Scheme 1. All-in-one immunoassay with a colorimetric detection using a HRP labeled with antibody capable of capturing a target antigen and TMB (3,3', 5,5’-tetramethylbenzidine) as a substrate.
Scheme 2. All-in-one immunoassay with a colorimetric detection using a HRP labeled with antibody capable of capturing a target antigen and ABTS (2,2'-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid)) as a substrate.
Scheme 3. All-in-one immunoassay with a colorimetric detection using a HRP labeled with antibody capable of capturing a target antigen and OPD (o-phenylenediamine dihydrochloride) as a substrate.
Scheme 4. All-in-one immunoassay with a colorimetric detection using a HRP labeled with antibody capable of capturing a target antigen and Amplex Red as a substrate.
Scheme 5. All-in-one immunoassay with a colorimetric detection using an ALP labeled with antibody capable of capturing a target antigen and pNPP (p-Nitrophenyl Phosphate, Disodium Salt) substrate.
Using pNPP (p-Nitrophenyl Phosphate, Disodium Salt) as a substrate of ALP labeled with an antibody, the All-in-One immunoassay with a colorimetric detection can be applied for the rapid quantification of a target antigen in a sample as shown in Scheme 5. For example, trace levels of D-dimer in human serum were quantified with the all-in-one immunoassay within 30 minutes. The detection limit (3.5 ng/ml) of the all-in-one immunoassay with ALP-labeled antibody using pNPP was slightly higher than that (1.2 ng/ml) of the all-in-one immunoassay with HRP-labeled antibody using TMB. However, the all-in-one immunoassay with ALP-labeled antibody using pNPP can also be applied for the quantification of D-dimer because the normal range of D-dimer in human serum is as high as 250 ng/ml.
Scheme 6. All-in-one immunoassay with a colorimetric detection using a β-galactosidase labeled with antibody capable of capturing a target antigen and ONPG (o-nitrophenyl-β-D-galactopyranoside) as a substrate.
Scheme 7. All-in-one immunoassay with a colorimetric detection using a glucose oxidase labeled with antibody capable of capturing a target antigen and TMB as a substrate.
The all-in-one immunoassay with a colorimetric detection can be operated with an antibody conjugated with other enzymes instead of HRP and ALP as shown in Schemes 6 and 7. The sensitivity of the all-in-one immunoassay with a colorimetric detection is determined by the property of an alternative enzyme (e.g., beta-galactosidase, glucose oxidase) instead of the conventional enzymes. Also, the incubation time for the enzyme reaction depends on the properties of the alternative enzyme. Substrates used for the all-in-one immunoassay with a colorimetric detection using a glucose oxidase labeled with an antibody are the same as those (e.g., TMB, ABTS, OPD, Amplex Red) using HRP labeled with an antibody.