Sop 2018

W.BRO.V.
SURENDRANATH REDDY MEMORIAL TRUST

MASONIC BLOOD BANK, 5/2, New Katpadi Road, Vellore - 4
ELISA TEST OF HIV-1 p 24 ANTIGEN AND ANTIBODIES HIV – 1
(INDLUDING SUBGROUP O & C ) AND HIV-2 IN HUMAN SERUM / PLASMA
PRINICIPLE
Microlisa HIV (Ag & Ab) is an enzyme immunoassay based on “Sandwich ELISA”. HIV envelop
proteins gp41, C terminus of gp120 for HIV-1 and gp36 for HIV-2 representing immunodominant
epitopes and anti HIV1 P24 antibodies are coated microtiter wells. Specimens and controls are added to
the microtiter wells and incubated. Where the specific antigens and antibodies to HIV-1 and HIV-2 binds
onto the surface of the wells. The unbound materials are washed Horseradish peroxide (HRP) conjugated
gp41, C ter of 120 of HIV-1 and gp36 of HIV-2 and anti HIV1 P 24 antibodies is added to bind the HIV
antigen antibody or anti-924 antibody- antigen complex if present in the sample. Finally substrate solution
containing chromogen and hydrogen peroxide is added to the wells and incubated.
A blue colour will develop in proportion to the amount of HIV-1 and / or HIV-2 antibodies and/or HIV-1
p24 antigen present in the specimen. The colour reaction is stopped by a stop solution.
PREPARATION OF WORKING CONJUGATE:
Dilute conjugate concentrate 1:100 in conjugate diluents. Always prepare a fresh dilution for easy assay
in a clean glass container (e.g for 8 wells enzyme conjuage 10 ul, conjugate diluents 1.0 ml). Mix the
solution thoroughly before use.
PREPARATION OF WORKING SUBSTRATE:
Mix TMB substrate and TMB diluents in 1:1 ratio to prepare working substrate. Always prepare a fresh
dilution for each assay in a clean glass container (e.g : for 8 wells TMB substrate 0.5 ml. TMB diluents
0.5 ml) Mix the solution thoroughly before use.
PREPARATION OF WORKING WASH BUFFER:
1 ml conc. Buffer with 24 ml water for 8 wells. Mix well before use. Wash buffer is stable for 2 months at
2-8 c
Page No. 21
W.BRO.V.SURENDRANATH REDDY MEMORIAL TRUST
TEST PROCEDURE :
Note :
Bring all the reagents and the sample to room temperature at least 30 min before the test.
Heamolysed sample/old sample should not be used. Avoid contaminations.
 Add 25 ul sample diluents in each well including controls.

 Add 100 ul negative control (three) A-1 B-1 C-1 respective wells.
 Add 100 ul positive control (Two) D-1 E-1 respective wells.
 Add 100 ul sample starting from F1.
 Apply cover and incubate at 37 C + 1 C for 60 mins
 While samples are incubated prepare working solution and working conjugate.
 Wash 6 times and blot the wells to remove the remaining wash solution/
 Add 100 ul of working conjugate in each well.
 Apply cover and incubate 37 C+ 1 C for 30 mins
 Wash 6 times and blot the wells to remove the remaining wash solution.
 Add 100 ul of working substrate in each well.
 Apply cover and incubate at room temperature for 30 mins in dark.
 Add 100 ul of stop solution.
 Read the absorbance at 450/630 nm within 30 mints in ELISA READER.
CALCULATIONS :
 NCx – Mean negative control
o Negative control must be <0.150 ( Acceptance criteria)
 PCx- Mean positive control
Postive control must be P1 > 0.50, P2 >0.400 ( Acceptance criteria)
 Cut off value = NCx + 0.20
INTERPRETATION OF RESULTS:
 Absorbents values less than the cut-off value are Non-reactive and may be considered as negative
for anti-HIV and HIV-1 P24 antigen.
 Absorbents values greater than or equal to the cut off value are reactive for anti-HIV and/or HIV-
1 antigen by Microlisa HIV (Ag & Ab).
Page No.22
NOTE :
Test value within 10% below the cut-off should be considered suspect for the presence of
antibodies/antigen should be re-tested by duplicate. If any one of the duplicate re-tested sample value is
equal or greater than the cut-off, Confimation test is recommended by other EIA assays / PCR/WESTER
BLOT.
Prepared by Approved by
NANCY GEETHA Dr.M.V. SURESH

