An ELISA test to detect antibodies against influenza A virus antigen (including avian influenza) in serum and plasma samples.

I Introduction

Avian Influenza Virus (AIV) nuclear protein (NP) is a core protein of AIV. Infected avian produce antibodies against these AIV antigens, which can be detected in an ELISA using an anti-species conjugate.

II Intended use

The AIV NP ELISA is designed to detect antibodies against these proteins. To this end recombinant NP proteins are attached to the solid phase. After washing the plates are incubated with the avian sera to be tested. The plates are washed after incubation to remove unbound materials. An anti-species conjugate is added to detect bound avian antibodies to AIV. After incubation and rinsing, the substrate is added and the optical density is measured at 450 nm.

III Principle

The test is based on the reaction of NP proteins with avian antibodies. To this end, NP expression proteins have been coated to a 96 well microtiter strip plate. The avian serum sample is added (diluted 1:100) to the wells of the coated plate. After washing, the bound avian antibodies are detected by an anti-species conjugate.Bound anti-species 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 anti-AIV antibodies in the serum sample.

IV Contents

• 5x12 x 8 microtiter strips
• 5 x strip holder
• 2 x 60 ml Buffer
• 1 x 60 ml Anti-species conjugate
• 1 x 2 ml Positive control
• 1 x 2 ml Negative control
• 5x 60 ml Wash-solution (200xconcentrated), dilute in de-ionized water before use!
• 1 x 40 ml Substrate A.
• 1 x 40 ml Substrate B.
• 1 x 40 ml Stop solution.
• 2 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 IX), 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 protocolI

• 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 equipment

• The 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 Test protocol

• Take out the test kit and bring to room temperature.
• Make a 1:10 dilution of the test sample in the buffer (90 µl buffer + 10 µl sample) in a separate round bottomed microtiter plate (not supplied).
• Take out the coated microtiter plateDispense 90 µl of buffer to all wells.
• Transfer 10 µl diluted sample to one well of the coated microtiter plate.
• Dispense 10 µl of the positive control to well D12 and E12.
• Dispense 10 µl of the negative control to well F12 and G12.
• Dispense 10 µl buffer to well H12.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 anti-species conjugate to all wells.
• Seal and incubate for 60 minutes at 37˚C.
• Wash according 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. 

IX Validation of the test

• To standardize the 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.

X 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.
• The OD of the positive control must be ≥ 1,000.Important

NOTE

• A positive ELISA result must be confirmed by virus isolation or haemagglutination inhibition test.

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

• For more ordering information