KAPVGMC MICROBIOLOGY LABORATORY STANDARD OPERATING PROCEDURE

(KML)
K.A.P. Viswanatham Government Medical SIGNAL HIV (IMMUNO DOT)
college
Tiruchirappalli – 620 001. Issued date : 01.01.2019

1.0 OBJECTIVE:
To describes the procedure for carrying out the SIGNAL HIV Flow through HIV 1+2
spot / Immunodot Test Kit, manufactured by Span diagnostics Ltd.,

2.0 SCOPE:
This standard operating procedure contains the procedure to be adopted for detection of
HIV 1 & 2 antibodies using SIGNAL HIV Flow through HIV 1+2 spot / Immunodot Test
Kit.

3.0 Principle:
The SIGNAL HIV Flow through HIV 1+2 spot / Immunodot Test Kit detects HIV
antibodies in the serum which bind to immobilized antigens on a porous immunofiltration
membrane to give a distinct pinkish purple dot.

4.0 RESPONSIBILITY:
It is responsibility of the lab technician to:
 Testing the blood sample for HIV antibody detection in the lab.
 Entering the details in the respective documents.
 Ensuring rejected samples are discarded properly.

5.0 DESIGNATED AREA:

Workbench of the SRL, Department of Microbiology, KAPVGMC, TIRUCHY .

6.0 DEFIINITIONS:
Negative control serum: Negative for HIV antibodies supplied by the manufacturer.
Positive control serum: Positive for HIV antibodies supplied by the manufacturer
External Quality control: Sample previously received and tested in the HIVTL/SRL.

Document Type : Standard Operating Procedure Issue No. : 11

Document No. : MICRO/KAPVGMC/SOP-SRL 25 Issue Date : 01.01.2019
Amendment No. : 00 Page No. :Page 1 of 5
Prepared by Reviewed by Approved by
 KAPVGMC MICROBIOLOGY LABORATORY
(KML)
K.A.P. Viswanatham Government Medical
college
Tiruchirappalli – 620 001.
STANDARD OPERATING PROCEDURE
SIGNAL HIV (IMMUNO DOT)
Issued date : 01.01.2019

7.0 MATERIALS REQUIRED:

EQUIPMENTS
 Refrigerator with freezer
 Micro pipette (20- 200ul)
 Timer
8.0 KIT COMPONENTS:
 SIGNAL HIV Flow through HIV 1+2 spot / Immunodot Test Kit
 Buffer solution
 Gold conjugate
 10ul dropper
 Controls
CONSUMABLES REGISTERS
 Tissue paper  Lab register
 Disposable gloves  Stock register
 Spirit & Cotton,  Work sheet
 Discarding bin with 1% Sodium
hypo chlorite
 Pipettes Tips (200 ul-1000 ul)
 Water
 SIGNAL HIV Flow through HIV
1+2 spot / Immunodot Test Kit
 Marker pen

9.0 STORAGE:
 SIGNAL HIV Flow through HIV 1+2 spot / Immunodot Test Kit should be stored in
Refrigerator at 2 - 8oC.
10.0 TEST PROCEDURE:
Do not open pouch until you are ready to test the sample.
 Universal precautions are strictly followed during the procedure.
 Prepare a sample work sheet as per the samples received

Document Type : Standard Operating Procedure Issue No. : 11

Document No. : MICRO/KAPVGMC/SOP-SRL 25 Issue Date : 01.01.2019
Amendment No. : 00 Page No. :Page 2 of 5
Prepared by Reviewed by Approved by
 KAPVGMC MICROBIOLOGY LABORATORY
(KML)
K.A.P. Viswanatham Government Medical
college
Tiruchirappalli – 620 001.
STANDARD OPERATING PROCEDURE
SIGNAL HIV (IMMUNO DOT)
Issued date : 01.01.2019