Quality Manager Medical Officer
Effective Date : 01-08-2015

Review Dat : 30-07-2017
Page No .23
ELISA TEST FOR HEPATITIS ‘B’ SURFACE ANTIGEN (HBsAg) IN
HUMAN SERUM / PLASMA
This system is very sensitive and specific and detects level of HBsAg as low as 0.5 ng/ml comparable to
the level of detection by radio immunoassay. Hence ELISA method is very sensitive test for HBsAg.
PRINICIPLE:
Anti- HBs is bound to the solid phase i.e. the polystyrene micro plate well were the test samples are
incubated in the antibody coated wells. The unreacted substances are washed thoroughly with buffer and
in antigens are present, it attaches to the surface of the wells. A second antibody with enzyme is used to
react with the trapped antigen. Again a washing step removes the unreacted enzyme antibodies that are
not bound to the antigen. The amount of enzyme left in the well is therefore is proportional to the amount
of antigen in the test sample. Enzyme activity is finally tested using an enzyme substrate. Depending on
the enzyme and enzyme substrate used, the assay is read visually/ELISA READER.
PREPRATION OF WORKING CONJUGATE:
Dilute conjugate concentrate 1: 50 in conjugate diluents. Always prepare a fresh dilution for each assay in
a clean glass container (e.g. for 8 wells, enzymes, conjugate 10ul, conjugate diluent 0.5 ml ). Mix the
Dilute TMB concentrate 1:100 in substrate solution (diluent). If any crystals are present resoulibze by
warming at room temperature ( e.g. for 8 wells TMB concentrate 10 ul, substrate diluents 1 ml). Mix the
solution thoroughly before use. Do not expose the substrate to strong light.
PREPARATION OF WORKING WASH BUFFER.:
2-8 C.
Page No.24
TEST PROCEDURE :
NOTE :
Bring all the reagents and the sample to room temperature at least 30 mins before the test. Heamolysed
sample / old sample should not be used. Avoid contaminations.
 Well A1 BLANL. B1 C1 Neg. Control, D1 E1 F1 Positive control.

 Add 50 ul Neg. and Positive controls in respective wells.
 Add 50 ul of sample in each well starti9ng from G1
 Add 50 ul of enzyme conjugate to each well. Mix gently for 2-3 secs.
 Cover & incubate at 37 C + 1 C for 60 mins
 Wash 5 times (300-350 ul with a soak time to 30 secs) and blot the wells to remove the remaining
wash solution.
 100 ul of working substrate solution is added to all wells including BLANK.
 Cover with aluminum foil and incubate at room temperature for 30 min in dark.
 Stop the reaction by adding 50 ul stop solutions to all wells. Mix gentely.
 Read the absorbance of the wells at 450/630 nm including BLANK using an ELISA READER.
CALCULATIONS:
 BLANK must be <0.100, mean PC absorbance must be >0.5, mean absorbance of Neg.Control
<0.150.
 Cut off value = Mean absorbance (O.D) of Neg. Control +0.1
INTERPRETATIONS OF RESULTS:
Values LESS than cut-off values are NEGATIVE
Values GREATER than / EQUAL to cut-off values are POSITIVE.


