a QC no. & aliquots of 30  µl in 5 to 6 sterile external screw cap vials are made and kept in a cryo vial box at -20 ºC in deep freeze no. DDF1.
2. These samples are used as internal quality control samples for each test run of ELISA. They are numbered as 1/1, 1/2 etc (ie QC no / vial no.)
3. One positive QC & one negative QC sample is run with each test of ELISA and its values are compared with previous values.
4. Positive and Negative controls provided with test kit are also tested with each run.
5. Results are noted and if any deviation is observed then run is considered invalid. It is investigated and appropriate corrective action is taken to identify the non conformity and correction is done.
6. Preserved samples are kept for 6 months after which they are discarded by autoclaving.

11. INTERFERENCE-

Turbid, Lipemic and Haemolysed samples interfere with results.

12. LABORATORY INTERPRETATION-

VALIDATION CRITERIA

            Positive Control: OD values ≥ 0.5

            Negative Control: OD values ≤ 0.18

Interpretation of Samples:

Positive - If the OD value of sample tested exceeds cut off value. (Cut off= OD of Negative control x 4).

Negative - If the OD value of the sample tested is less than cut off value. (Cut   off= OD of Negative control x 4).

13. ALERT CRITICAL VALUE – Not applicable

14. SAFETY PRECAUTIONS –

• Do not mouth pipette.
• Follow the safety precautions of Bio safety level 2. (Refer Biosafety manual, Issue no.1,section-12.1, page no.51)
• Handle the broken glassware carefully with gloved hands.
• All the spills should be handled with gloved hands. (Refer Biosafety manual, Issue no.1,section-12.5, page no.60)
• The preservative Sodium azide is carcinogenic, so handle with care and remains of wash buffer or reagents containing this should be poured in sink and flushed with lots of running water.
• Slowly mix samples or reagents to avoid the formation of aerosols.

15. VALIDATION OF RESULTS TO BE DONE BY – Technical and Doctor Staff

16. REVIEW, RECORDS, REMARKS AND RECOMMENDATION

• Register No. DR-02  Protocol for Dengue ELISA
• Register No. DR-01  Dengue sample receiving register

17. REFERENCE - Kit literature of NIV kit