HCV Gen4 Ag & Ab Microlisa

Microwell ELISA Test for the Detection of HCV Core antigen and anti-HCV antibodies in Human Serum/ Plasma

1. SUMMARY AND EXPLANATION OF THE TEST TMB 1 Bottle (10 ml)

Hepatitis C is an infection caused by Hepalitis C virus (HCV). HCV is an enveloped, positive sense Substrate To be diluted in TMB Diluent before use.
single stranded RNA virus belonging to the family flaviviridae. HCV infection can be accute and
TMB 1 Bottle (10 ml.)
chronic that leads to liver infection and damage.
Diluent Buffer solution containing H2O2 with preservative.
HCV antigen and anti-HCV antibodies can be screened in individuals through blood test post infection.
Control 1 Vial (1 ml)
Antibody development against viral protein may take time (upto 6 months) and infected individual
— Ready to use, normal human serum negative for HCV antibodies and
may spread infection in that period. So, the combination assay for screening of both anti-HCV
antigen, HIV-1& HIV-2 antibodies and HBsAg, containing sodium azide
antibodies and HCV Core antigen can reduce the window period and improve detection of infection.
as preservative.
2. INTENDED USE
Control-1 1 Vial (2 ml)
HCV Gen4 Ag & Ab Microlisa is an in vitro qualitative enzyme immunoassay for the detection of
+ Ready to use, inactivated and diluted human serum; Reactive for
anti-HCV antibodies and core antigen in human serum or plasma. It is intended for screening of
HCV antibodies, non-reactive for HIV-1, HIV-2 and HBsAg containing
blood donors and for clinical diagnostic testing.
sodium azide as preservative.
3. PRINCIPLE OF THE TEST Control-2 1 vial ( 1 ml) Lyophilized
HCV Gen4 Ag & Ab Microlisa test is a 4th Generation qualitative enzyme immunoassay. + HCV core antigen positive control, reconsititute with 1 ml of distilled
HCV recombinant antigen (NS3, NS4, NS5 and Core) and anti-HCV core antibodies are coated onto water before use.
microtiter wells. Specimens and controls are added to the microtiter wells and incubated. The plate Stop 1 Vial (12 ml.)
is then washed to remove unbound material. Horseradish peroxidase (HRP) conjugated to Anti- Solution Ready to use, 1N sulfuric acid
human IgG and anti-HCV antibodies is added to each well. This conjugate will bind to HCV antigen-
Plate Sealers Adhesive backed sheets for sealing microtiter plate/strips
antibody or anti-HCV antibody-antigen complex present. Finally substrate solution containing
chromogen and hydrogen peroxide is added to the wells and incubated. A blue colour will develop 8. ADDITIONAL MATERIAL AND INSTRUMENTS REQUIRED
in proportion to the amount of anti-HCV antibodies and/or HCV core antigen present in the specimen.  Micropipettes and microtips  Timer
The colour reaction is stopped by a stop solution. The enzyme substrate reaction is read by EIA  Elisa reader  Elisa washer
reader for absorbance at a wavelength of 450 nm. If the sample does not contain anti-HCV antibodies  Distilled or deionized water  Incubator 370C
and/or HCV core antigen then enzyme conjugate will not bind and the solution in the wells will be  Graduated Cylinders, for reagent dilution  Vor tex Mixer
either colourless or only a faint background colour develops.  Paper towels or absorbent tissue  Disposable gloves
4. DESCRIPTION OF SYMBOLS USED  Disinfectant Solution
The following are graphical symbols used in or found on J. Mitra diagnostic products and packing.
These symbols are the most common ones appearing on medical devices and their packing. They 9. SPECIMEN COLLECTION & PREPARATION
are explained in more detail in the European Standard EN ISO 15223-1:2021. 1. Only human serum or plasma samples should be used for the test. While preparing serum
samples, remove the serum from the clot as soon as possible to avoid haemolysis. Fresh
Manufactured By In vitro diagnostic
serum/plasma samples are preferred.
medical device
2. Specimens should be free of microbial contamination and may be stored at 2-8 0C for one
No. of tests See Instruction for use week, or frozen at -200C or lower. Avoid repeated freezing and thawing.
3. Use of heat inactivated, icteric hyperlipemic and haemolyzed samples should be avoided as
Lot Number Temperature may give erroneous results.
Batch Number Limitation
Manufacturing Date Caution - 10. SPECIMEN PROCESSING
See instruction for use (A) FROZEN SAMPLE
HCV Gen4 Ag & Ab Microlisa test is best used with fresh samples that have not been frozen and
Expiry Date Catalogue Number thawed. However most frozen samples will perform well if the procedure suggested below is
Do not use if package is damaged Keep away from sunlight followed.