Review Dat : 30-07-2017
Page No. 25
ELISA TEST FOR ANTIBODIES TO HEPATITIS ‘C” VIRUS IN HUMAN
SERUM / PLASMA
PRINICIPLE
The 3rd generation HCV Microlisa is highly sensitive technique which detects antibodies against HCV in
human serum / plasma. Since the HCV protein level are below the limits of detection the immune
diagnosis of HCV infection is based on host generated antibodies (anti –HCV) to viral proteints, Here a
combination of antigen with the sequence of both HCV structural and non-structural antigen i.e., E1, E2,
NS4, NS5. The microwells are coated with structural and non-structural HCV proteins. Antibodies to
HCV bind to the immobilized HCV antigens during incubation. Washing removes excess of unbound
Anti-HCV or human IgG. An enzyme conjugate anti human IgG with HRPO (Horse Radish Per Oxidase )
is added again the excess enzyme conjugate are washed were only the bond antigen-anti HCV-enzyme
conjugate complex if formed to which substrate solution is added. This leads to development of blue
colour which is indicative of the Ag-Ab reaction. A stop solution is added and the optical density of the
developed colour is read photometrically.
SAMPLE PREPARATION :
Dilute the serum sample with sample diluents (1:11 dilution ) in separate tube (200ul +20ul)
Microwell dilution : 100 ul of sample diluents + 10 ul of sample (direct)
PREPARATION OF WORKING CONJUGATE :
Dilute conjugate concentrate 1: 100 in conjugate diluents. Always prepare a fresh dilution for each assay
in a clean glass container (e.g : for 8 wells enzyme conjugate 10ul, conjugate diluents 1.0 ml). Mix the
PREPARATION OF WORKING SUBSTRATE”
Dilute TMB concentrate 1: 100 in substrate solution (diluents). If any crystals are present resoulibze by
warming at room temperate (e.g for 8 wells TMB concentrate 10 ul, substrate diluents 1 ml). Mix the
Dilute TMB concentrate 1:100 in substrate solution (diluents). If any crystals are present resoulibze by
warming at room temperature (e.g for 8 wells TMB concentrate 10 ul, substrate diluents 1 ml). Mix the
solution thoroughly before use. Do not expose the substrate to strong light.
PREPARTION OF WORKING WASH BUFFER:
2-8 C. Page No.26
TEST PROCEDURE :
NOTE :
Bring all the reagents and the sample to room temperature at least 30 mins before the test. Heamolysed
sample / Old sample should not be used. Avoid contaminations.
 Well A1 BLANK 100 ul sample diluent.

 Add 100 ul Neg. B1C1C1E1F1 positive controls in respective wells. (no dilution)
 Add 100 ul of sample diluent + 10 ul of sample in each well starting from G1
 Cover and incubates at 37 C + 1 C for 30 mins.
(300=350 ul with a soak time to 30 secs).
 Cover & incubate at 37 C + 1 C for 30 mins in dark .
(300 -350 ul with a soak time to 30 secs)
 Cover with aluminum foil and incubate at room temperature for 30 min in dark.
 Stop the reactive by adding 50 ul stop solution to all wells. Mix gently.
 Read the absorbance of the wells at 450/630 nm including BLANK using an ELISA READER.
CALCULATIONS
 BLANK must be <0.100, mean absorbance PC must be >0.5 absorbance of Neg. Control must be
-0.005 <NC<0.150
 Cut off value = 0.1 x mean absorbance (O.D) of pos. control +0.1
INTERPRETATIONS OF RESULTS:
Values LESS than cut-off value are NON-REACTIVE for Anti – HCV
Values GREATER than /EQUAL to cut-off value are REACTIVE for Anti – HCV


Review Dat : 30-07-2017 page No 27
Rapid Test for HIV - QUALPRO
PRINICIPLE
QUALPRO HIV utilizes the priniciple of immunochromatography, a unique two-site immunoassay on a
nitrocellulose membrane. Highly purified antigens – gp41, gp120 and p24-O fusion polypeptide,
representing HIV-1 and HIV-1 group “O” and synthetic peptide gp36 representing HIV-2 are stripped on
the membrane as two separate test bands. An assay control forms the third band. Similar antigens are also
coated on colloidal gold. A unique combination of synthetic peptides and recombinant antigens reduces
cross-reactivity and enable better discrimination between HIV-1 & HIV-2 samples. As the test specimen
flows through the membrane test assembly, the highly specific HIV-1/2 antigens colloidal gold conjugate
complexes with the HIV-1/2 specific antibodies in the specimen and travels on the membrane due to
capillary action along with the rabbit IgG-Colloidal gold conjugate. This complex moves further on the
membrane to the test region where it is immobilized by the HIV-1/2 antigens coated on the membrane at
two separate test regions for HIV-1 & HIV-2. This leads to the formation of coloured band(s). The
presence of coloured band(s) in the test regions indicated the presence of antibodies to HIV-1/2 in the
specimen. The unreacted conjugate and unbound complex. If any, along with rabbit IgG gold conjugate
move further on the membrance and are subsequently immobilized by the goat anti-rabbit IgG antibodies
coated on the membrance at the control region ©, forming a colured band. This control band acts as a
procedural control and serves to validate the results.
REAGENTS AND MATERIALS SUPPLIED.
A Individual pouched devices each comprising of

