KR20020030008A - Protein chip for diagnosis of osteoporosis - Google Patents

Abstract

PURPOSE: Provided is a protein chip for early diagnosis of osteoporosis including its existence, progressive state and its cause by attaching a monoclonal antibody for the various bone metabolism indicators related to the bone turnover process such as bone formation and bone resorption. CONSTITUTION: The measurement of each bone metabolism indicators comprises: attaching protein chips of monoclonal antibody on a base plate which is made of polystyrene, polyvinylchloride, polypropylene or glass; preparing the standard concentration curve by reacting antigen in various concentration with the above monoclonal antibody such as bone specific alkaline phosphatase, osteopontin, osteocalcin, calcitonin, osteoclastogenesis inhibitory factor, osteoprotegerin, TGF B3, calvindin, N-telopeptides, C -telopeptides, free pyridinoline and free deoxypyridinoline; and carrying out the concentration measurement of the bone metabolism indicators in th test sample for diagnosis.

Description

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

The present invention relates to a protein chip for early diagnosis of osteoporosis using various types of bone metabolism indicator.

Osteoporosis is a disease commonly found in both men and women, especially in men and women around the age of 50 and after menopause. It is mainly seen in women, and it is estimated that about 200 million people worldwide suffer from osteoporosis.

Osteoporosis is a symptomatic (Silent disease) with no signs or symptoms that can be detected early and found, and 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.

As an alternative method for diagnosing osteoporosis, a lot of researches are being conducted on the diagnosis of osteoporosis using biochemical markers.

In general, the bone of the human body undergoes a bone turnover process that repeats the bone formaion (Bone formaion) and bone resorption (Bone turnover).

Bone formation is the process of growing or remodeling bone through osteoblasts. Bone Specific alkaline phosphatase, Osteopontin, Osteocalcin, Calitonin, and Osteocle Proteins such as Osteoclastogenesis inhibitory factor (also called osteoprotegerin), 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, that is, bone metabolism indicators.

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.

On the other hand, protein chips, which have been actively researched and developed recently, are attracting attention as a technology for finding diseases of patients by accumulating proteins related to various diseases on a small substrate and reacting with body fluids or urine. However, if the cause is related to several types of protein, such as osteoporosis, it is impossible to make an accurate diagnosis only by detecting any one of them, and if the cause is not detected, proper treatment is more effective. There is a problem that becomes difficult.

In order to solve the above problems, the present invention provides a protein chip in which a monoclonal antibody is attached on the same substrate to various bone metabolism indices related to the bone conversion process including bone formation and bone resorption, and the presence of osteoporosis and The diagnosis of the causative agent is to be diagnosed accurately and quickly.

1 is a perspective view showing a protein chip according to the present invention.

Figure 2 is a graph showing the standard concentration curve for bone specific alkaline phosphatase according to the present invention.

Figure 3 is a graph showing the standard concentration curve for osteocalcin according to the present invention.

Figure 4 is a graph showing the standard concentration curve for calcitonin according to the present invention.

5 is a graph showing a standard concentration curve for free deoxypyridinolin according to the present invention.

Figure 6 is a graph showing the standard concentration curve for the free pyridinolin according to the present invention.

Figure 7 is a graph showing the standard concentration curve for the citelopeptide according to the present invention.

Figure 8 is a graph showing the standard concentration curve for the enteropeptide according to the present invention.

9 is a graph showing a standard concentration curve for TGF β3 according to the present invention.

10 is a graph showing a standard concentration curve for osteopontin according to the present invention.

FIG. 11 is a graph showing a standard concentration curve for the Osteoclestogenesis inhibitory factor (Osteoffotegerin) according to the present invention. FIG.

-Brief description of the symbols in the drawings

M1 to M8: Spot

R1 or R2: reference sample

Cc1 to Cc5: Standard Well

Tc1 to Tc3: powder wells

The present invention relates to a protein chip for accurately and quickly diagnosing osteoporosis using various kinds of bone metabolism indicators on the same substrate.

