RU2423707C2 - Method of human biopsy material and excrete analysis for chitinase-like protein ykl-40 - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: method of quantitative determination of human chitinase-like protein YKL-40 (another name - human cartilaginous glycoprotein-39, or HcGP-39) is offered. When implementing the method, colloidal chitin or heparin is sorbed in microplate wells, then solutions containing a certain amount of chitinase-like protein, and analysed samples are introduced in the wells. The amount of YKL-40 is analysed by the amount of an enzymatic reaction product formed by interaction of an enzymatic conjugate with YKL-40 antibodies against with a substratum of this enzyme.

EFFECT: method allows well reproducible and high sensitive YKL-40 analysis in various human biopsy materials and excretes.

2 cl, 2 ex, 1 tbl

Description

The invention relates to medical biochemistry, and in particular to methods for the quantitative determination of chitinase-like human protein YKL-40 (another name is human cartilage glycoprotein-39 or HcGP-39), and can be used in enzymology, microbiology, pharmacology, clinical biochemistry for quantitative assessments of this protein in various biopsies and human excreta.

Chitinase-like proteins are proteins similar in their primary structure to chitinase enzymes capable of cleaving chitin polyaminosaccharide. Like chitinases, chitinase-like proteins have a chitin-binding domain, but do not have enzymatic activity, that is, they do not cleave chitin due to an amino acid substitution in the catalytic center. YKL-40 has chitin and heparin binding domains. An analogue of the chitinase-like human protein YKL-40 was first found in the serum of non-lactating cows [1]. Subsequently, the ability to produce YKL-40 by chondrocytes, synovial cells, activated macrophages, neutrophils, and osteosarcoma cells (MG-63) was described [2-5]. Despite the fact that the value of YKL-40 for the human body remains unknown until now, a change in the gene expression of this protein during various pathological processes suggests that the chitinase-like protein plays a role in tissue remodeling. In clinical studies, it was shown that YKL-40 may be one of the markers of destructive activity in rheumatoid arthritis and osteoarthritis, liver fibrosis, adenocarcinoma of the lungs and large intestine, with a Th2-mediated immune response [6-9].

The most promising of modern methods for determining YKL-40 is the enzyme-linked immunosorbent assay due to its sensitivity, selectivity and the ability to automate the analytical process. To determine the concentration of YKL-40, a method was developed based on the enzyme-linked immunosorbent assay, which is the closest prototype to our invention [10]. However, the use of Fab fragments of monoclonal antibodies to YKL-40, which also requires an additional laborious technology of their sorption in the wells of strips, gives such commercial test systems a high cost.

The objective of the claimed invention is to develop a method that allows you to quantify chitinase-like protein YKL-40 in various biopsies and excreta of the person on the basis of the enzyme immunoassay, using the property of chitinase-like proteins to specifically bind to chitin and heparin.

The task is achieved by developing a determination method that involves sorption of colloidal chitin or heparin in the microplate wells, then adding solutions containing a known amount of chitinase-like protein to the microplate wells, and an analyzed sample, incubation (during which chitinase-like proteins by means of specific domain bind to colloidal chitin or heparin), pouring the contents of the wells, and then introducing the conjugate of the enzyme with antibodies against YKL-40 and a substrate of this about the enzyme and the calculation of the amount of protein by the amount of the resulting product of the enzymatic reaction.

The technical result of the claimed invention is the development of a simplified method for the quantitative determination of YKL-40 in various biopsies and human excreta by enzyme-linked immunosorbent assay, which differs from the prototype in the absence of the need to use monoclonal Fab fragments of antibodies to chitinase-like protein.

Example 1. Determination of the concentration of YKL-40. A tablet is prepared for sorption of colloidal chitin, exposing its inner surface to UV radiation for 2 minutes (lamp with a power of 20 to 100 watts). Immediately after UV irradiation, 100 μl of a solution of colloidal chitin in concentrations of 10-100 μg / ml are added to the wells. Cover and leave overnight at + 4 ° C. Three times the plate is washed with 0.15 M sodium phosphate buffer solution, pH 7.4, containing 0.05% Tween-20, pouring 150 μl into each well, then the tablet is dried by shaking out the remaining liquid. 100 μl of a solution containing a known amount of YKL-40 and a test sample (serum, lavage from the nasal mucosa, bronchoalveolar lavage fluid, lacrimal fluid) containing an unknown amount of chitinase-like protein are added to the wells of a row tablet. After incubating in an incubator for 1 h at + 37 ° C, washing twice with 0.15 M sodium phosphate buffer solution, pH 7.4, containing 0.05% tween-20, and draining the plate, 100 μl are added to each well a peroxidase conjugate with anti-YKL-40 antibodies in the same buffer at a selected dilution. After incubation in a thermostat for 1 h at + 37 ° C, twice washing with detergent and draining the plate, 100 μl of substrate buffer (10 mg of orthophenylenediamine in 25 ml of nitrate-phosphate buffer, pH 5.0, and 50 μl) are added to each well. 3% hydrogen peroxide). After 30 minutes of incubation in the dark, the reaction is stopped by adding 50 μl of 14% sulfuric acid to each well. The results of the reaction are taken into account using a spectrophotometer with a vertical beam by measuring light absorption at 492 nm. The concentration of YKL-40 is calculated from a standard curve. This method determined the concentration of YKL-40 in samples with a known content of chitinase-like protein. The results are shown in table 1.