1. Device Membrane test assembly ; stipped with HIV-1 AND HIV-2 specific antigens and goat
anti-rabbit IgG along with HIV specific antigen and rabbit IgG gold conjugate.
2. PIPETTE Disposable plastic sample Applicator.
3. Desiccant pouch
B. BUF sample Running Buffer. Buffer containing surfactant and preservatives.
C. Package insert.
STORAGE AND STABILITY
QUALPRO HIV is stable up to the expiry date mention on the label when stored at 4-30 C. Once the
pouch is opened, the membrane test assembly must be used immediately.
SAMPLE COLLECTION
1.QUALPROHIV uses human serum / Plasma as specimen

2. No special preparation of the patient is necessary prior to specimen collection by approved techniques.
3.Preferably use fresh sample. However, specimen may be stored refrigerated (2-8 C) for short duration.
For long storage, freeze at -20 C or below.
4. If serum is to be used as specimen, allow blood to clot completely. Centrifuge to obtain clear serum.
5. Repeated freezing and thawing of the specimens should be avoided.
6. Do not heat inactivate before use.
7. Do not use turbid, lipaemic and haemolysed serum / plasma.
8. Do not use haemolysed, clotted or contaminated specimens. Page No. 28
Test Procedure
1. Bring the sealed aluminium foil pouch of QUALPROHIV membrane test assembly to room
temperature.
2. Open a foil pouch by tearing along the “notch”
3. Remove the membrane test assembly and the sample applicator. Once opened, the membrane test
assembly must be used immediately.
4. Label the membrane test assembly with specimen identity.
5. Place the membrane test assembly on a flat horizontal surface.
6. Carefully dispense one drop (25 ul) of serum/plasma into the specimen well “S” using the sample
applicator provided.
7. Add there drops o sample running buffer inthr same well ‘S’
8. Obesereve the development of visible coloured band at test regions (‘1’ for HIV -1and/or ‘2’ for
HIV-2).
9. Positive results may be observed within 20 minutes.
10. The test should be considered invalid if the control band ‘C’ does not appear. The test is also
invalid if neither the control nor the test bands appear. Repeat the test with a new QUALPROHIV
membrane test assembly.
Interpretation of Results
Negative : A coloured band appears only in the control area marked “C”
HIV-1 Positive : A coloured band appears in the control area as well as in the area marked 1. The sample
is reactive for HIV-1.
HIV-2 Positive: A Coloured band appears in the control area as well as in the area marked “2”. The
sample is reactive for HIV-2.
HIV-1 & HIV-2 DUAL POSITIVE : A coloured band appears in the control area as well as in the areas
marked 1 & 2. This indicates a mixed infection.
INVALID : The test should be considered invalid if the control band ‘ C’ does not appear. The test is also
invalid if only the test band and no control band appear. Repeat the test with a new QUALPRO HIV
membrane test assembly.


Review Date : 30-07-2017 Page No. 29
Rapid Card Test Hepatitis – B
TEST PRINICPLE
The Benesphera Hepatitis – B test used solid phase immunochromatographic technology for the
qualitative detection of HBsAg in serum or plasma. The test is a two-site immunometric assay in
which a combination of monoclonal and polyclonal antibodies are used to selectively detect Hbs
Ag serum or plasma with a high degree of sensitivity. Each device has a reading window with a
an upper ‘Control’ region and the lower ‘Test’ region and a sample well. In the test procedure, 2
droups (70ul) each of serum or plasma sample is added to the sample well and allowed to
soaking. Read result within 20 minutes,. If HbsAg is present in the specimen, it will react with the
conjugate dye, which binds to the antibody on the membrane to generate a coloured line.
Presence of two coloured lines, one in the test zone and other in the control zone, indicates a
positive result, while the absence of the line in the test zone indicated a negative result.
PRECAUTIONS:
1.Allow the test device to reach room temperature (25-25 C) before testing.
2. Human specimens should be handled carefully as it is capable of transmitting infectious agents.
SPECIMEN COLLECTION AND STORAGE