 Take HIV Kit from refrigerator and leave in room temperature for 30 minutes before
testing.
 Bring all reagents and specimens to room temperature.
 Take required number of testing devices according to number of samples and controls to
be tested.
 Remove the test device from the foil pouch and place on a clean dry surface.
 Label the device with the control and sample PID numbers using a permanent marker.
 Place the devices on a flat surface.
 Add 2 drops of wash buffer solution to the well in the device.
 Add 1 drop of Test samples, negative and positive controls provided by the manufacturer
and a EQC sample previously received and tested by the laboratory should be added to
the test devices respectively.
 Use separate dropper for each sample.
 Add 2 drops of wash buffer solution to the well.
 Add 2 drops of signal reagent to the well.
 Add 3 drops of wash buffer solution.
 Record results within next 10 minutes.
 The materials and samples used in the procedure are discarded as per the biomedical
waste management guidelines.
11.0 INTERPRETATION OF RESULTS:
 Non-reactive: Only the control dot appears on the membrane. The absence of a test dots
at test region indicates a negative result.
 Reactive: Control dot and dot appearing on test region indicates positive result for HIV
1 &/ 2.
 Invalid Result: Absence of pinkish red dot at control “C” region with presence or
absence of dot at test “T” region indicates invalid result.
 Repeat the test using a new test device.
 Validity of the test:
Document Type : Standard Operating Procedure Issue No. : 11
Document No. : MICRO/KAPVGMC/SOP-SRL 25 Issue Date : 01.01.2019
Amendment No. : 00 Page No. :Page 3 of 5
Prepared by Reviewed by Approved by
 KAPVGMC MICROBIOLOGY LABORATORY
(KML)
K.A.P. Viswanatham Government Medical
college
Tiruchirappalli – 620 001.
STANDARD OPERATING PROCEDURE
SIGNAL HIV (IMMUNO DOT)
Issued date : 01.01.2019

 The validity of this test is dependent on the results obtained with the positive and negative
controls used in the test.
 Positive control should give a dot at ‘C’ along with dots at ‘T’ region.
 Negative control should give a dot only at ‘C’.

Note: The readings of all tests should be recorded by a minimum of two individuals. Weakly
reactive samples require adequate natural lighting for proper interpretation and should
therefore be read with appropriate lighting in the laboratory.

12.0 TROUBLE SHOOTING:

PROBLEM REASON SOLUTION
1. Flooding of sample  Adding more drops of  Add only as per the given
buffer or conjugate. procedure.
2. Weak intensity of  Very cold reagent  Bring the sample and buffer and
control line conjugate to room temperature if
stored lower temperature.
3. Poor sensitivity or weak  Frozen sample not mixed  Mix the sample well and
intensity of test and properly after thawing. centrifuge to remove particulate
control lines. matter before pipetting.
4. Half dot seen  Sample insufficiently added  Add sufficient volume.

13.0 REFERENCES:
 Kit Insert – SIGNAL HIV Flow through HIV 1+2 spot / Immunodot Test Kit,
manufactured by Span diagnostics Ltd.,
 National Guidelines on HIV testing, Ministry of Health and family Welfare, National
AIDS Control Organization, March 2007 & July 2015
 Manual on Quality Standards for HIV Testing Laboratories, Ministry of Health and
family Welfare, National AIDS Control Organization, March 2007.
 National Guidelines on Quality Management Systems in HIV Testing Laboratories,
Ministry of Health and family Welfare, National AIDS Control Organization, July 2015.

Document Type : Standard Operating Procedure Issue No. : 11

Document No. : MICRO/KAPVGMC/SOP-SRL 25 Issue Date : 01.01.2019
Amendment No. : 00 Page No. :Page 4 of 5
Prepared by Reviewed by Approved by
 KAPVGMC MICROBIOLOGY LABORATORY
(KML)
K.A.P. Viswanatham Government Medical
college
Tiruchirappalli – 620 001.
STANDARD OPERATING PROCEDURE
SIGNAL HIV (IMMUNO DOT)
Issued date : 01.01.2019

Document Type : Standard Operating Procedure Issue No. : 11

Document No. : MICRO/KAPVGMC/SOP-SRL 25 Issue Date : 01.01.2019
Amendment No. : 00 Page No. :Page 5 of 5
Prepared by Reviewed by Approved by