TITLE: Solid phase binding assay - Clusterin binding to Very Low-Density Lipoprotein Receptor (VLDLR)
AUTHORS: Patricia Yuste-Checa, Andreas Bracher, F Ulrich Hartl

[DESCRIPTION]
This protocol details how to monitor Clusterin binding to the Very Low-Density Lipoprotein Receptor (VLDLR) by Enzyme-linked immunosorbent assays (ELISA) adapted from Leeb et al. (2014).

[STEPS]
SECTION: Solid phase binding assay - Clusterin binding to Very Low-Density Lipoprotein Receptor (VLDLR)
1. Coat the corresponding wells of a 96-well plate (Nunc-Immuno MicroWell 96 well solid plate, MERCK) with 100 µL of TBS-C containing 10 1621 VLDLR ectodomain   at 4 °C.
 

SECTION: Solid phase binding assay - Clusterin binding to Very Low-Density Lipoprotein Receptor (VLDLR)
2. Wash the plate once with TBS-C.
 

SECTION: Solid phase binding assay - Clusterin binding to Very Low-Density Lipoprotein Receptor (VLDLR)
3. Add Blocking solution and incubate the plate for 120 min at Room temperature (25 ºC). The wells without receptor are now coated with BSA.

SECTION: Solid phase binding assay - Clusterin binding to Very Low-Density Lipoprotein Receptor (VLDLR)
4. Remove Blocking solution and apply a series of increasing concentrations of ligand diluted in Blocking solution, each in a final volume of 100 µL. Each ligand concentration should be added to one well with immobilized VLDLR and one well coated with BSA (Blocking solution) for ligand background binding. One well with VLDLR and one well coated with BSA should be incubated without ligand to determine the general plate background signal.
 


SECTION: Solid phase binding assay - Clusterin binding to Very Low-Density Lipoprotein Receptor (VLDLR)
5. Incubate 60 min at Room temperature (25 °C).

SECTION: Solid phase binding assay - Clusterin binding to Very Low-Density Lipoprotein Receptor (VLDLR)
6. Wash the plate three times with Blocking solution.
 

SECTION: Solid phase binding assay - Clusterin binding to Very Low-Density Lipoprotein Receptor (VLDLR)
7. Add the corresponding primary antibodies diluted 1/100 in Blocking solution and incubate 60 min at Room temperature (25 °C).
 




SECTION: Solid phase binding assay - Clusterin binding to Very Low-Density Lipoprotein Receptor (VLDLR)
8. Wash the plate three times with TBS-C Blocking solution.
SECTION: Solid phase binding assay - Clusterin binding to Very Low-Density Lipoprotein Receptor (VLDLR)
9. Add the corresponding secondary antibody (horseradish peroxidase (HRP) conjugated) diluted 1/10,000 in Blocking solution and incubate 60 min at Room temperature (25 °C).


SECTION: Solid phase binding assay - Clusterin binding to Very Low-Density Lipoprotein Receptor (VLDLR)
10. Wash the plate three times with TBS-C Blocking solution. 
SECTION: Solid phase binding assay - Clusterin binding to Very Low-Density Lipoprotein Receptor (VLDLR)
11. Add 100 µL per well of the HRP substrate 1-Step Ultra TMB ELISA Substrate Solutions (Thermo Fisher Scientific, 34028) to develop the plate and incubate until the desired color develops.
 


SECTION: Solid phase binding assay - Clusterin binding to Very Low-Density Lipoprotein Receptor (VLDLR)
12. Add 100 µL per well of quenching solution to stop the reaction.

SECTION: Solid phase binding assay - Clusterin binding to Very Low-Density Lipoprotein Receptor (VLDLR)
13. Measure absorbance at 450 nm.