Development and characterization of a latex turbidimetric immunoassay using rabbit anti-CRP single-chain Fv antibodies

In this study, we developed and demonstrated a latex turbidimetric immunoassay (LTIA) using latex beads immobilized with rabbit monoclonal single-chain variable fragments (scFvs) selected from an scFv-displayed phage library. Sixty-five different anti-c-reactive protein (anti-CRP) scFv clones were identified after biopanning selection using antigen-coupled multi-lamellar vesicles. By ranking antigen-binding clones using the apparent dissociation rate constant (appkoff) as a sorting index, scFv clones with a dissociation constant (KD free) ranging from 4.07 × 10-9 M to 1.21 × 10-11 M were isolated. Among them, three candidates (R2-6, R2-45, and R3-2) were produced in the culture supernatant at concentrations of 50 mg/L or higher in flask culture and maintained at considerably high antigen-binding activity in immobilized state on the CM5 sensor chip surface. All the scFv-immobilized latexes (scFv-Ltxs) prepared were well-dispersed in 50 mM MOPS at pH 7.0, without additives for dispersion, and their antigen-dependent aggregation was sufficiently detectable. The reactivity of scFv-Ltx to antigen differed among the scFv clones, in particular, R2-45 scFv-Ltx detected the CRP with the highest signal. Furthermore, the reactivity of scFv-Ltx varied significantly with salt concentration, scFv immobilization density, and the type of blocking protein. Particularly, antigen-dependent latex aggregation improved significantly in all rabbit scFv clones when scFv-Ltx was blocked with horse muscle myoglobin compared with conventional bovine serum albumin; while their baseline signals in the absence of antigen were fully stable. Under optimal conditions, R2-45 scFv-Ltx exhibited greater aggregation signals with antigen concentrations higher than those produced by conventional polyclonal antibody-immobilized latex for CRP detection in LTIA. The methodology for rabbit scFv isolation, immobilization, and antigen-dependent latex aggregation demonstrated in the present study can be applicable to scFv-based LTIA for various target antigens.