Fig. 5:

Examples of the most popular chemical reactions for antibody--NP conjugation. a) One-step procedure using unmodified antibodies and coupling agent EDC or EDC/NHS (sulfo-NHS for better solubility in aqueous media); b) Two-step process using monovalent linkers such as glutaraldehyde and unmodified antibodies; c) Two-step process using multivalent linkers such as dendrimers and unmodified antibodies; d) One-step process using modified antibodies (fragmented by (2S,3S)-1,4-bis(sulfanyl)butane-2,3-diol, known as 1,4-dithio-D-threitol (DTT) or 2-sulfanylethan-1-ol, also known as 2-mercaptoethanol (2-MEA) – extra step) and heterofunctional bi-linkers such as sulfo succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate [sulfo-SMCC] or sulfo- succinimidyl (4-iodoacetyl)aminobenzoate [sulfo-SIAB]; e) One or two steps procedure using oxidized antibodies (usually by periodates); the antibody-NP linkage is reversible, therefore further reduction with borohydrides can be advantageous as it generates a stable covalent bond; f) One-step reaction between streptavidin-coated NP (many types are commercially available) and biotinylated antibody (also many types commercially available). Other types of site-specific antibody immobilization methods such as via His-tagged antibodies and NTA-Ni coated NP or via Protein A/G – coated NP (for binding to Fc region of immunoglobulins) are less common and therefore not illustrated in this figure. The cartoons used in this illustration show a solid NP, however NP are also often fabricated from porous material.

© De Gruyter