Use of high pressure to accelerate antibody: antigen binding kinetics demonstrated in an HIV-1 p24: anti-HIV-1 p24 assay.

A major factor in the design of highly sensitive ligandbinding assays is the need to accelerate the interaction between analyte and binding partner (or capture reagent). In early immunoassay technologies, overnight incubations with exogenously supplied binding partners at low temperatures (typically 2–8 °C) were common. More recently, however, assay results are frequently required, for both medical and commercial reasons, within a relatively short time (;1–2 h). The need to accelerate the binding reaction is certainly becoming even more important as high-throughput instruments (where minutes are the more common time frame) become widely utilized. This problem is typically addressed in commercial instruments by adding a large excess of the exogenous binding partner or using less than optimum temperature conditions so as to drive the binding reaction as far as possible in an acceptable assay time. High hydrostatic pressure is a powerful tool for studying the structure and function of proteins [1, 2]. Very high pressures cause most proteins to denature because of irreversible changes in the secondary and tertiary structure [3]. At less than the denaturation pressure, the tertiary and secondary nature of proteins are reversibly affected by changes in hydrostatic pressure [3]. Although some commercial applications of high hydrostatic pressure in the field of biotechnology have been reported or proposed [4, 5], its use to control or modulate biomolecular interactions of commercial interest has received little attention to date. Here, we describe the use of high hydrostatic pressure to considerably accelerate the kinetics of the binding of antibody to an antigen. Recombinant HIV-1 IIIB gag p24 (HIV-1 p24) and rabbit anti-p24 HIV-1 IIIB IgG (anti-HIV-1 p24) were purchased from ImmunoDiagnostics. A solid-phase ELISA for detecting antibody to HIV-1 p24 was developed in-house. Polystyrene microtiter plates (HiBind; Corning/Costar) were coated with a 1 mg/L suspension of HIV-1 p24 antigen overnight at 4 °C in NaHCO3, pH 9.2. Unreacted sites were blocked with SuperBlock in phosphate-buffered saline (PBS; 150 mmol/L NaCl, 162 mmol/L Na2HPO4, 38 mmol/L NaH2PO4, pH 7.4)–Tris (Pierce Chemical Co.). Binding of anti-HIV-1 p24 to immobilized HIV-1 p24 was detected by using goat anti-rabbit IgG conjugated to horseradish peroxidase (HRP; Pierce Chemical), and the HRP substrate 2,29-azinobis(3-ethylbenzthiazolinesulfonic acid) (ABTS). Experiments to determine the effect of pressure on antibody–antigen binding kinetics were commenced by adding 125 mL of 0.2 mg/L HIV-1 p24 to 125 mL of 2 mg/L rabbit anti-HIV-1 p24 in PBS in a polypropylene microcentrifuge tube such that 100 mL of the antigen/antibody mixture contained 10 ng of antigen and 100 ng of antibody. Part (120 mL) of the reagent mixture was then inserted into a deformable plastic capsule [5] and overlaid with melting point bath oil (Sigma). The capsule was immediately placed in the reaction chamber of a highpressure apparatus (High Pressure Equipment Co., Erie, PA) maintained at ambient temperature (;22 °C). The pressure was then immediately increased to the desired value by means of a manually operated piston [5]. Control samples, in which either the antibody or the antigen was omitted, were subjected to the same experimental conditions. All experiments were performed at ambient temperature (;22 °C). After high pressure had been applied for the desired time, test samples were measured with the p24-coated microwell ELISA. Test sample (100 mL) was immediately removed from the capsule and placed in a well of the p24-coated microplate; 100 mL of nonpressurized test solution was tested in parallel. Test samples were shaken at ambient temperature in the microtiter wells for 1 h at room temperature and then washed three times with PBS containing 0.5 mL/L Tween-20 (PBS-T). The microplates were then shaken for 1 h at ambient temperature with 100 mL of a 1:2500 dilution of the goat anti-rabbit–HRP conjugate. The microtiter plate wells were then washed five times with PBS-T, 100 mL of ABTS was added to each well, and the absorbance was read at 405 nm after a 30-min incubation. To select appropriate reagent concentrations for experiments at high pressure, we performed an initial study with different ratios of antibody to antigen at atmospheric pressure. Antibody and antigen were mixed in polypropylene microcentrifuge vials, then held overnight at 4–6 °C to reach equilibrium binding before measurement in the ELISA. This study showed that (a) 100 ng of the anti-HIV-1 p24 antibody alone (with no p24 antigen) had an absorbance of ;1.1 A at 405 nm, and (b) binding of 10 ng of p24 antigen to the antibody during the overnight incubation resulted in nearly maximal inhibition of the subsequent binding of the antibody to the p24 antigen immobilized on the microtiter plate. From the initial data described above, we determined the kinetics of binding at atmospheric pressure in mixtures containing 100 ng of antibody and 10 ng of antigen per 100 mL. The antigen/antibody mixtures were incubated for different times at ambient temperature (;22 °C) and then assayed in the ELISA. The absorbance values measured in the ELISA were used to calculate the extent of the binding of the p24 antigen with the anti-p24 antibody in the competitive assay, the results for the antibody-only sample (highest absorbance) representing zero binding and the results for overnight incubation of the antigen with the antibody (lowest absorbance) indicating 100% binding. At atmospheric pressure, ;25% of maximal binding between antigen and antibody occurred in 1 h, 40% in 2 h, and 65% in 4 h, rounded to the nearest 5%. The binding achieved overnight (16 h) was used as the maximum binding. The results are given 610%, reflecting the uncertainty in estimating the degree of binding from calibration Technical Briefs