KR20010000464A - Protein chip for diagnosis of osteoporosis - Google Patents

Abstract

PURPOSE: A protein chip for diagnosing osteoporosis is provided, thereby osteoporosis can be diagnosed cost and time effectively just by carrying out the inspection of urine or blood of a patient. CONSTITUTION: The blood and urine of a patient suffering from osteoporosis generally have a high amount of proteins relating to the processes of bone formation and bone adsorption. The protein chip contains antibodies of the proteins selected from bone specific alkali phosphatase, osteopontin, osteocalcin, calcitonin, osteoclastogenesis inhibiting factor, TGF beta3, enteropeptide, cytelopeptide, free pyridinoline, free dioxypyridinoline and the antibody-supporting plate of the protein chip is made of polystylene, polyvinylchloride, polypropylene or glass. Osteoporosis can be diagnosed by adding urine or blood sample of a patient suffering osteoporosis into the protein chip then analysing the amount of the marker proteins.

Description

Protein chip for diagnosing osteoporosis {PROTEIN CHIP FOR DIAGNOSIS OF OSTEOPOROSIS}

The present invention relates to a protein chip for diagnosing osteoporosis using a biochemical marker.

Osteoporosis is a disease commonly found in both men and women, especially in men and women in their 50s and post-menopausal age. It is mainly seen in women, and it is estimated that about 200 million people worldwide suffer from osteoporosis.

Osteoporosis is a symptomatic disease with no signs or symptoms that can be diagnosed and detected early. There are many difficulties in early diagnosis, prevention and treatment.

Currently, the diagnosis of osteoporosis generally relies on bone mineral density (BMD), which measures bone density at specific sites using X-ray film or bone densitometry. Instead, by measuring a specific part of the human body there is a problem that the accuracy is not high enough to diagnose the progress of general osteoporosis based on this.

In order to solve the above problems, many studies on osteoporosis diagnosis using biochemical markers have been conducted.

In general, the bone of the human body undergoes a bone turn over process to repeat the bone formation (Bone Formation) and bone absorption (Bone Resorption).

Osteogenesis is the process of growing or remodeling bone through osteoblasts. Proteins such as Osteoclastogenesis inhibitory factor, TGF β3 and Calvindin are known to be involved.

Bone resorption is the process of decomposing minerals in bone to maintain a constant calcium ion concentration in the blood through osteoclasts (Osteoclasts), N-telopeptides, C-telopeptides, and free pyridines. Proteins such as free pyridinoline and free deoxypyridinoline are known to be involved.

Osteoporosis is a disease that progresses when an imbalance occurs in the above-described bone conversion process, and it is known that the amount of the protein is increased or decreased in the blood or urine of osteoporosis patients compared to the normal person. By applying this, there is an active research into a method for diagnosing osteoporosis using the proteins as biochemical markers.

However, the current method for diagnosing osteoporosis using biochemical markers requires specialized knowledge, so it remains at the laboratory level for the purpose of experiments.

Various types of antibodies or antigenic proteins can be integrated on a small substrate to detect specific antigens or antibody proteins in the blood or urine of a patient, and various diseases can be diagnosed using protein chips that measure the content.

In addition, a protein chip can diagnose various diseases at the same time with a small amount of analyte, and has the advantage of greatly reducing the diagnosis time and cost.

The present invention provides a protein chip in which antibodies against various types of antibody proteins involved in the bone conversion process are simultaneously integrated on a small substrate, thereby enabling a more accurate and comprehensive diagnosis of osteoporosis, and providing the maximum amount of protein on a minimum substrate. The results of the reaction with the analyte prepared from the patient's blood or urine are collected and analyzed so as to invest the minimum cost and time.

1 is a graph showing the results of absorbance measurements for osteospecific alkaline phosphatase

Figure 2 is a graph showing the absorbance measurement results for osteocalcin

Figure 3 is a graph showing the absorbance measurement results for calcitonin

4 is a graph showing absorbance measurement results for free deoxypyridinolin

5 is a graph showing absorbance measurement results for free pyridinolin

Figure 6 is a graph showing the absorbance measurement results for the citelopeptide

Figure 7 is a graph showing the absorbance measurement results for the entelopeptide

8 is a graph showing absorbance measurement results for TGF β3

The present invention relates to a protein chip for diagnosing osteoporosis using a biochemical marker.

More specifically, the presence of osteoporosis on the small substrate by the accumulation of antibodies to proteins involved in the bone conversion process including bone formation and bone resorption, and analytes prepared from the blood or urine of the patient as a biochemical marker and The present invention relates to a protein chip for early diagnosis of progress.