 Benesphera Hepatitis – B test can be performed on human serum or plasma only. Heparin
or EDTA may be used as an anticoagulant. Use of other anti-coagulants has not been
established.
 Remove serum or plasma from the clot of red cells as soon as possible to avoid
hemolysis. Only clear, nonhemolyzed specimens should be used. Specimens containing
any particulate matter may give inconsistent test results. Such specimens containing any
particulate matter may give inconsistent test results. Such specimens should be clarified
by centrifugation before testing.
 Testing should be performed as soon as possible after sample collection. Do not leave
samples at room temperature for prolonged periods.
 If specimens are to stored, they should be refrigerated at 2-8 C or frozen. For prolonged
storage, samples should be frozen and stored at -20 C. Specimens should not be
repeatedly frozen or thawed.
 Bring the specimens to room temperature prior to testing. The frozen specimens must be
completely thawed, thoroughly mixed and brought to room temperature prior to testing.
Page No. 30
Test Procedure:
1. Remove the testing device from the foil pouch by tearing at the ‘notch’ and place it on a
leveled surface.
2. Holding the sample dropper vertically, add 2 drops of the specimen into the sample well
marked “S’
3. Read results within 20 minutes.
Interpretation of the Results
1. Positive : Red- Purple bands appearing in the test (T) and the control (c ) zones, indicate the
presence of HBsAg in the specimen.
2. Negative : One red-purple band appearing in the control zone ( C) with no band in the test zone
(T), indicates that there is no HBsAg in the specimen or the concentration of HBsAg in the
specimen is below the detectable level.
3. Invalid : There is no colour band visible both in the test zone and in the control zone or there is a
visible band only in the test zone and not in the control zone. The test is considered to be invalid
either due to deterioration of the test or an improper testing procedure.
This test has ability to detect all 10 types of ‘ay’ & ‘ad’ subtypes.


Review Date : 30-07-2017
Page No. 31
Rapid Test for HCV Antibodies.
Prinicple
The Alere Trueline Rapid Test Kit for HCV Antibodies contains a membrane strip, which is pre-
coated with recombinant HCV capture antigen (core, NS3, NS4 and NS5) on test band region.
The protein A-colloid gold conjugate and serum sample moves along the membrane
chromatographically to the test region (T) and forms a visible line as the antigen-antibody protein
A gold particle complex forms with high degree of sensitivity and specificity. The Alere Trueline
Rapid Test Kit for HCV Antibodies test window has been clearly labeled. “T” for test line and “
C” for control line.. The control line is used for procedural control and should always appear if
the test procedure is performed correctly.
Materials provided.
The Alere Truline “ Rapid Test Kit for HCV antibodies contains following items to perform the
assay.
1. 30 Test devices individually foil pouched with a desiccant.
2. Assay diluent (3 x 2 ml / vial)
3. 30 ul capillary pipettes.
4. 1 instructions for use.
Sepcimen collection : Storage and Precaution

Whole Blood.
1. Collect the whole blood into the collection tube (containing anticoagulants such as heparin,
EDTA and sodium citrate ) by venipuncture.
2. If blood specimens are not immediately tested, they should be refrigerated at 2-8 C.
3. When stored at 2-8 C, the blood specimens should be used within 3 days.
4. For storage period longer than 3 days, freezing is recommended. They should be brought to
room temperate prior to use.
5. Using the blood specimens in the long-term keeping more than 3 days can cause non-specific
reaction.
2 Plasma or serum
1) Plasma collect the whole blood into the collection tube (containing anticoagulants such as
heparin, EDTA and sodium citrate) by venipuncture and then centrifuge blood to get plasma
specimen.
Serum collect the whole blood into the collection tube (Not containing anticoagulants such as
heparin, EDTA and sodium citrate) by venipuncture, leave to settle for 30 minutes for blood
coagulation and the centrifuge blood to get serum specimen of supernatant.
2) If plasma or serum specimens are not tested immediately they should be refrigerated at 2-8 C.
For storage period longer than 2 weeks, freezing is recommened. They should be bought to
room temperature prior to use.
3) Plasma or serum specimens containing a precipitate may yield inconsistent test results. Such
specimens must be clarified prior to assaying. Page No. 32
Procedure of the test.
1) Remove the test device from foil pouch, and place it on a flat, dry surface.
2) Usina a micropipette) Add 10Ul of serum, Plasma or whole blood specimen into the sample
wells(s)
3) Add 4 drops (About 120 ul) of assay diluents into sample wells (s)
4) Interpret test results in 5-20 minutes.
Interpreation of the test
1. A color band will appear at left section of the result window to show that the test is working
properly. This band is the control line (c )
2. Color band appears at the right section of the result window. This band is the test line.
Negative Result :
The presence of only one purple color band (Control Band ‘C’) in the result window indicates a
negative result.
Positive Result:
The presence of two color bands (“T” band and “C” band) within the Result window, no matter
which band appears first, indicates a positive result.
Invalid Result:
If the control banc “C” is not visible in the result window after performing the test, the result is
considered invalid. This specimen must be retested using a new test device.