Example 2. The determination of the concentration of YKL-40 is carried out analogously to example 1, but as a specific agent that binds a chitinase-like protein, heparin is adsorbed onto the plate at concentrations of 10-100 μg / ml.

LITERATURE

1. Rejman J.J., Hurley W.L. Isolation and characterization of a novel 39 kilodalton whey protein from bovine mammary secretions collected during the non-lacting period. Biochem. Biophys. Res. Comm. 1988. V. 150. P.329-334.

2. Hakala B.E., White C., Recklies A.D. Human cartilage gp-39, a magor secretory product of articular chondrocytes and synovial cells, is a mammalian member of a chitinase protein family. J. Biol. Chem. 1993. V.268. P.25803-25810.

3. Nyirkos P., Golds E.E. Human synovial cells secrete a 39 kDa protein similar to a bovine mammary protein expressed during the non-lactating period. Biochem. J. 1990. V.268. P.265-268.

4. Renkema G.H., Boot R.G., Au F.L. Chitotriosidase, a chitinase, and the 39-kDa human cartilage glycoprotein, a chitin-binding lectin, are homologues of family 18 glycosy hydrolases secreted by human macrophages. Eur. J. Biochem. 1998. V.251. P.504-509.

5. Volck B., Price P.A., Johansen J.S. YKL-40, a mammalian member of the chitinase family, is a matrix protein of specific granules in human neutrophils. Proc. Assoc. Am. Physicians. 1998. V.110. P.351-360.

6. Johansen J.S., Jensen H.S., Price P.A. A new biochemical marker for joint injury. Analysis of YKL-40 in serum and synovial fluid. Br. J. Rheumatol. 1993. V.32. P.949-955.

7. Johansen J.S., Moller S., Price P.A. Plasma YKL-40: a new potential marker of fibrosis in patients with alcoholic cirrosis? Scand. J. Gastroenterol. 1997. V.32. P.582-590.

8. Cintin C, Johansen J.S., Christensen I.J. Serum YKL-40 and colorectal cancer. Br. J. Cancer. 1999. V.79. P.1494-1499.

9. Chupp G.L., Lee C.G., Jarjour N. Chitinase-like protein in the lung and circulation of patients with severe asthma. 2007. V.357. P.2016-2027.

10. U.S. Patent No. 7230086

Table 1 Dependence of optical density (OD) on the concentration of YKL-40. Try OD _{492 nm} YKL-40, ng / ml Control, YKL-40, 0 ng / ml 0,060 0 Control, YKL-40.20 ng / ml 0,098 twenty Control, YKL-40, 50 ng / ml 0.149 fifty Control, YKL-40,100 ng / ml 0.243 one hundred Control, YKL-40,200 ng / ml 0.380 200 Control, YKL-40, 300 ng / ml 0.596 300 Serum 1 0,500 258 Serum 2 0,308 145 Serum 3 0.131 41 Nasal wash 1 0.116 32 Nasal Rinse 2 0.186 73 Nose Wash 3 0.297 139

Claims (2)

1. An enzyme-linked immunosorbent assay for determining the concentration of YKL-40, characterized in that colloidal chitin or heparin is adsorbed in the wells of the plate, then a solution of the test sample (serum, rinsing from the nasal mucosa, broncho-alveolar lavage fluid, lacrimal fluid) is introduced into the wells the incubation for the specific binding of chitinase-like protein and after drying the tablet and washing in the wells make the conjugate of the enzyme with antibodies against YKL-40 and after incubation and washing the substrate buffer, after which DYT calculation of the concentration of YKL-40. 2. An enzyme-linked immunosorbent assay for determining the concentration of YKL-40, characterized in that it contains a tablet, solutions of colloidal chitin or heparin, an enzyme conjugate with antibodies against YKL-40, and a substrate buffer.