Contains biological Material Contains biological Material Allow the frozen sample to thaw in a vertical position in the rack. Do not shake the sample. This
of Human Origin of Animal Origin allows particles to settle to the bottom. Centrifuge the sample at 10,000 rpm for 15 minutes to get
clear sample.
Country of Manufacture Keep Dry
(B) TRANSPORTATION
5. PACK SIZE If the specimen is to be transported, it should be packed in compliance with the current Government
 96 Tests regulations regarding transport of aetiologic agents.
6. STORAGE AND STABILITY
11. WARNING & PRECAUTION
Store all components at 2-80C when not in use. Expiry date on the kit indicates the date beyond
CAUTION: NO TEST METHOD CAN OFFER COMPLETE ASSURANCE THAT HUMAN BLOOD
which components should not be used.
PRODUCTS WILL NOT TRANSMIT INFECTION. ALL THE SAMPLES TO BE TESTED SHOULD BE
7. KIT & ITS COMPONENTS HANDLED CAREFULLY AS THOUGH CAPABLE OF TRANSMITTING INFECTION.
HCV Gen4 Ag & Ab 12 Strips (8 well each) 1. The use of disposable gloves and proper biohazardous clothing is STRONGLY RECOMMENDED
Microlisa Strip Plates Breakway microwells coated with HCV NS3, NS4, NS5 and Core while running the test.
antigen and anti-HCV core antibodies packed in a pouch with 2. In case there is a cut or wound in hand, DO NOT PERFORM THE TEST.
dessicant. 3. Do not smoke, drink or eat in areas where specimens or kit reagents are being handled.
Sample 1 Bottle (15 ml.) 4. Tests are for in vitro diagnostic use only and should be run by competent person only.
Diluent Buffer containing protein stabilizers and antimicrobial agents as 5. Do not pipette by mouth.
preservative.
6. All materials used in the assay and samples should be decontaminated in 5% sodium
Enzyme Conjugate 1 Vial (1.6 ml.) hypochlorite solution for 30-60 min. before disposal or by autoclaving at 1210C at 15psi for 60
Concentrate (11X) Anti-human IgG and anti-HCV antibodies labelled with horseradish min. Do not autoclave materials or solution containing sodium hypochlorite. They should be
peroxidase with protein stabilizers. disposed off in accordance with established safety procedures.
Conjugate 1 Bottle (15 ml.) 7. Wash hands thoroughly with soap or any suitable detergent, after the use of the kit. Consult a
Diluent Buffer containing stabilizers and preservatives. physician immediately in case of accident or contact with eyes, in the event that contaminated
material are ingested or come in contact with skin puncture or wounds.
Wash Buffer 1 Bottle (50 ml)
Concentrate (25X) PBS with surfactant. Dilute 1:25 with distilled water before use. 8. Spills should be decontaminated promptly with Sodium Hypochlorite or any other suitable
disinfectant.
9. Controls contain Sodium Azide as a preservative. If these material are to be disposed off 4. Preparation of Working Conjugate:
through a sink or other common plumbing systems, flush with generous amounts of water to Dilute conjugate concentrate 1:11 in conjugate diluent. Do not store working conjugate. Prepare a
prevent accumulation of potentially explosive compounds. In addition, consult the manual fresh dilution for each assay in a clean glass vessel. Determine the quantity of working conjugate
guideline "Safety Management No. CDC-22", Decontamination of Laboratory Sink Drains to solution to be prepared from table given below. Mix solution thoroughly before use.
remove Azide salts" (Centre for Disease Control, Atlanta, Georgia, April 30, 1976.)
No. of Strips 1 2 3 4 5 6 7 8 9 10 11 12
10. Stop solution contains sulfuric acid. If sulfuric acid comes in contact with the skin, wash
No. of Wells 8 16 24 32 40 48 56 64 72 80 88 96
thoroughly with water. In case of contact with eyes, flush with excess of water.
Enzyme Conjugate 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1.1 1.2
11. ELISA Reader & micropipettes used in testing should be calibrated at regular interval to
Concentrate (ml)
ensure accurate results.
