3.2.1 Anti HIV Testing (ELISA Method)

Model SOP
Standard Operating Procedure
Name of the facility / activity : Anti HIV Testing of Blood unit (by Elisa
Method)
SOP no. Effective Date Pages Prepared by Authorised by

3.2.1 27-11-2000 5
Version Review Period Date of Review Reviewed by Number of copies
VI 2 years 01-01-2015 10
LOCATION : TTI Testing Laboratory
SUBJECT : Anti HIV Testing
FUNCTION : Sample tested for Anti HIV antibodies by ELISA method.
DISTRIBUTION: Supervisor in charge of TTI Testing Laboratory
Master File
1. SCOPE & APPLICATION:

Anti HIV antibodies testing is carried out on all bag samples before these are released
for transfusion. Pre-donation samples of pheresis donors are also tested.
2. RESPONSIBILITY:
It is the responsibility of technician from TTI Testing lab; to carry out the test and
report as required.
3. MATERIALS REQUIRED:
 Reagent kit
 Mircopipettes and disposable pipette tips
 Timer
 EIA reader
 EIA Washer
 Incubator 370C
 Vortex Mixer
 Glassware
 Distilled water.
Prepared By Approved By
Specimen – clotted blood sample of the donor.
4. PROCEDURE
Preparation of the reagents

1. Bring all the reagents to room temperature for 30 minutes before use.
2. Take the required number of strips from sealed HiV Antigen-antibody coated
microplate, and the remaining strips must be kept at 2-80C with a silica gel
(desiccant) in an aluminium pouch.
3. Preparation of Washing Solution
Make a 1:20 dilution of Washing Solution with distilled or de-ionized water (to the
extent of required amount for example, add 10 ml of concentrated Washing
Solution to 190 ml distilled or deionized water). Washing Solution may be
crystallized at cool storage condition. If crystallized, use it after thawing at 37 0C
water-bath maintained at 370C.
4. Preparation of Conjugate
Make a 1:51 dilution of Conjugate concentrate with Conjugate Diluent to the
extent of required amount, 10 minutes before use (Refer Table 1. Shake well
before use.
Table 1.
Strips 1 2 4 6 8 10 12
required
Conjugate diluents (ml) 1 2 4 6 8 10 12
Concentrated conjugate 20 40 80 120 160 200 240
(µl)
5. Preparation of Substrate
Make a 1:101 dilution of TMB Chromogen in DMSO with Substrate Buffer, 5 to 10
minutes before use (Refer Table 2). Avoid exposure to light. Substrate should be
used only after thawing at 370C, if crystallized.
Table 2.
Strips
1 2 4 6 8 10 12
Required
Substrate
1 2 4 6 8 10 12
Buffer (ml)
TMB
10 20 40 60 80 100 120
Chromogen (µl)
STORAGE AND SHELF LIFE OF PREPARED REAGENTS

Prepared Reagent Storage Expiry date
Wash Solution 2-80C 1 week
Substrate Room temperature 4 hours
ASSAY PROCEDURE
1. Take the required numbers of strips and fix them to frame.
2. Pipette 100 µl of Negative Control into each well of 1A to 1B, 100 µl of antibody
Positive Control into well of 1C, 100 µl of Antigen Positive Control into well 1D and
pipette 100 µl of each sample into the remaining wells.
 Take care not to mix or splash contents out of wells while using microplate
shaker.
3. Incubate at 37 ± 10 C for 60 minutes after sealing the plate with cover sealer.
4. Before the last 10 minutes of 1st incubation, make a 1:51 dilution of Conjugate
with Conjugate Diluent.
5. Aspirate the contents from all the wells and wash each one 5 times with 300 µl of
diluted Washing Solution. (300 µl/well/time)
6. Invert the plate and tap it on absorbent paper to remove the remaining Washing
Solution, and then, pipette 100 µl of prepared diluted Conjugate into each well.
7. Incubate the plate at 37 ± 10 C for 30 minutes after sealing it with plate sealer.
8. Before the last 5 to 10 minutes of second incubation, make a 1:101 dilution of
Substrate with Substrate Buffer.
9. Aspirate the contents from each of the wells and wash each one 5 times with 300
µl of diluted Washing Solution (300 µl/well/time)
10. Invert the plate and tap it on absorbent paper to remove the remaining Washing
Solution. And then, pipette 100 µl of prepared substrate into each well and
incubate at controlled room temperature (23 ± 20 C) for 30 minutes. Avoid
exposure to light.
11. Pipette 100 µl of Stop Solution into each well and tap the plate gently to
homogenize the colouring materials.
12. Read the absorbance at 450 nm (reference wavelength at 620 nm ) against an air
blank within 30 minutes after addition of Stop Solution.
QUALITY CONTROL
 The average absorbance (PCx) of both the Positive Controls should be greater
than or equal to 1.0.
 The average absorbance (NCx) of the Negative Control should be less than or
equal to 0.100 and greater than – 0.005.
If the results are outside the above range, the test should be conducted again.
INTERPRETATION OF RESULTS
1. Calculation of the cut off value.
a) Calculation the Negative Control mean (NCx)
Negative control 1 absorbance = 0.045
Negative control 2 absorbance = 0.043
Negative control mean (NCx) = (0.045 + 0.043) / 2 = 0.044
 If 3 negative controls were tested, calculate a mean value of 3 Negative
Controls.
b) Calculate the cut-off value
Cut off value = NCx + 0.200 = 0.044 + 0.200 = 0.244
2. Interpretation
Sample with absorbance greater that or equal to the cut off value are considered
reactive to anti-HIV and HIV antigen. Samples with absorbance less than the cut off
value are considered non reactive to anti-HIV and HIV antigen.
 If the samples are considered reactive, the test should be conducted two
more times.
In case the re-tests show non reactive result the samples are considered negative, and
on the other hand, If one of the re-tests shows reactive results the smaples are
considered positive.
 The sample considered positive shall be tested again by Western blot and etc.
for final judgement.
5. DOCUMENTATION
Paste the print out in the HIV register and also record the following details:
(a) The date on which the test is run.
(b) The name of the kit used.
(c) Lot No and expiry date of the kit.
(d) Initials of the Technologist who performed the test and the Supervisor who
verified the results.
(e) The reactive units are marked in red.
Transfer the record to donor records.
6. REFERENCE:
1. Kit package insert.
2. Technical Manual of American Association of Blood Banks – 15th Edition,
2005.
7. END OF DOCUMENT.

3.2.1 Anti HIV Testing (ELISA Method)

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Model SOP

Standard Operating Procedure

SOP no. Effective Date Pages Prepared by Authorised by

1. SCOPE & APPLICATION:

Preparation of the reagents

STORAGE AND SHELF LIFE OF PREPARED REAGENTS

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