The protein chip of the present invention is characterized by attaching monoclonal antibodies to various kinds of bone metabolism indices related to the bone conversion process including bone formation and bone resorption on the same substrate.

In addition, the protein chip of the present invention is a monoclonal antibody for measuring the concentration of various types of bone metabolism indicator contained in the sample on the same substrate, and a monoclonal antibody for preparing a standard concentration curve according to the concentration change of a specific antigen into each group Formed and attached together.

Using the protein chip of the present invention, the concentration of antigen contained in the blood or urine of a patient, that is, various kinds of bone metabolism indicators is simultaneously measured to comprehensively and accurately diagnose the presence and progression of osteoporosis.

Hereinafter, the present invention will be described in detail.

The substrate of the protein chip of the present invention is preferably used as polystyrene (Polystylene) as a substrate material. The reason for using the polystyrene substrate is that protein adhesion is very easy and the adhesion efficiency is about 50%.

In the present invention, a polystyrene substrate is mainly used as a substrate, but in addition to this, polyvinyl chloride, polypropylene, etc. may be used according to the properties of proteins attached to the substrate, and avidin is used on a conventional glass plate. It is also possible to attach the desired protein.

A protein consisting of a B-domain (Protein A) of the protein (hereinafter referred to as "B-ProA") is dispensed on the substrate and coated or spherical proteins are BSA (Bovine serum albumin) and OVA (Ovalbumin). And after dispensing KLH (Keyhole limpet hemocyanine), GAD (Gluaraldehyde), GMBS (N- [g-Maleimidobutyryloxy] succinimide seter), Sulfo-GMBS, etc. as a binder (Cross-reagent) of the monoclonal antibody on the substrate Adhesion efficiency and orientation can be improved.

Protein A (Protein A) is known to have a specific high binding force with the antibody, the binding sites of the protein A to bind the antibody E, D, A, B and C-domain (Domain) of these areas B Domains have been reported to have high theoretical potential as binding sites with antibodies.

The monoclonal antibodies to various kinds of bone metabolism indicators are attached to the substrate treated with B-ProA or spherical protein, and bone metabolism indicators used in the present invention are as follows.

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

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.

Monoclonal antibodies can be prepared by various methods from the bone metabolism index as described above. For example, the hybridoma technique by Koehler and Milstein ( Nature 256: 495-497 (1975), the B-cell hybridoma technique (Kosber et al. Immuno Today) 4:72 (1983); Cote et al. Proc Natl Acad Sci 80: 2026-2030 (1983) and EBV-hybridoma technique (Cole et al. Monoclonal Antibidies and Canser Therapy , Alan R. Liss Inc, New York NY , pp 77-96 (1985) and the like.

In the present invention, monoclonal antibodies were prepared as shown in the following table.

Metabolic index purchase Production request Bone-Specific Alkali-Phosphatase Fitzgerland IND., USA Osteopontin Chemicon, USA Osteocalcin DSL, USA TGF β3 R & D, USA Osteoclestogenesis Inhibitor Abcam, USA Calcitonin Fitzgerland IND., USA Calvin Dean Abcam, USA Entelopeptides Abcam, USA Citelopeptide Abcam, USA Free pyridinolin Metra Biosystems, USA Free deoxypyridinolin Metra Biosystems, USA

Each monoclonal antibody as described above is attached to a certain concentration (Spot: M1 to M8) on the substrate at a certain concentration, but the same multiple wells (Well: Cc1 to the same group with several monoclonal antibodies attached as a group) Cc5, Tc1 to Tc3).

Some of the wells (Cc1 to Cc5) are reacted with monoclonal antibodies attached to each spot while varying the concentration of antigen for a particular monoclonal antibody from low to high concentrations. Subsequently, the Toluene PAB-biotin conjugate and Streptavidin-PE (R-phycoerithrin) conjugate were reacted in order to measure the fluorescence intensity according to the reaction degree, and a standard concentration curve according to the change of antigen concentration in the concentration range of the specific antigen was prepared.

Samples to be analyzed are reacted with the remaining wells Tc1 to Tc3, and the concentrations of specific antigens contained in the sample are calculated using the color development and the standard concentration curve prepared in the same manner as described above.

