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.
Highlights
In human body fluids and tissues, adenosine deaminase is present as two isozymes: ADA1 and ADA2
ADA1 exists in two molecular forms: the small monomeric adenosine deaminase (ADA1-S, 41 kDa) and the large adenosine deaminase (ADA1-L, 298 kDa), which is composed of Adenosine deaminase small form (ADA1-S) and adenosine-binding protein (dipeptidyl-dipeptidase IV (DPPIV) or CD26) [3,4,5,6]
Hens immunized with calf adenosine deaminase (cADA) emulsified with Freund’s complete and incomplete adjuvant showed no signs of pain or stress over the course of 22 weeks
Summary
In human body fluids and tissues, adenosine deaminase (adenosine aminohydrolase, EC 3.5.4.4) is present as two isozymes: ADA1 and ADA2. Both isozymes play an essential role in purine nucleoside metabolism catalyzing the irreversible deamination reaction of adenosine or 2′deoxyadenosine to inosine or 2′deoxyinosine. These two isozymes are kinetically distinguishable by their reaction with EHNA ((+)-erythro-9-(2-hydroxy-3-nonyl)adenine), a specific inhibitor of ADA1 [1]. The small globular ADA1-S molecule is formed by a characteristic parallel α/βbarrel motif (TIM barrel fold) with a zinc cofactor located in the catalytic pocket [7]. The protein-protein contact between these two molecules occurs through two hydrophobic loops in the β-propeller domain of DPPIV and two hydrophilic α-helices within ADA, which is why even after the formation of the complex both enzymes remain catalytically active [8]
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