Molecular conjugate

A method for detecting a molecule of interest in a cell or tissue sample, comprising:

• contacting the cell or the tissue sample with a primary antibody specifically recognizing the molecule of interest in the cell or the tissue sample to provide the cell or the tissue sample bound with the primary antibody
• contacting the cell or tissue sample with a conjugate comprising
• an antibody capable of specifically recognizing the primary antibody,
• a detectable label comprising an enzyme including about 17-25 maleimide groups introduced into the enzyme by NHS-PEG-maleimide linkers, each linker being covalently bonded to an amino group of the enzyme, and
• a plurality of hydrazide thiol linkers, each hydrazide thiol linker comprising one or more linking atoms positioned between a hydrazide group and a thiol group, wherein the hydrazide thiol linker is selected from mercaptobutyric acid hydrazide (MBH), mercaptobutyric acid carbohydrazide (MBCH), mercaptoacetamido-mercaptobutyric acid hydrazide (MAMBH), thiohexanamidomercaptobutyric acid hydrazide (THMBH), N,N′-(6-hydrazinyl-6-oxohexane-1,5-diyl)bis(2-mercaptoacetamide) (BTAL), bisthiohexanamidohydrazidolysine (BTHL), N-(1,5-dihydrazinyl-1,5-dioxopentan-2-yl)2-mercaptoacetamide (TAGD), thiohexamidoglutamic acid dihydrazide (THGD), a PEG-based hydrazide-thiol linker, a multifunctional hydrazide thiol linker, a PEG-based multifunctional hydrazide thiol linker, a polyacrylamide hydrazide thiol linker, and combinations thereof, the hydrazide group of each hydrazide thiol linker being covalently bonded to an oxidized glycosylated Fc portion of the antibody and the thiol group of one of the hydrazide thiol linkers being covalently bonded to one of the maleimide groups on the enzyme;
• and
• detecting a signal generated by the conjugate that are bound to the primary antibody indicating the presence of the molecule of interest in the cell or tissue sample.