ELISA-based protein-PAMP interaction assay

doi:10.1038/nprot.2007.481

Method Article

ELISA-based protein-PAMP interaction assay

https://doi.org/10.1038/nprot.2007.481

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Biological techniques

Biochemistry

Computational biology and bioinformatics

metHb

hemocyanin

PAMPs

ELISA

The interaction between HMC or metHb with LPS or LTA are examined by ELISA using LPS- or LTA-immobilized plates and probed with anti-hemocyanin or anti-hemoglobin antibody. To test the specificity of the ELISA, we further examined whether or not pre-incubation of HMC or Hb with LPS or LTA could reduce the ELISA-readout in a dose-dependent manner.

Incubate aliquots of 100 μl LPS or LTA which is resuspended in 0.01 M pH 7.4 PBS in a 96-well microtitre plate (Maxisorp®, NUNC, Denmark) at room temperature overnight.
Rinse the wells three times with 300 μl of PBS each.
Block the wells with 150 μl of PBS with 2% (w/v) BSA at 37 °C for 2 h.
Rinse the wells three times with 300 μl of PBS each.
Add to each well, 100 μl of the serially diluted protein (HMC or Hb in 2% BSA) in PBST (PBS containing 0.05% v/v Tween 20) and incubate at 37°C for 1 h.
Rinse the wells three times with 300 μl of PBST each.
Add to each well, 100 μl of primary antibody (rabbit-anti HMC or rabbit-anti Hb), which is diluted to 1:500 with 2% BSA in PBST and further incubate at 37 °C for 30 min.
Rinse the wells three times with 300 μl of PBST each.
Add to each well, 100 μl of the horseradish peroxidase-conjugated goat-anti-rabbit antibody (DAKO, Japan), which is diluted to 1:1000 in 2% BSA in PBST, and incubate at 37 °C for 30 min.
Rinse the wells three times with 300 μl of PBST each.
Immediately before using, dissolve peroxidase substrate tablet (ABTS, 2,2’-azino-bis[3-ethylbenzthiazoline-6-sulfonic acid], Roche) in substrate solution (Roche) as indicated by the manufacturer’s manual.
Add to each well, 50 μl of the peroxidase substrate and incubate for 10 min at room temperature.
Read the OD at 405 nm.
To verify the specificity of the protein-PAMP interaction observed in the ELISA, further examine whether or not pre-incubation of HMC or Hb with LPS or LTA could reduce the ELISA-readout in a dose-dependent manner.
a) Choose a concentration of either HMC or Hb that shows an ELISA-readout(A405)at the linear range.

b) Incubate the HMC or Hb with 0-200 μg/ml LPS or LTA in 2% BSA in PBST at 37 °C for 1 h.

c) Then transfer the reaction mixture to the LPS- or LTA- immobilized microtitre plate.

d) Conduct the ELISA as described in steps 5-13.

e) The reduction of the readout of HMC or Hb pre-incubated with LPS or LTA respectively, demonstrates the specificity of the protein-PAMP interaction.

As shown in Figure 8, the typical ELISA readout of HMC-LPS interaction is A405 = 0.1-0.4; and that of Hb-LPS interaction is A405=0.4-1.6

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ELISA-based protein-PAMP interaction assay

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