Conjugate 1 2 3 4 5 6 7 8 9 10 11 12
12. PRECAUTIONS FOR USE
Diluent (ml)
Optimal assay performance requires strict adherence to the assay procedure described in the
manual. 5. Preparation of working substrate solution :
1. Do not use kit components beyond the expiration date which is printed on the kit. Mix TMB substrate and TMB Diluent in 1:1 ratio to prepare working substrate.
2. Bring all the reagents & samples to room temperature (20-30oC) before use. No. of Strips 1 2 3 4 5 6 7 8 9 10 11 12
3. Do not combine reagents from different batches, as they are optimised for individual batch to
No. of Wells 8 16 24 32 40 48 56 64 72 80 88 96
give best results.
4. Avoid microbial contamination of reagents. The use of sterile disposable tips is recommended TMB Susbstrate (ml) 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0
while removing aliquots from reagent bottles. TMB Diluent (ml) 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0
5. Due to interchange of caps the reagents may get contaminated. Care should be taken while
handling the reagents to avoid contamination of any sort. Do not store working substrate. Prepare a fresh dilution for each assay in a clean plastic/glass
6. Mark the test specimen with patient’s name or identification number. Improper identification vessel. Determine the quantity of working substrate solution to be prepared from table. Mix solution
may lead to wrong result reporting. thoroughly before use. Discard unused solution. A deep blue color present in the substrate solution
7. Use freshly collected, clean serum samples for assay. Try to avoid turbid, lipemic serum or indicates that the solution has been contaminated and must be discarded.
plasma samples.
14. WASH PROCEDURE:
8. Use a separate tip for each sample and then discard it as biohazardous waste. 1. Incomplete washing will adversely affect the test outcome.
9. All pipetting steps should be performed with utmost care and accuracy. Cross contamination
2. Aspirate the well contents completely into a waste container. Then fill the wells completely
between reagents and samples will invalidate results.
with wash buffer (300 - 350 µl) avoiding overflow of buffer from one well to another and allow
10. Do not allow microwells to dry once the assay has started.
to soak (approx. 30 seconds). Aspirate completely and repeat the wash and soak procedure 4
11. Run negative and positive controls in each assay to evaluate validity of the kit. additional times for a total of 5 washes.
12. Prevent evaporation during sample incubation by covering the strips with strip sealer. Remove
3. Automated washer if used should be well adjusted to fill each well completely without over
sealer before washing.
filling.
13. Distilled or deionised water must be used for wash buffer preparation.
14. Thorough washing of the wells is critical to the performance of the assay. Overflowing of 4. Tap upside down on absorbant sheet till no droplets appear on the sheet, taking care not to
reagents or washing to adjacent wells must be prevented during washing, which may lead to dislodge the wells.
incorrect results due to carry over effect.
15. TEST PROCEDURE
15. Prepare working substrate solution just 10 minutes prior to adding in the wells. Once the assay has started, complete the procedure without interruption. All the reagents should be
16. If blue colour or white particles appear in working substrate solution then do not use it. Take dispensed in the centre of the well and the tip of the pipette should not touch the wall of the
fresh containers and tips and prepare it again. microwell.
17. Use separate tips for TMB substrate and TMB diluent. Fit the stripholder with the required number of HCV Gen4 Ag & Ab Microlisa strips. The sequence
18. Avoid strong light exposure during the assay. of the procedure must be carefully followed. From well A-1, arrange all controls in a horizontal or
