ELISA-based assay for determination of influenza virus-host receptor binding specificity

Authors：

Category

Sugar binding proteins

Protocol Name

Authors

Suzuki, Yasuo *
Health Science Hills, College of Life and Health Sciences, Chubu University

Sriwilaijaroen, Nongluk
(1) Health Science Hills, College of Life and Health Sciences, Chubu University, (2) Faculty of Medicine, Thammasat University (Rangsit Campus)

*To whom correspondence should be addressed.

KeyWords

Ganglioside Receptor ELISA Influenza virus

Reagents

●	Ethanol
●	Blocking solution: 1 g of defatted bovine serum albumin (BSA) in 100 mL of PBS
●	Dilution buffer: 0.5 g of defatted BSA in 100 mL of PBS
●	Substrate solution: 2 mg of o-phenylenediamine (Sigma-Aldrich, St. Louis, MO) is dissolved in 10 mL of 100 mM citrate-phosphate buffer (pH5.5) before addition of 3.3 μL of 30% H₂O₂. This coloring solution should be mixed freshly before use.

Instruments

●	96-well Polysorp flat-bottomed plates
●	37°C incubator
●	ELISA plate reader

Methods

ELISA-based assay for determination of influenza virus-host receptor binding specificity

1)	Dissolve ganglioside (3–50 pmol/10 μL/well) in ethanol or another organic solvent that has no effect on a plastic plate (F96 polysorp; Nalge Nunc International) and dilute serially two-fold with ethanol.	Comment 0

2)	Evaporate ethanol at 37°C for several minutes and dry completely with warm air.	Comment 0

3)	Add 250 μL of blocking solution (PBS with 1% of defatted BSA) to each well and incubate for 2 h at 37°C.	Comment 0

4)	Wash the wells five times with PBS, add 100 μL of 2⁵ HAU of influenza virus in PBS, and keep at 4°C overnight with gentle shaking.	Comment 0

5)	Wash unbound virus five times with cold PBS, add 100 μL of primary antibody directed to the virus diluted in 0.5% defatted BSA, and keep for 2 h at 4°C.	Comment 0

6)	Wash five times with PBS to get rid of free primary antibody, add 100 μL of HRP-conjugate secondary antibody in 0.5% defatted BSA, and keep for 2 h at 4°C.	Comment 0

7)	Wash away unbound conjugate five times with PBS, add 100 μL of coloring solution, and keep at room temperature until a yellow color develops.	Comment 0

8)	Stop the reaction by adding 50 μL of 1 N H₂SO₄. This acidic solution will change color from yellow to orange.	Comment 0

Record the absorbance at 490 nm with reference wavelength of 630 nm. Before plotting a graph, subtract OD₄₉₀ from OD₆₃₀ to obtain the actual values of virus binding activity.

Comment 0

Notes

If purified, gangliosides can be coated on wells of a flat-bottomed microtiter plate and the ELISA technique can be employed for quantitative detection of virus binding activity to the gangliosides. The gangliosides and influenza virus materials are obtained from the same sources as those of the TLC test, but a smaller amount (2⁵ HAU, influenza virus: 3–50 pmol/well, ganglioside) can be detected by ELISA. The other reagents are similar to those of TLC except for the reagents mentioned above.

Influenza viruses should be handled in the facility of Biological Safety Level 2 or 3. Therefore, handling of the viruses should be under the control of national law.

Figure & Legends

Fig. 1. Receptor binding specificity of highly pathogenic avian influenza virus, A/KongKong/213/03 (H5N1) isolated from human

The direct binding activity of viruses to sialylparagloboside (IV³(Neu5Ac)Lc4Cer, IV⁶(Neu5Ac)Lc4Cer) was determined as described in this protocol. A/HongKong/213/03 (H5N1) bound to both Neu5Acα2-3paragloboside (IV³(Neu5Ac)Lc4Cer), and Neu5Acα2-6paragloboside (IV⁶(Neu5Ac)Lc4Cer) but A/Duck/HongKong/200/01 isolated from duck bound preferentially to Neu5Acα2-3paragloboside (IV³(Neu5Ac)Lc4Cer) and A/Memphis/1/71 isolated from human bound preferentially to Neu5Acα2-6paragloboside (IV⁶(Neu5Ac)Lc4Cer).

Copyrights

Date of registration：2014-10-20 16:31:08