In situ protein detection with enhanced specificity using DNA-conjugated antibodies and proximity ligation

• Antibodies are important tools in anatomical pathology and research, but the quality of in situ protein detection by immunohistochemistry greatly depends on the choice of antibodies and the abundance of the targeted proteins. Many antibodies used in scientific research do not meet requirements for specificity and sensitivity. Accordingly, methods that improve antibody performance and produce quantitative data can greatly advance both scientific investigations and clinical diagnostics based on protein expression and in situ localization. We demonstrate here protocols for antibody labeling that allow specific protein detection in tissues via bright-field in situ proximity ligation assays, where each protein molecule must be recognized by two antibodies. We further demonstrate that single polyclonal antibodies or purified serum preparations can be used for these dual recognition assays. The requirement for protein recognition by pairs of antibody conjugates can significantly improve specificity of protein detection over single-binder assays.

Ämnesord

NATURVETENSKAP  -- Biologi (hsv//swe)

NATURAL SCIENCES  -- Biological Sciences (hsv//eng)

MEDICIN OCH HÄLSOVETENSKAP  -- Medicinska och farmaceutiska grundvetenskaper -- Immunologi inom det medicinska området (hsv//swe)

MEDICAL AND HEALTH SCIENCES  -- Basic Medicine -- Immunology in the medical area (hsv//eng)

TEKNIK OCH TEKNOLOGIER  -- Industriell bioteknik -- Annan industriell bioteknik (hsv//swe)

ENGINEERING AND TECHNOLOGY  -- Industrial Biotechnology -- Other Industrial Biotechnology (hsv//eng)

Nyckelord

antibody conjugate

APEX 1 protein

calvasculin

DNA conjugated antibody

immunoglobulin G antibody

polyclonal antibody

protein

protein A

protein G

rabbit antiserum

reagent

trefoil factor 1

unclassified drug

animal tissue

antibody affinity

antibody labeling

antibody specificity

Article

assay

click chemistry

controlled study

DNA strand

genetic transcription

immunohistochemistry

in situ proximity ligation assay

limit of detection

mRNA expression level

nonhuman

priority journal

protein analysis

protein expression

protein expression level

protein purification

tissue microarray

tissue section

Publikations- och innehållstyp

ref (ämneskategori)

art (ämneskategori)