19. Ensure that the microwell strips are levelled in the strip holder. Before reading, wipe the vertical configuration. Configuration is dependent upon reader software.
bottom of the microwell strips carefully with soft, absorbent tissue to remove any moisture. 1. Add 100 µl sample diluent in each well.
20. In case of any doubt the run should be repeated.
2. Add 50µl Negative Control in A-1 well.
13. PREPARATION OF REAGENTS 3. Add 50µl PC-1 in B-1, C-1 & D-1 wells and PC-2 in E1wells.
Prepare the following reagents before or during assay procedures. Reagents and samples should 4. Add 50 µl sample in each well starting from F-1. Homogenise the sample/controls and diluent
be at room temperature (20-300C) before beginning the assay and can remain at room temperature with atleast one aspiration or with microplate shaker for five seconds.
during testing. Return reagents to 2-80C after use. All containers used for preparation of reagents 5. Apply cover seal.
must be cleaned thoroughly and rinsed with distilled or deionized water. Prewarm the incubator to 6. Incubate at 370C + 20C for 90 min. + 5 min.
37ºC.
7. While the samples are incubating, prepare Working Wash Solution and Working Conjugate as
1. HCV Gen4 Ag & Ab Microlisa Strip:
specified in Preparation of Reagents.
Bring foil pack to room temperature (20-300C) before opening to prevent condensation on the
8. Take out the plate form the incubator after the incubation time is over and, wash the wells 5
microwell strips.
times with Working Wash solution according to the wash procedure given in the previous
a. Break-off the required number of strips needed for the assay and place in the well holder. Take
section (wash procedure).
the strip holder with the required number of strips, taking into account that one negative &
three positive control-1 and one positive control-2 should be included in the run while opening 9. Add 100 µl of Working Conjugate Solution in each well.
the fresh kit. However for one or two strips, one negative, two positive control-1 and one 10. Apply cover seal.
positive control-2 and for more strips at least one negative, three positive control-1 and one 11. Incubate at 370C + 20C for 30 min. + 2 min.
positive control-2 should be included in each subsequent runs. 12. Aspirate and wash as described in step no. 8.
b. Unused wells should be stored at 2-8oC, with dessicant in an aluminium pouch with clamp & rod.
13. Add 100 µl of working substrate solution in each well.
Microwells are stable for 30 days at 2-8ºC from the date of opening of sealed pouch, when
14. Incubate at room temperature (20 - 300C) for 30 min. in dark.
stored with desicant along with clamp & rod.
Caution: Handle microwell strip with care. Do not touch the bottom exterior surface of the 15. Add 100 µl of stop solution and wait for 3 minutes.
wells. 16. Read absorbance at 450 and 630 nm (reference wavelength 600 - 650 nm) within
30 minutes in an ELISA READER.
2. Preparation of working Postive Control-2:
Gently tap the vial of positive control-2 (Lyophilized) on work bench to remove any substance
from rubber cap, carefully remove the cap and add 1 ml distilled water into lyophilized antigen 16. SUMMARY OF PROCEDURE
vial. Put the cap and let it stand for 10 minutes. Mix solution thoroughly before use. The
Add Sample Sample Diluent
working antigen is stable at -20ºC for 60 days (only 6 freeze thaw of liquid antigen are
Diluent 100 µl
allowed at -20ºC).
3. Preparation of Wash Buffer: Add control 50 µl
a) Check the buffer concentrate for the presence of salt crystals. If crystals are present in the & samples
solution, resolubilize by warming at 370C until all crystals dissolve.
Cover the plate & 90 minutes at 37oC
b) Prepare at least 50 ml. (2ml. concentrated buffer with 48 ml. water) of buffer for each microlisa
incubate
strip used. Mix well before use.
c) Mix 20 ml. 25X wash buffer concentrate with 480 ml. of distilled or deionized water. Working