Page No. 33
Rapid Test for Syphilis
Introduction
A rapid chromatographic immunoassay for the qualitative detection of antibodies (IgG and
IgM) to Trepenema pallidum (TP) in serum / plasma / whole blood to aid in the diagnosis of
syphilis.
Intended Use
The Aspen Syphilis Rapid Test Device (Serum / Plasma/ whole blood) is a rapid
chromatographic immunoassay for the qualitative detection of antibodies (IgG and IgM) to
Treponema Pallidum (TP) in whole blood, serum or plasma to aid in the diagnosis of syphilis.
Priniciple
The Syphilis rapid test device is a rapid qualitative membrane Device based immunoassay for
the detection of TP antibodies (IgG and IgM) in serum / plasma/ whole blood. In this test
procedure, recombinant syphilis antigen is immobilized in the test line region (T) of the
device. After a specimen is added to the specimen well (S) of the device, it reacts with
syphilis antigen coated particles in the test. This mixture migrates chromatographically along
the length of the test device and interacts with the immobilized syphilis antigen. The double
antigen test format can detect both IgG and IgM in specimens. If the specimen contains TP
antibodies, a colored line will appear in the test line region (T) indicating a positive result. If
the specimen does not contain TP antibodies a colored line will not appear in this region,
indicating a negative result. To serve as a procedural control, a colored line will always
appear in the control line ( c) region, indicating that proper volume of specimen has been
added and membrane wicking has occurred.
Reagents
The test device contains syphilis antigen coated particles and syphilis antigen coated on the
membrane.
Page No.34
Specimen collection and Preparation
The Aspen syphilis rapid test device can be performed using Serum/Plasma / Whole blood.
Testing should be performed immediately after the specimens have been collected. Do not
leave the specimen at room temperature for prolonged periods. Sepcimens may be stored at 2-
8 C for up to 3 days. For long term storage, specimens should be kept below -20C
Bring specimen to room temperature prior to testing frozen speciments must be completely
thawed and mixed well prior to testing. Specimen should not be frozen and thawed
repeatedly.
Test Procedure.
Note : Bring the test device, specimen and buffer to the room temperature if stored at 2-8 C
Take out the test device from the pouch and place on a clean & flat surface.
1. Add 2 drops (50ul) of serum / Plasma / Whole blood to the specimen well of test device
using dropper / pipette.
2. Add 1 drop of buffer (40ul) Read result at 10 minutes. ( Do not interpret the result after
30 minutes)
Interpretaion
Negative : Pink / Purple line at C Only
Positive : Pink / purple line at C & T
Invalid : If control line does not appear, the test is invalid.
In this case, Please repeat the test using another device following the test procedure correctly.


