Development of Adenosine Deaminase-Specific IgY Antibodies: Diagnostic and Inhibitory Application

Abstract

Adenosine deaminase (ADA) is currently used as a diagnostic marker for tuberculous pleuritis. Although ADA has been suggested as a potential marker for several types of cancer, the importance of each of ADA isoforms as well as their levels and enzymatic activities in tumors need to be further investigated. Herein we developed avian immunoglobulin Y highly specific to human ADA via hens immunization with calf adenosine deaminase. The obtained antibodies were used for the development of a sensitive double-egg yolk immunoglobulin (IgY) sandwich ELISA assay with an ADA detection limit of 0.5 ng/ml and a linearity range of up to 10 ng/ml. Specific, affinity-purified IgYs were able to recognize human recombinant ADA and ADA present in human cancer cell lines. In addition, antigen-specific IgY antibodies were able to inhibit catalytic activity of calf ADA with an IC₅₀ value of 47.48 nM. We showed that generated IgY antibodies may be useful for ADA detection, thus acting as a diagnostic agent in immunoenzymatic assays.

Keywords: Adenosine deaminase (ADA); Affinity purification; Anti-adenosine deaminase antibody; ELISA; Egg yolk immunoglobulin (IgY); Enzyme inhibition.

Fig. 1

Production of antigen-specific IgY antibodies during the course of immunization. Anti-cADA IgY production (triangles) and avidity (circles) (a). For the indirect ELISA, plates were coated with cADA protein (0.5 μg/ml). The time of booster injections is indicated by arrowheads. b For Western blot analysis, cADA was used at an amount of 50 ng/lane. In both experiments, anti-cADA antibodies were used at 1:100 dilution and rabbit anti-IgY antibodies were used at 1:5000. The ELISA results are expressed as the OD₄₉₀ ^* ± SEM values obtained after normalization to the background absorbance obtained for the control antibodies

Fig. 2

Anti-cADA IgY antibody titer. For indirect ELISA, the plate was coated with cADA antigen (0.5 μg/ml, 100 μl/well) (a). For Western blot analysis, 50 ng cADA/lane was used (b). In both experiments, IgY antibodies were used in serial dilutions from 50 to 0.1 μg/ml. Rabbit anti-IgY-HRP conjugate was used in a 1:5000 dilution. For Western blot analysis, membrane strips corresponding to 0 were incubated with control IgY antibodies at a concentration of 50 μg/ml. The results of ELISA are presented as an ELISA index (EI) ± SEM, where EI = OD_sample/OD_control

Fig. 3

The detection limit of calf adenosine deaminase. For determination of the cADA detection limit, Western blot with target antigen from 50 to 0.1 ng/lane was performed. The membrane was incubated with IgY antibodies (10 μg/ml), followed by rabbit anti-IgY conjugate dilution 1:5000 (a). For ELISA, the plate was coated with 1 to 0.005 μg/ml of native calf ADA protein and incubated with antigen-specific and control IgY antibodies (25 μg/ml). Detection of cADA-IgY antibody complexes was performed with rabbit anti-IgY IgG antibodies conjugated with HRP (1:5000) (b). For ELISA, the results are presented as an ELISA index (EI) ± SEM, where EI = OD_sample/OD_control

Fig. 4

Affinity chromatography purification of anti-cADA IgY antibodies. a Standard SDS-PAGE electrophoresis in non-reducing conditions, followed by silver staining. Fractions: FT—flowthrough 25 times diluted in PBS, W10—PBST with EDTA buffer, pH 7.4 (10th wash), P5—PBS with EDTA buffer, pH 7.4 (5th wash), K1–K5 fractions after elution with citrate buffer, pH 2.5. b Dot-blot analysis of obtained anti-cADA affinity-purificated antibodies. Strip I—anti-cADA crude IgY antibodies isolate, II—anti-cADA affinity-purified IgY antibodies, III—control IgY antibodies, IV—buffer only. All IgYs were used at a concentration of 1 μg/ml. Protein-antibody complexes were detected with anti-IgY IgG HRP conjugates diluted 1:5000

Fig. 5

Sandwich-type ELISA assay for calf adenosine deaminase detection. The plate was coated with anti-cADA affinity-purified IgY antibodies and control IgY antibodies (2.5 μg/ml) followed by incubation with cADA at concentrations ranging from 500 to 0.05 ng/ml. For detection, affinity-purified anti-cADA IgY biotin-labeled antibodies were used at a concentration of 2.5 μg/ml. The complexes were detected with streptavidin conjugate with HRP (1: 5000 dilution). Symbols represent mean ± SD from two independent experiments performed in duplicate for each point and are expressed as the OD₄₉₀ ^* values obtained after subtraction of the background values

Fig. 6

The detection of human recombinant adenosine deaminase by anti-calf adenosine deaminase IgY antibodies. Human ADA (10, 100, and 1000 times diluted; reducing conditions) was resolved on SDS-PAGE, followed by silver staining. SDS-PAGE (reducing conditions, 4–12% Tris-glycine) of calf (1 ng/well) and human (50 times diluted) ADA followed by nitrocellulose electrotransfer was performed. The membrane was incubated with anti-cADA and control IgY antibodies (10 μg/ml). Protein-antibody complexes were detected with anti-IgY IgG HRP conjugates diluted 1:5000

Fig. 7

Double IgY sandwich ELISA for sensitive detection of adenosine deaminase. Anti-cADA and control IgY were used as coating antibodies (2.5 μg/ml in carbonate buffer). cADA in the concentration range from 10 to 0.1 ng/ml and hADA in a dilution range from 10 to 10,000 were used, followed by incubation with anti-cADA IgY antibodies modified with biotin at a concentration of 2.5 μg/ml. Complexes were detected with the use of sterptavidin HRP conjugate (1:5000). Results are expressed as the OD₄₉₀ ^* ± SEM values obtained after subtraction of the values recorded for the control antibodies

Fig. 8

The detection of adenosine deaminase in human cell lines. For determination of the ability of IgY antibodies to detect ADA in human cell line lysates, cADA at a concentration range from 500 to 62.5 ng/lane (as a positive control) and cell lysates (total protein concentration from 40 to 2.5 μg/lane) were resolved on SDS-PAGE (4–12%, Tris-glycine). After electrotransfer, the membrane was incubated with specific and control IgY antibodies (1 μg/ml), followed by an incubation with rabbit anti-IgY conjugate diluted 1:5000

Fig. 9

Evaluation of anti-calf adenosine deaminase affinity-purified IgY antibodies as an inhibitor of adenosine deaminase. IgY antibodies (specific and control) at final concentrations ranging from 111.11 to 9.54 nM were mixed with native cADA (final concentration of 0.04 U/ml) in PBS buffer, pH 7.2. After 1 h incubation at 37 °C, adenosine in PBS was added (final concentration 1.25 mM) and the deamination reaction was monitored (A₂₆₀). For calculation of the IC₅₀, a value variable slope equation model was applied. Symbols represent mean ± SD from two independent experiments performed in duplicate for each point