Antibody conjugates
Antibody/signal-generating moiety conjugates are disclosed that include an antibody covalently linked to a signal-generating moiety through a heterobifunctional polyalkyleneglycol linker. The disclosed conjugates show exceptional signal-generation in immunohistochemical and in situ hybridization assays on tissue sections and cytology samples. In one embodiment, enzyme-metallographic detection of nucleic acid sequences with hapten-labeled probes can be accomplished using the disclosed conjugates as a primary antibody without amplification.
1. A method for detecting a molecule of interest in a tissue section or a cytology sample, comprising:
contacting the tissue section or cytology sample with an antibody-polyethylene glycol (PEG) linker-signal-generating moiety conjugate comprising an antibody covalently linked to at least 3 signal-generating moieties, wherein each signal-generating moiety is linked to the antibody through a monodisperse,
heterobifunctional PEG linker to provide an antibody-PEG linker-signal-generating moiety conjugate having the formula
wherein Ab is an antibody, SM is an enzyme signal-generating moiety, n=4 to 12 and s=3 to 10; and
detecting a signal generated by the antibody-PEG linker-signal-generating moiety conjugate that indicates the presence of the molecule of interest in the sample.
2. The method of claim 1 , wherein the enzymes are horseradish peroxidase or alkaline phosphatase.
3. The method of claim 1 , further comprising contacting the tissue section or cytology sample with a water-soluble metal ion and a redox-inactive substrate of the enzymes that is converted to a redox-active agent by the enzymes, which redox-active agent reduces the metal ion causing it to precipitate.
4. The method of claim 3 , wherein the enzymes are alkaline phosphatase.
5. The method of claim 1 , wherein the signal-generating moieties are oxido-reductase enzymes and the method further comprises contacting the tissue section or cytology sample with a water soluble metal ion, an oxidizing agent and a reducing agent.
6. The method of claim 5 , wherein the oxido-reductase enzymes are horseradish peroxidase.
7. The method of claim 1 , wherein the antibody comprises an anti-antibody antibody.
8. The method of claim 1 , wherein the antibody comprises an anti-hapten antibody, and the method further comprises contacting the tissue section or cytology sample with a hapten-labeled antibody or a hapten-labeled nucleic acid sequence.
9. The method of claim 1 , further comprising contacting the tissue section or cytology sample with a chromogenic, fluorogenic and/or luminogenic compound.
10. The method of claim 1 , wherein s=3 to 6.
11. The method of claim 10 , wherein s=3 to 4.
12. The method of claim 11 , wherein SM is horseradish peroxidase or alkaline phosphatase.
13. A method for detecting a molecule of interest in a tissue section, comprising: contacting the tissue section with an antibody-PEG linker-signal-generating moiety conjugate having the formula:
wherein Ab is an antibody, SM is a signal-generating moiety selected from alkaline phosphatase or horseradish peroxidase, n=4 to 12 and s=3 to 6, and wherein the PEG linker that linked the signal-generating moiety SM to the antibody Ab in the antibody-PEG linker-signal-generating moiety conjugate is a monodispersed PEG linker;
contacting the conjugate with a chromogenic substrate of alkaline phosphatase or horseradish peroxidase; and,
detecting a chromogen generated by the antibody-PEG linker-signal-generating moiety conjugate from the chromogenic substrate that indicates the presence of the molecule of interest in the sample.