An ELISA test to detect influenza virus A antigen in tissue culture samples and swab material

Cat.No. BI-C0012 Influenza A Antigen ELISA

I Introduction

For diagnosis of influenza virus (IV) type A. the demonstration of influenza virus is the most commonly used method. Possible false-negative results caused by naturally occurring variants of the virus is minimized in this assay, since two monoclonal antibodies directed against two different well conserved epitopes of NP (Nucleoprotein) were used in the assay.

II Intended use

The influenza virus type A ELISA is designed to detect these viral proteins. To this end monoclonal antibodies are attached to the solid phase. The plates are incubated with the sample to be tested. The plates are washed after incubation to remove unbound materials. A monoclonal antibody against influenza conjugated with biotine is added to detect bound influenza virus to the antibodies on the solid phase. The plates are washed after incubation to remove unbound materials. Enzyme conjugated streptavidine is added to detect bound biotine conjugate. After incubation and washing, the substrate is added and the optical density is measured at 450 nm.

III Principle

The test is based on the reaction of antibodies with NP proteins. To this end, monoclonal antibodies have been coated to a 96 well microtiter strip plate. The sample is added (in a titration pure, 1:10, 1:30 and 1:90 diluted) to the wells of the coated plate.After washing, the bound influenza virus is detected by an anti-influenza biotine conjugate.After washing, the bound anti-influenza conjugate is detected by streptavidine HRPO conjugate.Bound streptavidine conjugate is made visible by adding substrate/chromagen mix.The intensity of the colour reaction in the wells is directly correlated to the concentration of influenza virus in the sample.

IV Contents

• 12 x 8 microtiter strips
• 1 x strip holder
• 1 x 12 ml Biotine conjugate
• 1 x 12 ml Buffer
• 1 x 12 ml Streptavidine conjugate
• 1 x 0,5 ml Positive control
• 1 x 0,5 ml Negative control
• 1 x 60 ml Wash-solution (200xconcentrated), dilute in de-ionized water before use!
• 1 x 8 ml Substrate A.
• 1 x 8 ml Substrate B.
• 1 x 8 ml Stop solution.
• 1 x Plastic cover seal.

V Handling and storage of specimens

The ELISA should be stored at 4-8 ˚C. An unopened package can be used until the expiry date. An opened package can be used if the requirements, mentioned in the validation
(see XI), are fulfilled. If not fulfilled the test can no longer be used. Avoid repeated freezing and thawing as this increases non-specific reactivity. Samples may be used fresh or may be kept frozen below -20˚C before use. Positive and negative controls may be stored after reconstitution in aliquots at -20˚C and used until the expiry date. 

VI Wash protocol

In ELISA’s, un-complexed components must be removed efficiently between each incubation step. This is accomplished by appropriate washing. It should be stressed that each washing step must be carried out with care to guarantee reproducible inter- and intra-assay results. It is essential to follow the washing procedures outlined below. Washing may be done manually or with automatic equipment. Automatic washing equipment usually gives better results.

Manual washing

The wash-solution is 200x concentrated, dilute in de-ionized water before use!Empty each well by turning the microtiter plate upside-down followed by a firm vertical downward movement to remove the contents of the wells. Fill all the wells with 250 µl of diluted washing solution.This washing cycle (2 and 3) should be carried out at least 4 times!Turn the plate upside down and empty the wells with a firm vertical downward movement.Place the inverted plate on absorbent paper towels and tap the plate firmly to remove any residual washing solution in the wells.Take care that none of the wells dries out before the next reagent is dispensed.Washing with automatic equipmentThe wash-solution is 200x concentrated, dilute in de-ionized water before use! When automatic plate washing equipment is used, check that all wells are aspirated completely and that the washing solution is correctly dispensed, reaching the rim of each well during each rinsing cycle. The washer should be programmed to execute at least 4 washing cycles!

VII Precautions

Handle all biological materials as though capable of transmitting infectious diseases.Do not pipette by mouth.Do not eat, drink, smoke, prepare foods or apply cosmetics within the designated work area.TMB is toxic by inhalation, through contact with skin or when swallowed, observe when handling the substrate.Do not use components past the expiry date and do not mix components from different serial lots together.Optimal results will be obtained by strict adherence to this protocol. Careful pipetting and washing throughout this procedure are necessary to maintain precision and accuracy.Each well is ultimately used as an optical cuvette. Therefore, do not touch the under-surface of the microtiter plate and protect it from damage and dirt.

VIII Swab material

It is advised to test swab material from nose and throat undiluted or 1:1 diluted (in an eppendorf vial).(ELISA Buffer can be ordered additional to dilute this samples)

IX Fecal material

It is advised to test fecal material diluted 1:1 (in an eppendorf vial) or in titration 1:1, 1:2, 1:4 and 1:8 (ELISA Buffer can be ordered additional to dilute this samples)

 X Test protocol

• Take out the test kit and bring to room temperature.
• Take out the coated microtiter plate
• Take 4 wells for each sample and for the control 1 strip.
• Dispense 135 µl of buffer to all second wells.
• Dispense 100 µl of buffer to all third and fourth wells.
• Dispense 115 µl of sample to the first well.
• Transfer 15 µl sample from the first well to the second.Mix well by using a 50 µl pipet.
• Transfer 50 µl diluted sample to the third well and mix well with the same pipette.
• Transfer 50 µl of the third well to the fourth well and mix well with the same pipette.
• Remove 50 µl from the fourth well.
• Dispense 115 µl of positive control to the first well.
• Transfer 15 µl positive control from the first well to the second.
• Mix well by using a 50 µl pipet.
• Transfer 50 µl of the second well to the third well and mix well with the same pipette.
• Transfer 50 µl of the third well to the fourth well and mix well with the same pipette.Remove 50 µl from the fourth well.
• Dispense 115 µl of negative control to the first well.
• Transfer 15 µl negative control from the first well to the second.
• Mix well by using a 50 µl pipet.
• Transfer 50 µl of the second well to the third well and mix well with the same pipette.
• Remove 50 µl from the third well.The fourth well is a blanc.
• Seal and incubate for 60 minutes at 37˚C.
• Wash the microtiter plate with washing solution according to the washing protocol.
• The provided washing solution must be diluted 200x in de-ionized water!
• Dispense 100 µl of biotin conjugate to all wells.
• Seal and incubate for 60 minutes at 37˚C.
• Wash according washing protocol.
• Dispense 100 µl of streptavidin conjugate to all wells
• Seal and incubate for 20 minutes at 37˚C.
• Wash according to washing protocol.
• Mix equal parts of substrate A and substrate B while gentle shaking.
• Prepare immediately before use!
• Dispense 100 µl of substrate solution to each well.
• Incubate for 10-15 minutes at room temperature (21˚C).
• Add 50 µl of stop solution to each well; mix well.
• Read the absorbency values immediately (within 10 minutes!) at 450nm reference 620nm.

XI Validation of the test

• To standardize the influenza virus type A ELISA positive and negative controls have to be tested.
• The positive control should give an extinction of ≥ 1,000 OD measured at 450 nm using 620 nm as reference.
• The OD (450 nm) of the negative control must be ≤ 0,300.XII Interpretation of test results
• A sample is considered positive when the measured extinction is higher than 2 times the OD of the negative control.
• When a sample is negative > sample <. 2 x negative, it should be tested again within 2-4 weeks.
• It is advised to keep animals isolated during this periode The OD of the positive control must be ≥ 1,000.Important

NOTE: A positive ELISA result must be confirmed by virus isolation or haemagglutination test.

For ordering, please go to DR.WANG, Fax: +49 7071 792022, E-Mail: info@normae.de 

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