Preparation and Characterization of mAbs-LTF ICs

Preparation and characterization of a mouse mAbs against huLTF (M860) in this laboratory have been reported in our previous work.35 (link) For preparation of LTF-IC, huLTF (2 µg/mL, Sigma-Aldrich) and M860 (2 µg/mL, mAbs of LTF), purified with protein G antibody affinity chromatography (GE Healthcare Life Sciences), or LTF-Abs were mixed in a sterile tube with gentle rotation at 37°C for an hour. ICs between LTF and M860 were separated from the uncoupled Ab and antigen using Sephadex Superfine G-75 column. The elutions of IC were pooled, desalted and concentrated. Endotoxin was removed by polymyxin B coupled beads repeatly and the level of endotoxin in IC was below 1 EU/mg which was detected by Chromogenic LAL Endotoxin Assay Kit (Genscript). ICs between ovalbumin (OVA) (Sigma-Aldrich) and M562 (mAbs of OVA) were used as control and prepared similarly.

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Dong H., Yang Y., Gao C., Sun H., Wang H., Hong C., Wang J., Gong F, & Gao X. (2020). Lactoferrin-containing immunocomplex mediates antitumor effects by resetting tumor-associated macrophages to M1 phenotype. Journal for Immunotherapy of Cancer, 8(1), e000339.

Publication 2020

protein G antibody affinity chromatography Chromogenic LAL Endotoxin Assay Kit

Affinity chromatography Antibody Antibody affinity Antibody g Antigen Assay Chromatography Chromogenic Endotoxin Mabs Mouse Ovalbumin Polymyxin b Protein Protein g Sephadex g 75 Sterile

Corresponding Organization : Soochow University

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Variable analysis

independent variables

Concentration of huLTF (2 µg/mL)
Concentration of M860 (2 µg/mL)
Mixing of huLTF and M860 in a sterile tube with gentle rotation at 37°C for an hour

dependent variables

Formation of immune complexes (ICs) between huLTF and M860

control variables

Protein G antibody affinity chromatography (GE Healthcare Life Sciences) used to purify M860
Sephadex Superfine G-75 column used to separate ICs from uncoupled Ab and antigen
Polymyxin B coupled beads used to remove endotoxin from ICs
Chromogenic LAL Endotoxin Assay Kit (Genscript) used to detect endotoxin level in ICs
ICs between ovalbumin (OVA) and M562 (mAbs of OVA) used as control

positive control

ICs between ovalbumin (OVA) and M562 (mAbs of OVA)

negative control

Not explicitly mentioned

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