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. Author manuscript; available in PMC: 2019 Jun 1.

Published in final edited form as: Bone. 2018 Mar 14;111:36–43. doi: 10.1016/j.bone.2018.03.013

First incubation 1: 50 μl patient sample incubated with an assay buffer containing a basic polymer to dislodge anionic binding partners like heparin and BMP proteins than can occlude sclerostin’s C-terminal surface, making it more accessible to conjugate recognition. Second incubation: N-terminal specific murine monoclonal capture antibody coupled to paramagnetic particles is added. Upon binding sclerostin, it orients sclerostin such that the C-terminal portion of sclerostin is distal to the capture particle’s surface. A sequential separation and wash cycle is then performed to remove unbound sclerostin fragments and other non-specific serum/plasma proteins. Third incubation: Sandwich formation is achieved via addition of an ABEI-labelled polyclonal goat functionally anti-sclerostin C-terminal tail specific conjugate. Following a final separation and wash cycle, developer is added and emitted light photons [relative light units (RLU)] are measured and converted to pg/mL via a master standard curve. Total time to results is approximately 65 minutes.

1B. Specificity of the LIAISON sclerostin CLIA components overlaid upon a Clustal O(1.2.4) alignment of sclerostin and SOSTDC1 primary sequences. The capture monoclonal antibody is specific to a 16-mer located within the N-terminal tail (Gln₁ to Ser₅₆) of sclerostin. The tracer polyclonal antibody is conjugated to ABEI. Its functional C-terminal tail specificity is inferred from, and based upon, three observations: 1) The distal orientation of sclerostin’s C-terminal portion consequent to N-terminal capture specificity of the PMP; 2) SOSTDC1’s C-terminal portion (Gly₁₁₇ to Ser₂₀₆) that aligns with the C-terminal thrombin peptide of sclerostin (Gly₉₉ to Tyr₁₉₀) is twice as basic as its N-terminal portion (2.33fold [21.7%/9.2%] which is similar to sclerostin, whose C-terminal fragment is 2.23 fold [25%/11.2%] more basic than its N-terminal fragment; and 3) SOSTDC1, which has 47.1% direct homology to sclerostin’s post-thrombin digest C-terminal Loop2-Loop3 fragment domain (Gly₉₉ to Arg₁₄₉) and only 19.5% homology to the post-thrombin digest C-terminal tail domain (Phe₁₅₀ to Tyr₁₉₀), does not inhibit the assay when added to the third incubation step. Collectively these observations make it more likely that the conjugate’s specificity is predominantly towards the C-terminal tail.

1C. Primary attributes of sclerostin and SOSTDC1. Intact molecule: sclerostin (Gln₁ to Tyr₁₉₀) has 35.9% direct homology with SOSTDC1 (M₁ to Ser₂₀₆). Gly₉₉ – Arg₁₄₉ and Phe₁₅₀ – Tyr₁₉₀ comprise the Loop2–Loop3 and C-terminal portions of the C-terminal thrombin fragment, respectively. Percent basic (arginine/lysine) residue content of sclerostin secondary structural elements (i.e. Loops 1, 2 and 3) along with the N- and C-terminal thrombin fragments (and the aligned portions from SOSTDC1) demonstrate a similar relative basic aspect to the C-terminal portion of each molecule.

First incubation 1: 50 μl patient sample incubated with an assay buffer containing a basic polymer to dislodge anionic binding partners like heparin and BMP proteins than can occlude sclerostin’s C-terminal surface, making it more accessible to conjugate recognition. Second incubation: N-terminal specific murine monoclonal capture antibody coupled to paramagnetic particles is added. Upon binding sclerostin, it orients sclerostin such that the C-terminal portion of sclerostin is distal to the capture particle’s surface. A sequential separation and wash cycle is then performed to remove unbound sclerostin fragments and other non-specific serum/plasma proteins. Third incubation: Sandwich formation is achieved via addition of an ABEI-labelled polyclonal goat functionally anti-sclerostin C-terminal tail specific conjugate. Following a final separation and wash cycle, developer is added and emitted light photons [relative light units (RLU)] are measured and converted to pg/mL via a master standard curve. Total time to results is approximately 65 minutes.

1B. Specificity of the LIAISON sclerostin CLIA components overlaid upon a Clustal O(1.2.4) alignment of sclerostin and SOSTDC1 primary sequences. The capture monoclonal antibody is specific to a 16-mer located within the N-terminal tail (Gln₁ to Ser₅₆) of sclerostin. The tracer polyclonal antibody is conjugated to ABEI. Its functional C-terminal tail specificity is inferred from, and based upon, three observations: 1) The distal orientation of sclerostin’s C-terminal portion consequent to N-terminal capture specificity of the PMP; 2) SOSTDC1’s C-terminal portion (Gly₁₁₇ to Ser₂₀₆) that aligns with the C-terminal thrombin peptide of sclerostin (Gly₉₉ to Tyr₁₉₀) is twice as basic as its N-terminal portion (2.33fold [21.7%/9.2%] which is similar to sclerostin, whose C-terminal fragment is 2.23 fold [25%/11.2%] more basic than its N-terminal fragment; and 3) SOSTDC1, which has 47.1% direct homology to sclerostin’s post-thrombin digest C-terminal Loop2-Loop3 fragment domain (Gly₉₉ to Arg₁₄₉) and only 19.5% homology to the post-thrombin digest C-terminal tail domain (Phe₁₅₀ to Tyr₁₉₀), does not inhibit the assay when added to the third incubation step. Collectively these observations make it more likely that the conjugate’s specificity is predominantly towards the C-terminal tail.

1C. Primary attributes of sclerostin and SOSTDC1. Intact molecule: sclerostin (Gln₁ to Tyr₁₉₀) has 35.9% direct homology with SOSTDC1 (M₁ to Ser₂₀₆). Gly₉₉ – Arg₁₄₉ and Phe₁₅₀ – Tyr₁₉₀ comprise the Loop2–Loop3 and C-terminal portions of the C-terminal thrombin fragment, respectively. Percent basic (arginine/lysine) residue content of sclerostin secondary structural elements (i.e. Loops 1, 2 and 3) along with the N- and C-terminal thrombin fragments (and the aligned portions from SOSTDC1) demonstrate a similar relative basic aspect to the C-terminal portion of each molecule.