Strong and oriented immobilization of single domain antibodies from crude bacterial lysates for high-throughput compatible cost-effective antibody array generation

Abstract

Antibody microarrays are among the novel class of rapidly emerging proteomic technologies that will allow us to efficiently perform specific diagnoses and proteomic analysis. Recombinant antibody fragments are especially suited for this approach but their stability is often a limiting factor. Camelids produce functional antibodies devoid of light chains (HCAbs) of which the single N-terminal domain is fully capable of antigen binding. When produced as an independent domain, these so-called single domain antibody fragments (sdAbs) have several advantages for biotechnological applications thanks to their unique properties of size (15 kDa), stability, solubility, and expression yield. These features should allow sdAbs to outperform other antibody formats in a number of applications, notably as capture molecules for antibody arrays. In this study, we have produced antibody microarrays using direct and oriented immobilization of sdAbs, produced in crude bacterial lysates, to generate a proof-of-principle of a high-throughput compatible array design. Several sdAb immobilization strategies have been explored. Immobilization of in vivo biotinylated sdAbs by direct spotting of bacterial lysate on streptavidin and sandwich detection was developed to achieve high sensitivity and specificity, whereas immobilization of "multi-tagged" sdAbs via anti-tag antibodies and a direct labeled sample detection strategy was optimized for the design of high-density antibody arrays for high-throughput proteomics and identification of potential biomarkers.

Figure 1. Functional sdAbs are efficiently produced in the cytoplasm of E. coli

A) MC38-CEA (■, ●) or MC38 (▲) cells were incubated with serial dilutions of anti-CEA sdAb produced in cytoplasm (■) or periplasm (●) of E. coli. Captured antibodies were detected by a mouse anti-6his mAb followed by a goat against mouse FITC-conjugated mAb. Cells were analyzed by flow cytometry assay on FACScalibur. B) Biotinylated Nef antigen (5 nM) coated on streptavidin-plate was incubated with serial dilution of anti-Nef sdAb produced in the cytoplasm (■) or periplasm (●) of E. coli or anti-CEA sdAb produced in the cytoplasm of E. coli (▲). Captured antibodies were detected by a mouse anti c-myc mAb followed by a goat anti-mouse HRP-conjugated mAb. Standard deviation represents two experiments performed in triplicates.

Figure 2. In vivo biotinylation and multi tags strongly improve immobilization of sdAbs

A) Serial dilutions of pure anti-Nef sdAb biotinylated in vivo (●), in vitro (■) or unbiotinylated (▲) were coated on streptavidin plate and incubated with Nef at 5 nM. The captured antigen was detected with a mouse anti-Nef antibody followed by a goat anti-mouse HRP-conjugated mAb. B) Protein G bead were coated with 1 μg/ml of 9E10 mAb. Serial dilutions of pure sdAbs with three (●), one (■) or no (▲) myc tag were incubated with the bead, followed by biotinylated Nef at 5 nM. The captured antigen was detected with a HRP-conjugated streptavidin. Standard deviation represents two experiments performed in triplicates.

Figure 3. Bacterial lysates are a good source of capture antibody

Streptavidin beads were coated with sdAb against Nef biotinylated in vivo pure (●) (1 μg/ml) or in bacterial lysate (▲) (50 nl/wells) or sdAb against Nef unbiotinylated in bacterial lysate (▼) and incubated with serial dilution of Nef. The captured antigen was detected with a mouse anti-Nef antibody followed by a goat anti-mouse HRP-conjugated mAb. Standard deviation represents two experiments performed in triplicates.

Figure 4. In vivo biotinylated and trimyc-tagged sdAbs in bacterial lysate allow a sensitive antigenic detection on slide or beads

A) Immobilization of sdAbs using trimyc tag allows the direct use of bacterial lysates. Epoxy bead were coated with 9E10 (1 μg/ml) and incubated with bacterial lysate (50 nl/wells) containing sdAbs with three (●), one (■) or no (▲) c-myc followed by serial dilutions of biotinylated Nef. The captured antigen was detected with HRP-conjugated streptavidin. B) Immobilization of sdAbs using trimyc allows direct spotting of bacterial lysate. Nitrocellulose slides were coated with 9E10 (9E10) or PBS (Ø). Bacterial lysates containing sdAbs fused to three, one or no c-myc were spotted. Serial dilutions of biotinylated Nef were incubated and the captured antigen was detected with Alexa705-conjugated streptavidin. C): Streptavidin beads were coated with bacterial lysate (50 nl/wells) containing anti-Nef sdAbs biotinylated in vivo (●, ▲, ◆) or not (■, ▼, ○) and incubated with serial dilutions of Nef or Alexa488-conjugated Nef (◆, ○). The captured antigen was detected with a mouse anti-Nef antibody followed by a goat anti-mouse HRP (●,■) or Alexa488-conjugated mAb (▲, ▼). D) Nitrocellulose slides were incubated with streptavidin (S) or PBS (Ø). Bacterial lysates containing sdAbs biotinylated in vivo or unbiotinylated were spotted. Serial dilutions of Nef were incubated and the captured antigen was detected with a mouse anti-Nef antibody followed by a goat anti-mouse Alexa705-conjugated mAb. Standard deviation represents two experiments performed in triplicates.

Figure 5. Immobilization of in vivo biotinylated sdAbs allows a sensitive detection of CEA in patient sera using a bead assay

Streptavidin beads were coated with bacterial lysates (0.5 μl/wells) containing in vivo biotinylated (●,■, ▼, ▲) or unbiotinylated (◆) anti-CEA sdAbs and incubated with serial dilutions of patient sera (●: S1 CEA 276 ng/ml, ■: S2 CEA 769 ng/ml, ▲: S3 CEA 178 ng/ml, ▼: S4 CEA < 5ng/ml). The captured antigen was detected with a mouse anti-CEA antibody (35A7) followed by a goat anti-mouse HRP-conjugated mAb. Standard deviation represents two experiments performed in triplicates.