The small substrate may be fixed to a desired protein by using avidin (avidin) on a conventional glass plate, in the present invention, it is preferable to use polystyrene (Polystylene) as a substrate material. The reason for using the polystyrene substrate is that the protein is very easy to attach and the adhesion efficiency is about 50%.

The substrate in the present invention mainly uses a polystyrene substrate, but in addition to this, polyvinyl chloride or polypropylene may also be used depending on the nature of the protein attached to the substrate.

The substrate described above is used after the following process.

Eight grooves having a width of 1 mm, a length of 2 mm, and a depth of 1.5 mm are formed side by side at 1 mm intervals on a polystyrene substrate having a width of 1.5 cm and a length of 1.5 cm. When the protein to be analyzed is integrated in each groove with a diameter of approximately 400 nm and an interval of 500 nm, it is possible to accumulate 10 kinds of proteins in a length of 1 cm. That is, about 80 kinds of proteins can be integrated in one substrate.

It is known that the amount of bone turnover related proteins in the blood or urine of osteoporosis patients is higher than normal. Therefore, the presence and progression of osteoporosis can be diagnosed early by integrating an antibody against such a protein onto a small substrate and analyzing the content of the marker protein in an analyte prepared from the blood or urine of the patient.

Proteins used as biochemical markers for diagnosing osteoporosis in the present invention are as follows.

1. Bone-specific alkaline phosphatase, osteopontin, osteocalcin, calcitonin, osteoclassogenase inhibitors, TGF β3, and calvindine, proteins involved in the bone formation process of bone growth and remodeling through osteoblasts.

2. Entelopeptides, citopeptides, free pyridinolin, and free deoxypyridinolin, which are involved in the bone resorption process that breaks down minerals in the bone to maintain a constant calcium ion concentration in the blood through osteoclasts.

Hereinafter, the present invention will be described in detail with reference to preferred embodiments of the present invention.

Example 1 Preparation of Protein Chip

Material of substrate: The substrate was produced using polystyrene.

Protein substrate attachment: Each antibody protein is mixed with 50mM sodium bicarbonate (pH9.6), Tris-Cl 20mM (pH8.5), phosphate buffer 10mM (pH7.2) and aliquoted to the substrate and allowed to stand at room temperature for 2 hours. Reaction was fixed. After the reaction, the substrate was washed two times or more with distilled water, and then a blocking buffer was dispensed, and then reacted at room temperature for 10 minutes to prepare protein chips.

The blocking buffer is phosphate buffer containing 0.1% bovine plasma albumin (BSA), 0.05% Tween 20, and the above-described antibody protein was prepared as shown in the table below.

<Table 1> Cover antibody

Monoclonal antibodies purchase Production request Bone-Specific Alkali-Phosphatase Fitzgerald IND, USA Osteopontin Chemicon, USA Osteocalcin DSL, USA TGF <beta> 3 R & D, USA Osteoclassogenics Suppressors Abcam, USA Calcitonin Fitzgerald IND, USA Calvin Dean Abcam, USA Entelopeptides Abcam, USA Citelopeptide Abcam, USA Free pyridinolin Metra Biosystems, USA Free deoxypyridinolin Metra Biosystems, USA

Example 2 Reaction with Analyte

The analyte (blood or urine of the patient and the normal person) was diluted in the blocking buffer and dispensed to the protein chip of Example 1, and reacted at room temperature for 2 hours. After the reaction, the protein chips were washed three times with distilled water, and the blocking buffer was dispensed and reacted at room temperature for 10 minutes.

<Example 3> Reaction result analysis

Labeled antibody (Antybody-enzyme conjugate) to the protein chip of Example 2 was mixed with the blocking buffer and dispensed and reacted for 2 hours at room temperature. After completion of the reaction, the protein chips were washed three times with distilled water, and the blocking buffer was dispensed and reacted at room temperature for 10 minutes. Then, para-nitrophenyl phosphate was added as a substrate and reacted at room temperature for 1 hour. .

After the reaction, the protein chip was analyzed by measuring absorbance at a specific wavelength range through an analyzer. Absorbance measurement results for the labeled antibody are shown in the table below. The graphs of the measurement results are shown in the accompanying drawings. (FIGS. 1, 2, 3, 4, 5, 6, 7, and 8).

