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
MCB MCB
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