One-step Preparation of a VHH-based Immunoadsorbent for the Extracorporeal Removal of β2-microglobulin

Abstract

Dialysis-related amyloidosis (DRA), which has been widely recognized to be associated with the accumulation of β2-microglobulin (β2-m) in blood, is one of the most common complications in patients receiving long-term dialysis treatment. The most significant side-effect of existing hemodialysis sorbents for the removal of β2-m from blood is the loss of vital proteins due to non-specific adsorptions. Although the traditional antibodies have the capability to specifically remove β2-m from blood, high cost limits their applications in clinics. Single domain antibodies derived from the Camelidae species serve as a superior choice in the preparation of immunoadsorbents due to their small size, high stability, amenability, simplicity of expression in microbes, and high affinity to recognize and interact with β2-m. In this study, we modified the anti-β2-m VHH by the formylglycine-generating enzyme (FGE), and then directly immobilized the aldehyde-modified VHH to the amino-activated beads. Notably, the fabrication is cost- and time-effective, since all the preparation steps were performed in the crude cell extract without rigorous purification. The accordingly prepared immunoadsorbent with VHHs as ligands exhibited the high capacity of β2-m (0.75 mg/mL). In conclusion, the VHH antibodies were successfully used as affinity ligands in the preparation of novel immunoadsorbents by the site-specific immobilization, and effectively adsorbed β2-m from blood, therefore opening a new avenue for efficient hemodialysis.

Keywords: VHH; formylglycine-generating enzyme; hemodialysis; β2-microglobulin.

Figure 1

Acetic acid-assisted precipitation of host cell proteins. Lane M is the protein molecular weight marker; 50% acetic acid solution was added dropwise into the crude cell extract. The supernatant treated with different pH values were analyzed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and gray scanning. After precipitation, the supernatant was readjusted to pH 9 (Lanes 4–9). The lysate, which directly adjusted from pH 7 to pH 9, was set as a control (Lanes 7–9).

Figure 2

Validation of the site-specific modification of VHHs. SDS-PAGE analysis of Aldtag-VHH and the lucifer yellow CH (LY-CH) labeled products. Lane M, molecular weight markers; lane 1, native Aldtag-VHH; lane 2, Aldtag-VHH incubated with LY-CH; lane 3, FGE modified Aldtag-VHH incubated without LY-CH; lane 4, FGE modified Aldtag-VHH incubated with LY-CH; lane 5, crude cell extract was pretreated with 50% acetic acid to pH 4 with adjustment to pH 9 with 1 M NaOH; lane 6, crude cell extract pretreated with 1 M NaOH (adjusted directly to pH 9).

Figure 3

Optimization of FGE-catalytic conditions of VHH catalysis in the crude cell extract. Four factors were investigated, including (A) the molar ratio of VHH and FGE, (B) incubation time, (C) the concentration of DTT added to the catalytic buffer, and (D) the catalytic temperature. The products of VHH incubated with FGE under different conditions were analyzed by SDS-PAGE, and the catalytic efficiency was calculated using the software of ImageJ. Data are presented as means ± SDs, n = 3. Error bars are within symbol size if not visible.

Figure 4

Affinities of the unmodified (A) and modified VHHs (B) binding β2-m investigated by Biacore T200. For surface plasmon resonance spectroscopy (SPR)-based affinity measurements, β2-m was covalently coupled on a CM5-chip. Kinetic measurements were performed by injecting seven concentrations ((162.8, 81.40, 40.70, 20.35, 10.17, 5.087, 2.544) × 10⁻⁸ M) of purified VHHs. The obtained data sets were evaluated using the 1:1 Langmuir binding model.

Figure 5

The static adsorption performance of the VHH-based adsorbent in plasma. (A) The isothermal adsorption curve of the sorbent at room temperature; qEX represents the test data while qTH represents the theoretical data. (B) The rearranged linearization curve. The slope of the curve was the maximal adsorptive capacity of the gel, and the intercept of curve was the opposite number of Kd. Y represents the equilibrium concentration of β2-m; X represents c/q (see Equation 2 in Method 3.6).

Figure 6

Adsorption of β2-m from β2-m enriched human serum using VHH-based immunosorbent fabricated through site-specific reductive amination conjugation. (A) Chromatography profile. (B) SDS-PAGE analysis of the chromatographic fractions. Lane 1, native β2-m; Lane 2, fraction eluted with 1% SDS; Lane 3, human serum. HSA represents the human serum albumin.

Figure 7

Changes in concentration of serum components before and after the adsorption balance in vitro. T.P, total protein; Alb, albumin; IGF, insulin-like growth factor; TBA, total bile acid; IL-6, interleukin 6; IL-8, interleukin 8; IL-2R, interleukin 2 receptor.

Figure 8

The saturation adsorption capacity (SAC) of the VHH-based immunoadsorbent preserved at 4 °C for one month (each experiment was performed three times).

Figure 9

Scheme of the preparation of a VHH based immunoadsorbent: (a) 1,4-butanediol diglycidyl and (b) 3,3′-diaminodipropylamine were used to prepare the amino-activated matrix based on the CL-6B agarose beads. Aldehyde-modified VHHs were then coupled to the matrix via the reductive amination.