Table 2 Bone Specific Alkaline Phosphatase Sample Experiment

sample Dilution factor Absorbance Sample concentration (U / L) result One 1: 2 0.37 10.1 n / d 1: 3 0.56 23.0 2 1: 2 0.91 25.0 n / d 1: 3 0.34 14.2 3 1: 2 0.22 6.1 p / d 1: 3 0.14 5.7

* Reference Range: 6.9-26.4 (U / L)

<Table 3> Osteocalcin Sample Experiment

sample gender Dilution factor Absorbance Sample concentration (ng / ml) result One male 1: 5 0.31 8.9 n / d 1:10 0.11 6.1 2 female 1: 5 0.30 8.7 n / d 1:10 1.56 9.0 3 female 1: 2 0.28 3.2 p / d 1: 4 0.12 2.8

* Reference range: Female 3.7-10.0 ng / ml

Male 3.4-9.1 ng / ml

<Table 4> Calcitonin Sample Experiment

sample Dilution factor Absorbance Sample concentration (pg / ml) result One 1: 2 0.20 41 n / d 1: 3 0.20 65 2 1: 2 0.21 52 n / d 1: 3 0.21 79 3 1: 2 0.18 18 p / d 1: 3 0.17 12

* Reference range:> 20 pg / ml

Table 5 Free Dioxypyridinolin Sample Experiments

sample gender Dilution factor Absorbance Sample concentration (nmol / l) result One male 1: 5 1.26 3.3 n / d 1:10 1.43 4.1 2 female 1: 5 1.02 5.1 n / d 1:10 1.23 7.0 3 female 1: 5 0.66 8.0 p / d 1:10 1.04 9.8

* Reference range: Female 3.0-7.4 (nM / mM)

Male 2.3-5.4 (nM / mM)

<Table 6> Pyridinolin Sample Experiment

sample gender Dilution factor Absorbance Sample concentration (nmol / l) result One female 1: 5 0.40 1.3 n / d 1:10 0.21 1.39 2 female 1: 5 0.45 1.47 n / d 1:10 0.27 1.8 3 female 1: 5 0.92 3.0 p / d 1:10 0.53 3.5

* Reference range: Female 1.29-1.81 (nmol / l)

Male 1.21-1.97 (nmol / l)

Table 7 Cyclopeptide Sample Experiment

sample gender Dilution factor Absorbance Sample concentration (ng / ml) result One female 1: 2 0.50 78 n / d 1: 3 0.31 73 2 male 1: 2 0.60 95 n / d 1: 3 0.51 121 3 female 1: 3 0.68 53.7 p / d 1: 2 0.97 76.5

* Reference range: Female 69-147 (ng / ml)

Male 76-163 (ng / ml)

<Table 8> Entelopeptide Sample Experiments

sample gender Dilution factor Absorbance Sample concentration (ug / mmol) result One female 1: 2 0.67 231 n / d 1: 3 0.77 398 2 male 1: 2 0.70 243 n / d 1: 3 0.74 385 3 female 1: 3 1.29 446 p / d 1: 2 0.92 478

* Reference range: 215-415 (ug / mmol)

<Table 9> TGF β3 Sample Experiment

sample Dilution factor Absorbance Sample concentration (ng / l) result One 1: 2 0.78 2.1 n / d 1: 3 1.11 2.0 2 1: 2 0.55 1.5 n / d 1: 3 0.69 2.8 3 1: 2 1.85 5.0 p / d 1: 3 3.32 9.0

* Reference range: 1-3 ng / ml

-Interpretation and diagnosis of reaction results

After preparing a standard curve measuring the degree to which each labeled antibody reacts with the reference antigen by concentration, the absorbance by the reaction between the antigen in the analyte and the antibody attached to the substrate is used to measure the absorbance in the sample. Calculate the concentration of the antigen.

Diagnosis is made by comparing the concentration of antigen in the analyte calculated by the method described above with the physiological concentrations of normal and patient.

As described above, the present invention provides a protein chip incorporating various types of proteins related to a bone conversion process including bone formation and bone resorption on a small substrate to diagnose the presence and progression of osteoporosis at an early stage. can do.

In addition, the present invention enables a faster and more accurate diagnosis by using various types of proteins as biochemical markers, and provides a protein chip that is more compact and has a higher fixing efficiency than conventional protein chips.

Claims (3)

A protein chip for diagnosing osteoporosis, characterized by integrating antibodies against proteins involved in the bone conversion process on a small substrate. The method according to claim 1, wherein the osteospecific alkaline phosphatase, osteopontin, osteocalcin, calcitonin, osteocytogenesis inhibitor, TGF β3, calvindine, entelopeptide, cetopeptide, free pyridinolin, free deoxypyri A protein chip comprising an antibody selected from and integrated with one or more proteins of dinoline. The protein chip according to claim 1 or 2, wherein the substrate is made of polystyrene or polyvinylchloride, polypropylene, glass, or the like.