AG275, AG294 and AG274 antibodies recognize the hen egg-white lysozyme by ELISA

Authors

  • Benjamin Adomako
  • Vincent Mendes Ferreira
  • Garance Michel
  • Marin Ollagnon
  • Yekaterina Prutyanova
  • Julien Schaer
  • Emma Siebenmann
  • Bethania Tamrat
  • Mathieu Vigneau
  • Maxime Zholdokov
  • Philippe Hammel
  • Stéphane Durual
  • Anja Maag
  • Maria Lung
  • Patricia Lopez
  • Tess Arbez
  • Luca Baltazar
  • Jade Berlincourt
  • Marc Bobillier
  • Diana Boeva
  • Agnès Bury
  • Alexis Calomeni
  • Hania Farhat
  • Eva Garrido
  • Hugo Gillet
  • Tasnim Khadam-Al-Jame
  • Cyril Guilhen cyril.guilhen@unige.ch UNIGE

DOI:

https://doi.org/10.24450/journals/abrep.2022.e671

Abstract

The recombinant antibodies AG275, AG294 and AG274 detect the hen egg-white lysozyme by ELISA.

Introduction

Lysozyme is an antibacterial enzyme widely distributed in vertebrates, invertebrates, plants, and microbes. It is also found in a large variety of animal secretions and tissues such as saliva, mucus, or avian eggs, and it is secreted by polymorphonuclear leukocytes among other cells (Mason and Taylor, 1975). Lysozyme hydrolyzes the 1,4-beta-linkages between N-acetylmuramic acid and N-acetylglucosamine in the peptidoglycan of Gram-positive bacteria (Chipman and Sharon, 1969). Three recombinant antibodies (AG275, AG294 and AG274) detect the hen egg-white lysozyme (HEWL) (Uniprot P00698) by ELISA. Two other tested antibodies (AE913 and AE915) did not.

Materials & Methods

Antibodies: ABCD_AE913, ABCD_AE915, ABCD_AG274, ABCD_AG275 and ABCD_AG294 antibodies (ABCD nomenclature, http://web.expasy.org/ abcd/) were produced by the Geneva Antibody Facility (http://unige.ch/medecine/antibodies/) as mini-antibodies with the antigen-binding portion fused to a rabbit IgG Fc. The synthesized scFv sequences (GeneArt, Invitrogen) correspond to the sequences of the variable regions joined by a peptide linker (GGGGS)3 (see Table 1 for clone names and references). HEK293 suspension cells (growing in FreeStyle™ 293 Expression Medium, Gibco 12338) were transiently transfected with the vector coding for the scFv-Fc of each antibody. Supernatants (see Table 1 for individual yields) were collected after 4 days.

ABCD Clone Reference Yield ( mg /L)
AE913 L2,5 Davies et al.,1995 100
AE915 L1 Davies et al., 1995 10
AG274 D44.1 Braden et al., 1994 5
AG275 HyHEL-63 Li et al., 2000 70
AG294 D1.3 Holmes et al., 1998 80
Table 1. Clone number, reference and production yields for the antibodies used in this study.

Protocol: The whole procedure was carried out at room temperature. HEWL (5 µg/mL; Sigma L6876) was incubated in high protein-binding capacity 96-well plates (50 µl/well) (Thermo Fisher Scientific 44-2404) for 30 minutes. The support was blocked 20 minutes in PBS containing 4% (w/v) BSA. Each well was rinsed three times with 100 μL of washing buffer (PBS + 0.1% (w/v) BSA + 0.05% (w/v) Tween20), then incubated for 30 minutes with 50 µl of antibody-containing supernatant diluted in washing buffer as indicated (Fig. 1). After rinsing 3 times (100 µl washing buffer), wells were incubated with horseradish peroxidase-coupled goat anti-rabbit IgG (Sigma A8275, dilution 1:1000, 50 μl per well) for 30 min. After 5 rinses, Tetramethylbenzidine (TMB) substrate (Sigma T5569) was added (50 μl per well). The reaction was stopped by the addition of 25 μl of 2 M H2SO4. The absorbance (OD) was measured at 450 nm.

Results

Antibodies AG275, AG294 and AG274 bound in a concentration-dependent manner to the HEWL, but not to the BSA negative control (Fig. 1). AE913 and AE915 did not recognize the HEWL by ELISA.

Figure 1. AG275, AG294 and AG274 bound specifically to the hen egg-white lysozyme (HEWL), but not to the BSA control (shown only for AG275; the other background curves were superimposed), as detected by ELISA.

Conflict of interest

The authors declare no conflict of interest.

References

Braden BC, Souchon H, Eiselé J-L, et al. Three-dimensional structures of the free and the antigen-complexed Fab from monoclonal anti-lysozyme antibody D44.1. J Mol Biol. 1994; 243(4):767-81. PMID: 7966295.

Chipman DM, Sharon N. Mechanism of lysozyme action. Science. 1969; 165(3892):454-65. PMID: 4893486.

Davies EL, Smith JS, Birkett CR, Manser JM, Anderson-Dear DV, Young JR. Selection of specific phage-display antibodies using libraries derived from chicken immunoglobulin genes. J Immunol Methods. 1995; 186(1):125-35. PMID: 7561141.

Holmes MA, Buss TN, Foote J. Conformational correction mechanisms aiding antigen recognition by a humanized antibody. J Exp Med. 1998; 187(4):479-85. PMID: 9463398.

Li Y, Li H, Smith-Gill SJ, Mariuzza RA. Three-dimensional structures of the free and antigen-bound fab from monoclonal antilysozyme antibody HyHEL-63. Biochemistry. 2000; 39(21):6296-309. PMID: 10828942.

Mason DY, Taylor CR. The distribution of muramidase (lysozyme) in human tissues. J Clin Pathol. 1975; 28(2):124-32. PMID: 1092717.

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Published

2022-02-17 — Updated on 2022-02-17

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1.
Adomako B, Mendes Ferreira V, Michel G, Ollagnon M, Prutyanova Y, Schaer J, Siebenmann E, Tamrat B, Vigneau M, Zholdokov M, Hammel P, Durual S, Maag A, Lung M, Lopez P, Arbez T, Baltazar L, Berlincourt J, Bobillier M, Boeva D, Bury A, Calomeni A, Farhat H, Garrido E, Gillet H, Khadam-Al-Jame T, Guilhen C. AG275, AG294 and AG274 antibodies recognize the hen egg-white lysozyme by ELISA. Antib. Rep. [Internet]. 2022 Feb. 17 [cited 2023 Dec. 10];5(1):e671. Available from: https://oap.unige.ch/journals/abrep/article/view/671

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