Page No. 35
Rapid test for Malaria
INTRODUCTION
Malaria is a serious parasitic disease characterized by fever. Chills and anaemia and is caused
by a parasite that is transmitted human to human by the bite of infected female Anopheles
mosquitoes. There are four major species of malarial parasite. P falciparu, p.vivax, p.ovale
and p.malariae. In an infected human body, the parasites sporozotES. HRPII is present only
in P.falciparu, whereas pLDH is present in all four species and may be used as pan or all-
species as a means to differentiate P.falciparum from P.vivaz and other species. So the
ErbaQik Malaria Ag Pf/Pv (HRP11/Pldh) Test Device (Whole Blood) is designed for the
differential diagnosis between p.falciparum and p.vivax.
Priniciple of Test
ErbaQik Malaria Ag Pf/Pv (HRPII/pLDH) is based on adopted dual color system. The Test
device contains a membrane strip pre-coated with the antibodies specific to Histidine Rich
Protein II (HRP II) of P.Falciparun on PF test line and Lactate Dehydrogenase ( Pldh)
specific antibodies conjugated to colloidal gold, are also dispensed on conjuate pad. The
conjugates react with HRP II and pLDH in the sample. The antigen – antibody complex
forms a visible purple/black band on Pf and / or Pv test lines separately while moving along
the membrane. On the control line, Anti-Bovine serum Albumin antibody (BSA) is coated,
which captures BSA conjugated gold particle to appear as red band. The control line, red
coloured, is used as procedural control and should always appear if the test procedures is
performed correctly.
Specimen Collection and Storage.
1. Collect the whole blood into the collection tube (containing EDTA, citrate or Heparin )
by venipuncture.
2. Using the specimen older than 3 days can cause non-specific reaction.
3. When stored at 2-8 C the whole blood sample should be used within 3 days.
Page No. 36
Procedure of the Test
1. Allow all kit components and specimens to come to room temperature prior to testing.
2. Remove the test device from foil pouch and place it on a flat, dry surface.
3. With a 5 ul disposable dropper provided or micropipette (5 ul) draw whole blood
specimen up to the notch and then transfer drawn whole blood into the small sample well
(S).
4. Add 3-4 drops of buffer solution into the buffer wells (B)
5. Wait for 30 minutes and read result.
INTERPRETATION OF THE RESULTS
Negative : The presence of one Red band (“C” Control line ) within the result window
indicates a negative result.
Positive- P.falciparum (pf): The appearance of two color bands ( purple/black) “Pf” Test line
and Red “C” Control line )
Positive – P.Vivax (pv) : The appearance of two color bands (Purple/Black “Pv” and Red “C”
Control line ) within the result window indicates a Pv Positive.
Mixed infection (Pf and Pv) : Three bands (Purple /Black “Pf”, “Pv” test line and Red “C”
Control line ) within the result window indicates mixed infection.
Invalid : If the control band fails to appear within the result window, the result is considered
invalid. The directions may not have been followed correctly or the test may have
deteriorated . It is recommended that the specimen be retested.
Note : There is no meaning attributed to line colour intensity or width.


Page No. 37

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W.BRO.V.

SURENDRANATH REDDY MEMORIAL TRUST

PREPARATION OF WORKING CONJUGATE:

PREPARATION OF WORKING SUBSTRATE:

PREPARATION OF WORKING WASH BUFFER:

Heamolysed sample/old sample should not be used. Avoid contaminations.

 Add 25 ul sample diluents in each well including controls.

NANCY GEETHA Dr.M.V. SURESH

Effective Date : 01-08-2015

PREPRATION OF WORKING CONJUGATE:

PREPARATION OF WORKING SUBSTRATE:

PREPARATION OF WORKING WASH BUFFER.:

 Well A1 BLANL. B1 C1 Neg. Control, D1 E1 F1 Positive control.

Values LESS than cut-off values are NEGATIVE

Values GREATER than / EQUAL to cut-off values are POSITIVE.

NANCY GEETHA Dr.M.V. SURESH

Effective Date : 01-08-2015

Microwell dilution : 100 ul of sample diluents + 10 ul of sample (direct)

PREPARATION OF WORKING CONJUGATE :

PREPARATION OF WORKING SUBSTRATE”

PREPARATION OF WORKING SUBSTRATE:

PREPARTION OF WORKING WASH BUFFER:

 Well A1 BLANK 100 ul sample diluent.

NANCY GEETHA Dr.M.V. SURESH

Effective Date : 01-08-2015

REAGENTS AND MATERIALS SUPPLIED.

A Individual pouched devices each comprising of

STORAGE AND STABILITY

1.QUALPROHIV uses human serum / Plasma as specimen

NANCY GEETHA Dr.M.V. SURESH

Effective Date : 01-08-2015

SPECIMEN COLLECTION AND STORAGE

Interpretation of the Results

NANCY GEETHA Dr.M.V. SURESH

Effective Date : 01-08-2015

Sepcimen collection : Storage and Precaution

Procedure of the test.

Interpreation of the test

NANCY GEETHA Dr.M.V. SURESH

Effective Date : 01-08-2015

Negative : Pink / Purple line at C Only

Positive : Pink / purple line at C & T

Invalid : If control line does not appear, the test is invalid.

NANCY GEETHA Dr.M.V. SURESH

Effective Date : 01-08-2015

Specimen Collection and Storage.

INTERPRETATION OF THE RESULTS

Note : There is no meaning attributed to line colour intensity or width.

NANCY GEETHA Dr.M.V. SURESH

Effective Date : 01-08-2015

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