Simple, Rapid Chemical Labeling and Screening of Antibodies with Luminescent Peptides

Abstract

Sensitive and selective detection assays are essential for the accurate measurement of analytes in both clinical and research laboratories. Immunoassays that rely on nonoverlapping antibodies directed against the same target analyte (e.g., sandwich enzyme-linked immunosorbent assays (ELISAs)) are commonly used molecular detection technologies. Use of split enzyme reporters has simplified the workflow for these traditionally complex assays. However, identifying functional antibody pairs for a given target analyte can be cumbersome, as it generally involves generating and screening panels of antibodies conjugated to reporters. Accordingly, we sought a faster and easier reporter conjugation strategy to streamline antibody screening. We describe here the development of such a method that is based on an optimized ternary NanoLuc luciferase. This bioluminescence complementation system is comprised of a reagent-based thermally stable polypeptide (LgTrip) and two small peptide tags (β9 and β10) with lysine-reactive handles for direct conjugation onto antibodies. These reagents enable fast, single-step, wash-free antibody labeling and sensitive functional screening. Simplicity, speed, and utility of the one-pot labeling technology are demonstrated in screening antibody pairs for the analyte interleukin-4. The screen resulted in the rapid development of a sensitive homogeneous immunoassay for this clinically relevant cytokine.

Figure 1

Peptide label design. (A) The ternary Nluc system possesses two small peptides that correspond to strands 9 (β9, purple) and 10 (β10, blue) of the 10-strand luciferase β-barrel. The remaining strands (1–8) represent a complementary 17 kDa polypeptide (LgTrip). (B) Chemical labels derived from ternary Nluc peptides contain lysine-reactive N-hydroxysulfosuccinimidyl ester handles appended to the N-terminus of β9 or β10 peptide via a poly(ethylene glycol) (PEG) linker of varying length.

Scheme 1. Synthetic Route for Chemical Modification of Ternary Nluc Peptides with sulfo-NHS

(1) sulfo-NHS; (2) bis-PEG-acid; (3) bis-PEG-sulfo-NHS; (4) NH₂ peptide; and (5) labeling product.

Figure 2

Labeling kinetics. A 10-fold molar excess of the label (67 μM) was added to 1 mg/mL (6.7 μM) αIL-6 antibodies in PBS. Antibodies 5IL6 and 505E were conjugated to β9 and β10, respectively. Minus antibody controls (i.e., label only) were run in parallel. Excess Tris (final [Tris] = 100 mM) was added to quench reactions. Time = 0 values were taken immediately after label addition. (A) Crude, unwashed β9 and β10 conjugate reaction mixtures from the indicated time points were assayed in cell culture media at 62.5 ng/mL (4.2 nM) antibody. Conjugate pairs were assayed in the presence (+IL-6) and absence (−IL-6) of 10 ng/mL IL-6 analyte. Mean relative light units (RLU) values (n = 2) of a representative experiment are shown with variability expressed as standard deviation (SD). (B) Assay dynamic range (S/B) was calculated for each time point by dividing the (+) IL-6 signal by the (−) IL-6 signal from panel (A).

Figure 3

Conjugate cleanup improves assay performance but is optional for streamlining antibody labeling-to-assay workflow. Conjugation reactions were run for 1 h in PBS and quenched (final [Tris] = 100 mM). Conjugates were either set aside as crude reaction products or washed via desalting columns. β9 and β10 antibody conjugates were assayed at 62.5 ng/mL against a titration of IL-6 analyte in cell culture media. (A) Raw RLU and (B) signal over background (S/B) are plotted for the same data set. Mean values ± SD are shown (n = 3).

Figure 4

General workflow for conjugation and assay of antibody pairs using ternary Nluc peptide labels.

Figure 5

Rapid screen of IL-4-specific antibodies. A panel of six αIL-4 antibodies was conjugated to β9 and β10 labels and assayed in cell culture media ±50 ng/mL IL-4 analyte. Results of the screen are shown according to (A) light output (RLU) in the presence of the analyte and (B) assay dynamic range (S/B).

Figure 6

Characterization of IL-4 antibody screen hits Ab6-β9 and Ab5-β10. Washed and crude antibody conjugates were assayed at 250 ng/mL against a titration of the IL-4 analyte in cell culture media. Mean RLU values (n = 3) are shown with variability expressed as SD.