Wash 5 Cycles
Wash Buffer is stable for 2 months when stored at 2-80C.
 Prepare No. of 1 2 3 4 5 6 7 8 9 10 11 12 2. In establishing HCV infection or, in evaluating patients with Hepatitis C symptoms, HCV Gen4 Ag
Strips
working Enz. conc. (ml) 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1.1 1.2 & Ab Microlisa testing alone cannot be used to diagnose Hepatitis C even if antibodies and/or HCV
Conj. 1 2 3 4 5 6 7 8 9 10 11 12
conjugate Diluent (ml) antigen are present in human serum or plasma.
3. This is only a screening test. A negative test result at any time does not preclude the possibility
Add Conjugate 100 µl
of exposure to, or infection with HCV.
4. All samples detected positive must be confirmed by using other anti-HCV ELISA/CLIA or a
Cover the plate & 30 minutes at 37oC
confirmatory test i.e. RIBA or PCR. Therefore for a definitive diagnosis, the patient’s clinical history,
incubate
symptomatology as well as serological data should be considered. The results should be reported
Wash 5 Cycles only after complying with the above procedure.
5. Some samples show cross reactivity for HCV antibodies. Following factors are found to cause false
Prepare No. of 1 2 3 4 5 6 7 8 9 10 11 12 positive test results: Naturally occurring antibodies, HIV positive, HBV positive, Dengue, syphilis, RA
Strips
Chromogenic TMB Subs. 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 and anti-mouse antibodies etc.
( ml )
Substrate TMB Diluent (ml.) 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0
20. PERFORMANCE CHARACTERISTICS
Add Substrate 100 µl I) In-house Evaluation:
Ia) Sensitivity and Specificity studies were carried out on samples fresh, as well as frozen, from
Incubate in dark 30 minutes at Room Temp. low risk as well as high risk group. Performance of the test with reference to sensitivity and
specificity was evaluated in-house with the panel of 91 negative samples and 28 HCV positive (6
Add Stop Solution 100 µl and wait for 3 minutes. HCV Antigen & 22 HCV Antibody) samples.
The results of all the positive and negative samples were compared with commercially available
Read Results 450 nm. and 630 nm. licensed test kit; Monolisa HCV Ag & Ab Ultra.
The results of the in-house study done are as follows:
17. CALCULATION OF RESULTS No. of Status HCV Gen4 Monolisa HCV
Abbreviations Samples Ag & Ab Microlisa Ag & Ab Ultra
NC - Absorbance (O.D.) of the Negative Control
Positive Negative Positive Negative
PC-1x - Mean Positive Control-1 absorbance
PC-1 - Absorbance (O.D.) of the Positive Control-1 28 HCV Positive 28 - 28 -
PC-2 - Absorbance (O.D.) of the Positive Control-2 91 HCV Negative 1 90 - 91
TEST VALIDITY: Sensitivity: 100% Specificity: 98.9%
Negative Control Acceptance Criteria:
NC absorbance (O.D.) must be < 0.200. If it is not so, the run is invalid and must be repeated. Ib) Specificity Testing:
The HCV Gen4 Ag & Ab Microlisa kit specificity is checked by using 553 clinical patient samples and 12
Positive Control Acceptance Criteria: potentially cross-reacting specimen; HIV, HBsAg, Syphilis, RA, Dengue and ante-natal samples. The
1. PC-1 absorbance (O.D.) must be > 0.600 specificity on all above samples tested is 100%
2. PC-2 absorbance (O.D.) must be > 0.600
Ic) Performance with sero-conversion panel:
If PC-1 and PC-2 absorbance is not in defined limit, the run is invalid and must be repeated. The HCV Gen4 Ag & Ab Microlisa kit performance is checked using 2 sero-conversion panels. The kit
Result Calculation detects HCV infection earlier as compared to an anti-HCV antibody detection kit (anti-HCV kit) and results
The absorbance (O.D.) of each sample is compared with cut-off value to detect the presence or are as follows:
absence of antibodies to HCV or/and HCV capsid antigen. 2 seroconversion panels anti-HCV kit HCV Gen4 Ag & Ab Microlisa
1. Calculate the mean absorbance (O.D.) observed for HCV antibody Positive Control-1 (PC-1) (32 samples)

