Sandwich ELISA


Abstract 

This protocol describes the detection of antigens in solution using nanoCLAMPs covalently immobilized to maleimide coated plates via their lone C-terminal Cys as capture antibodies, and a secondary (detection) enzyme-conjugated antibody to a known epitope on the antigen. Antigen pulled from solution by the nanoCLAMP is bound by the detection enzyme-conjugated antibody, which is chromogenically developed by adding the appropriate enzyme substrate. Note: because nanoCLAMPs contain a N-terminal 6His tag, anti-His antibodies are incompatible with this assay.

Instructions 

1 Rinse Pierce Maleimide Activated Plates, 8 well strips (Thermo cat# 15150) with 200 ul Maleimide Coupling Buffer (MCB). Repeat this step for a total of 4 times
In between rinses, shake the solution from the plate, then pat the plate on a paper towel twice, in a fresh location each time
2 Dilute nanoCLAMPS to 1 – 5 ug/ml in MCB-TCEP (MCB with 1 mM TCEP, prepared fresh). Allow nanoCLAMPs to reduce for 30 minutes
3 Remove last MCB wash from plate and add 100 ul of diluted nanoCLAMP per well and incubate for 2 hours at 4°C
Incubation can range from 2 hours to overnight. If incubating overnight, place plate in airtight bag with a wet paper towel to limit evaporation
4 Dump protein from plates and wash with 200 ul Maleimide Wash Buffer, MWB, (100 mM NaH2PO4, 150 mM NaCl, 0.05% Tween20, pH 7.2). Repeat this step for a total of 4 times
5 Block unreacted maleimide groups with 200 ul of 10 ug/ml L-Cys in MCB (MCB-Cys) for 1 hour
6 Wash wells with 200 ul MWB. Repeat this step for a total of 3 times
7 Block wells with 200 ul 2% M-PBS-T (20 mM NaH2PO4, 150 mM NaCl, pH 7.4, 2% dry milk, 0.05% Tween20) for 2 hours
8 Rinse blocked plates with 200 ul PBS-T. Repeat this step for a total of 3 times
9 If storing, dump the block, pat dry on paper towel and allow plates to dry upside down on a paper towel for 2 hours
Store in dessicated chamber or plastic bag overnight at 4°C
10 Add 100 ul solution containing antigen in 2% M-PBS-T, and incubate for 1 hour
11 Dump the antigen solution and rinse the wells with 200 ul PBS-T. Repeat this step for a total of 4 times
12 Dilute secondary antibody to unique epitope on antigen per manufacturers recommendations in 2 – 4% M-PBS-T, and add 100 ul per well and incubate for 1 hour
13 Dump the secondary antibody and wash the wells with 200 ul PBS-T. Repeat this step for a total of 4 times
14 Add 100 ul TMB Ultra and incubate for 30 minutes
15 Stop the reaction with 50 ul 2 N H2SO4 and measure the A450

Solution(s) Used
Use / Nickname Solution
PBS-T PBST RJS
MWB MWB
MCB-TCEP MCB-TCEP pH 7.2
MCB MCB pH 7.2
H2SO4, 2 N Sulfuric Acid, 2 N
2% M-PBS-T 2% M-PBS-T

Outside Material(s) Used
Vendor Outside Material Catalog Number Usage Rate
ThermoFisher Scientific 1-Step™ Ultra TMB-ELISA Substrate Solution (Thermo) 34028 0.1 ml per well
ThermoFisher Scientific Pierce Maleimide Activated Plates (8 well strips) 15150
Applications Using This Protocol
Outcome Title
Successful SMT3 (yeast SUMO) sandwich ELISA with immobilized SMT3-A1(Cys)
Successful NusA sandwich ELISA with immobilized nusA-A1(Cys)
Successful Maltose binding protein (MBP) sandwich ELISA with immobilized malE-A1(Cys)
Successful GFP sandwich ELISA with immobilized GFP-A1(Cys)
Successful Avidin sandwich ELISA with immobilized AVD-A1(Cys)