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. Author manuscript; available in PMC: 2017 Oct 27.
Published in final edited form as: Methods Mol Biol. 2012;844:15–30. doi: 10.1007/978-1-61779-527-5_2

Table 1.

Schematic depicting the time arrangement of a simplified sequential ELISA protocol of exemplary targets analyzed in three subsequent cycles

Time (h) Previous day capture incubation overnight
Regular ELISA cycle # 1 Sequential ELISA cycle # 2 Sequential ELISA cycle # 3
0.0 Blocking
1.0 Samples
2.0 Incubation Blocking
3.0 Detection ⇒TRANSFER SAMPLESa TO CYCLE # 2 ⇒Samples
4.0 Incubation Incubation Blocking
5.0 Streptavidin–HRP Detection ⇒TRANSFER SAMPLESa TO CYCLE # 3 ⇒Samples
5.5 TMB Incubation Incubation
6.0 Reading Incubation Incubation
7.0 Streptavidin–HRP Detection ⇒REMOVE AND STORE SAMPLESb
7.5 TMB Incubation
8.0 Reading Incubation
9.0 Streptavidin–HRP
9.5 TMB
10.0 Reading
a

Samples are removed and transferred to the storage plate (master mix) before washing and detection step

b

Samples are removed before washing, transferred to the master mix, and stored overnight at 4°C for consecutive cycles