For example: Number of positive samples 7 21

PC-1 Absorbance
- 1.012 B1 Well The sensitivity for HCV Antigen Positive samples is 100% and Specificity is also 100% on 32
- 1.023 C1 Well samples tested.
- 1.015 D1 Well II) The performance of HCV Gen4 Ag & Ab Microlisa has been evaluated by National Institute of
Total : 3.050 3 Wells Biologicals, India. The result obtained of 3 different lots are as follows:
PC-1x = 3.050/3 = 1.016
Lot Details Sensitivity Specificity
Note: If one of the HCV antibodies positive control (PC-1) individual values differs by more than 30% from LOT 1 100% 100%
the mean value, disregard that value and calculate again with the two remaining positive control values.
LOT 2 100% 99.8%
Cut-off value LOT 3 100 % 100%
Cut-off value is determined by using the following formula:
Cut-off Value = PC-1x X 0.27 Precision: Within-run and between-run precisions have been determined by testing 10 replicates of four
Where PC-1x is mean absorbance (O.D) of Positive Control-1. samples: one HCV core antigen and two HCV antibody positive( one weak and one strong) and one negative
e.g. 1.016 x 0.27 = 0.219 samples. The C.V.(%) of all the samples were within 10%.

18. INTERPRETATION OF RESULTS 21. LIMITED EXPRESSED WARRANTY DISCLAIMER

1. Test specimens with absorbance value less than the cut off value are non-reactive and may be The manufacturer limits the warranty to the test kit, as much as that the test kit will function as an
considered as negative for anti-HCV antibodies and/or HCV Core antigen. in-vitro diagnostic assay within the limitations and specifications as described in the product
2. Specimens with absorbance value equal to or greater than the cut off value are considered instruction-manual, when used strictly in accordance with the instructions contained therein. The
initially positve by the criteria of HCV Gen4 Ag & Ab Microlisa. Original specimen should be manufacturer disclaims any warranty expressed or implied including such expressed or implied
retested in duplicate before the final interpretation. warranty with respect to merchantability, fitness for use or implied utility for any purpose. The
3. Test specimens with absorbance value within 10% below the cut off should be considered manufacture’s liability is limited to either replacement of the product or refund of the purchase
suspect for the presence of HCV antibodies and/or antigen and should be retested in duplicate. price of the product and in no case liable to for claim of any kind for an amount greater than the
4. If both duplicate retest sample absorbance value is less than cut off value, the specimen is purchase price of the goods in respect of which damages are likely to be claimed.
considered negative. The manufacturer shall not be liable to the purchaser or third parties for any injury, damage or
5. If any one of the duplicates retest sample absorbance value is equal to or greater than the cut economic loss, howsoever caused by the product in the use or in the application there of.
off or both duplicate retest value are equal to or greater than the cut off, the specimen is
considered positive by the criteria of HCV Gen4 Ag & Ab Microlisa . Further confirmation by 22. REFERENCES
other EIA assays or confirmation assays including RIBA or PCR is recommended. 1. Hepatitis C virus core antigen assayan alternative method for hepatitis C diagnosis, Lijuan
6. Specimens which are not repeatedly positive, may have shown colour due to: Wang*, Hong Lv* and Guojun Zhang. Annals of Clinical Biochemistry 2017, Vol. 54(2) 279–
a) Carry over of a highly reactive sample due to contamination of pipette tips. 285
b) Substrate contamination
c) Inadequate wash or aspiration during wash procedure. 2. Simultaneous Detection of Hepatitis C Virus (HCV) Core Antigen and Anti-HCV Antibodies
Improves the Early Detection of HCV Infection ,Syria Laperche,1* Nadine Le Marrec,1 Annie
19. LIMITATIONS OF THE ASSAY Girault,1 Franc¸oise Bouchardeau,1Annabelle Servant-Delmas,1 Miche‘le Maniez-Montreuil,2
1. HCV Gen4 Ag & Ab Microlisa assay is designed for testing antibodies against HCV and HCV Pierre Gallian,3Thierry Levayer,4 Pascal Morel,5 and Nicole Simon6 : JOURNAL OF CLINICAL
Core antigen in human serum and plasma. Other body fluids and pooled samples are not MICROBIOLOGY, Aug. 2005, p. 3877–3883
recommended in this assay. Any result derived from the test of any other body fluid or from test of
pooled serum/plasma may not be interpreted correctly based on the current criteria.
3. Role of hepatitis C virus core antigen assay in hepatitis C care in developing countryOuafa PROBLEM POSSIBLE CAUSE SOLUTION
Kallala1,2* , Saoussen Kacem, Imene Fodha Bruno Pozzetto and Trabelsi Abdelhalim1, Kallala
4. Poor a) Washing problems. Check all 8 ports/ manifold
et al. Egyptian Liver Journal (2021).
reproducibility for uniform flow of wash
4. Hepatitis C core antigen testing to diagnose active hepatitis C infection among buffer. If there are blockage,
haemodialysis patients Xue Zheng Wong1, Chye Chung Gan1, Rosmawati Mohamed1, clen the ports.
