Development of an enzyme-linked immunoassay for the quantification of YKL-40 (cartilage gp-39) in guinea pig serum using hen egg yolk antibodies.
An indirect competition immunoassay for the quantification of YKL-40 (cartilage gp-39, Chondrex) in guinea pig serum has been developed using egg yolk antibodies (IgY). The immune response of hens to YKL-40 was verified by immunoblot analyses. Highly specific antibodies were obtained 30 days after the first injection. The ELISA was developed in 96-well microtiter plates with quadruplicate determinations for each point. The assay was based on the ability of YKL-40 present in serum to displace the binding of antibodies to the coated antigen. An inhibition mixture containing standard YKL-40 or guinea pig serum, diluted 1/5, and primary antibodies, diluted 1/5000, was allowed to equilibrate for 2 h at room temperature and dispensed for 16 h at 4 degrees C in wells coated with 1 microg/ml of YKL-40. Detection was achieved by the addition of rabbit anti-chicken antibodies conjugated to peroxidase followed by tetramethylbenzidine. Specificity was assessed by parallelism between a dilution curve of serum and standard YKL-40. The sensitivity of detection was 10 ng/ml. Intra- and interassay coefficients of variation were both 8.7%. The analytical recovery was 101.5+/-5.4% (mean+/-standard deviation (SD), n=9). The YKL-40 concentration in serum from 12 adult guinea pigs was 330+/-216 ng/ml (mean+/-SD) with a lower value of 164 ng/ml and an upper value of 982 ng/ml. In contrast to the rat, a dilution curve of rabbit serum gave parallelism with the guinea pig standard, suggesting recognition of a similar epitope. Possible applications of the assay in the guinea pig include disease models where YKL-40 is overexpressed and could be used as a marker, i.e. osteoarthritis, rheumatoid arthritis, cancer, liver fibrosis, atherosclerosis and more generally, pathologies with increased tissue remodeling.
De Ceuninck
F
F
Division of Rheumatology, Institut de Recherches Servier, 11 rue des Moulineaux, 92150, Suresnes, France. frederic.deceuninck@fr.netgrs.com
Pastoureau
P
P
Agnellet
S
S
Bonnet
J
J
Vanhoutte
P M
PM
D054392:Adipokines; D000818:Animals; D002645:Chickens; D000071451:Chitinase-3-Like Protein 1; D004530:Egg Yolk; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods* / Q000592:standards; D006023:Glycoproteins / Q000097:blood* / Q000276:immunology; D006168:Guinea Pigs; D015151:Immunoblotting / Q000379:methods; D007136:Immunoglobulins / Q000276:immunology*; D037102:Lectins; D011817:Rabbits; D051381:Rats
D054392:Adipokines; C084533:CHI3L1 protein, human; D000071451:Chitinase-3-Like Protein 1; D006023:Glycoproteins; C016512:IgY; D007136:Immunoglobulins; D037102:Lectins