Rosnawati Yahya2, Shubash Ganapathy3,Soek Siam Tan4 and Soo Kun Lim1, Wong et al. BMC
b) Uncalibrated pipettes or Use only calibrated pipettes
Nephrology (2020) 21:480.
tips not well fitted, improper with well fitted tips & pipette
5. Simultaneous detection of hepatitis c virus antigen and antibodies in dried blood spots, pipetting/ dispensing. carefully without bubbling.
C.P.U. Brandãoa, B.L.C. Marquesb, V.A. Marquesb, C.A. Villela-Nogueirac, K.M.R. Do Ód,
c) Interference in optical Clean or dry the bottom of
M.T. de Paulaa, L.L. Lewis-Ximenezb, E. Lampeb, J.A. Sá Ferreirae, L.M. Villar b,” , Journal of
pathway due to Air bubbles. microwells, check for bubbles
Clinical Virology 57 (2013) 98– 102
and repeat the readings.
23. TROUBLE SHOOTING CHART
5. False Positive a) Beside 3a, b, c, d, e & f Check the calculation
PROBLEM POSSIBLE CAUSE SOLUTION incorrect interpretation and part given in the insert and
1. No colour a) Any one reagent has been Follow the procedure calculation of final results correctly interpret.
developed at the added in wrong sequence. meticulously & repeat assay. b) High incubator temperature, Check incubator temperature,
end of assay b) Inactivated Enzyme Check storage of enzyme incorrect timing or pipetting procedure & repeat assay.
conjugate conc., improper conjugate and it should be free 6. False Negative/ a) Inadequate addition Follow the procedure
storage of any contamination. low O.D. of of substrate/conjugate meticulously & repeat assay.
c) Microplate inactivated, Keep unused strips in aluminium Positive control & solution.
due to improper storage poly pouch with the dessicant positive sample
pouch inside and proerly closed b) Kit expired, reagent of Check the expiry of the kit
with clamp & rod. different kit used. before use.
d) Inactivated substrate, Use freshly prepared substrate c) White particles in working Discard the substrate and
improper storage solution. Recheck procedure, substrate solution. prepare the working substrate
or preparation repeat assay again in fresh tube.
e) Omission of any step in Follow the procedure d) Uncalibrated pipettes, Use only calibrated pipettes
test procedure meticulously & repeat assay. improper pipetting. with well fitted tips & pipette
carefully without bubbling.
f) Incorrect (low) incubator Check incubator temperature,
temperature, timing or pipetting procedure & repeat assay e) Deterioration of Enzyme Check storage of Enzyme
conjugate conjugate. It shall be stored
g) Improper preparation of Check procedure & repeat assay
at 2-8ºC.
enzyme conjugate (dilution error)
f) Deteriotion of working Check reconstitution procedure
improper mixing of reagents.
antigen or improper preparation and storage. It should be used
h) Kit deterioration Check storage of the kit and of working antigen within 60 days at -20ºC and
should be stored at 2-8ºC. freeze thaw should not be more
2. High O.D. value a) Plate not stopped after Follow the procedure than 6 times.
of Negative 30 minutes of additing meticulously & repeat assay.
f) Stop solution is added Follow the test procedure
control stop solution
before 30 minutes. Reaction meticulously.
b) Same microtip used Change micropipette tips while terminated before 30 minutes.
for Positive and negative addition of negative/ positive
g) O.D. taken at incorrect Read O.D. values at 450 nm
controls control
wavelength. and 630 nm.
c) Nonspecific attachment/ If plates get scratches/
binding of other reagent while aberrations during washing, h) Incorrect (low) incubator Check incubator temperature,
addition of next step. non specific proteins may bind temperature, timing or pipetting procedure & repeat assay
while addition of next step.
3. Too much colour a) Contaminated substrate Check substrate (TMB Diluent)
in all wells of the it should be colourless. If
plate (high blue in colour then discard
background) and use clean disposable
container.
b) Contaminated washing Check the container and quality
solution (1X). Poor quality of water used for dilution.
of water used for diluting Use of distilled water is
wash buffer conc. preferred.
c) Over incubation of Follow the procedure
substrate and delay in meticulously.
addition of stop solution.
d) Insufficient washing. Check wash device and clean
i) Washing not consistent probes of manifold. fill the well
due to blockage of probes close to the top.
ii) Filling volume not After washing, blot the
sufficient. microwells on absorbent
iii) Insufficient no. of wash tissue. Follow wash protocol
cycles. meticulously
iv) Contaminated wash
R-00

device
VER-01

e) Use of wash solution Use only HCV Gen4 Ag & Ag

from other manufacturer. Microlisa wash solution. in vitro diagnostic Reagent, not for medicinal use
f) Working substrate not Incubate the plate in dark after J. Mitra & Co. Pvt. Ltd.
protected from light addition of substrate. A 180-181, Okhla Ind. Area, Ph-1, New Delhi-110 020, INDIA
Rev. Date: Jul.-22

Ph: +91-11-47130300, 47130500

MN/EAC/093

e-mail: [email protected] Internet: www.jmitra.co.in