Hereinafter, the present invention will be described in detail with reference to the accompanying drawings and a preferred embodiment of the present invention.

Example 1 Preparation of Protein Chip

The substrate was made of polystyrene and coated with B-ProA or spherical proteins BSA (Bovine serum albumin), OVA (Ovalbumin), and KLH (Keyhole limpet hemocyanine) in order to increase the adhesion efficiency and orientation of the antibody on the substrate. Antibodies were immediately attached onto B-ProA-treated substrates, and GAD (Gluaraldehyde), GMBS (N- [g-Maleimidobutyryloxy] succinimide seter), Sulfo-GMBS, etc., were used as cross-reagents on spherical protein-coated substrates. Through the binding to the antibody.

As shown in FIG. 1, M1 to M8 represent analytes to be detected and quantified, that is, spots of monoclonal antibodies to respective bone metabolism indicators. R1 or R2 denotes a portion attaching a reference sample for determining whether the protein chip is operated. Cc1 to Cc5 is to obtain the standard concentration curve of each bone metabolism indicator, and reacts with monoclonal antibodies attached to each spot by changing the concentration of antigens for specific monoclonal antibodies from Cc1 to Cc5 from low to high concentrations. By measuring the fluorescence intensity according to the degree, a standard concentration curve according to the change of the antigen concentration in the concentration range of a specific antigen was prepared. Similarly, the standard concentration curves for 10 bone metabolism indices from M1 to M10 can be obtained simultaneously.

Tc1 to Tc3 are for measuring the concentration of specific antigens contained in the sample to be analyzed. Like Cc1 to Cc5 described above, the antibody to each bone metabolism indicator reacts with the sample to be analyzed and the fluorescence intensity according to the reaction degree. The concentration of the specific antigen contained in the sample was calculated using the standard concentration curve prepared above.

Osteoporosis is diagnosed by identifying the comprehensive information on the causes and progression of osteoporosis by comparing the concentrations of bone metabolism indicators contained in the sample calculated as described above with those of normal individuals determined through clinical experiments. do.

In the present embodiment and drawings, three samples (Tc1 to Tc3) were analyzed using eight bone metabolism indicators (M1 to M10), but more types of protein chips using bone metabolism indicators and samples will be possible.

Experimental Example 1 Measurement of Response Result of Each Bone Metabolism Index

20 nL of the monoclonal antibody for each bone metabolism index was dispensed to the spots of M1 to M8 on the substrate and then incubated for 3 hours at room temperature. After culturing, the protein chip was washed with PBST (Phosphate buffered saline-0.08% tween20) for 15 minutes, reacted with 5% BSA-PBS (Blocking agent) for 16 hours, and then washed with PBST for 15 minutes.

Dispense 180 ul of bone metabolism markers to be used as antigens for specific monoclonal antibodies at the positions of Cc1 to Cc5 on the protein chip substrate, and analytes (blood or urine of patients and normal persons) at the positions of Tc1 to Tc3. After diluting at a predetermined rate, the cells were incubated for 2 hours, and washed three times with PBST for 5 minutes.

After 200ul of toluene PAb-biotin conjugate was added and incubated for 1 hour, washed three times with PBST for 5 minutes, and then reacted for 15 minutes with Streptavidin-PE (R-phycoerithrin) conjugate, followed by 15 minutes with PBST.

After drying the protein chip in the dark room, the reaction result was calculated by measuring the fluorescence intensity.

Experimental Example 2 Analysis of Reaction Result

The degree of reaction for each concentration of the antigen reacted with each antibody in the well Cc1 to Cc5 on the protein chip substrate was measured, and a standard concentration curve according to the change of the antigen concentration in the concentration range of the specific antigen was prepared. The reaction degree for each concentration is shown in the table below, and the standard concentration curve is as shown in the accompanying drawings.

The reaction levels of analytes and monoclonal antibodies in the wells Tc1 to Tc3 on the protein chip substrate were measured, and the concentrations of specific antigens, ie, bone metabolism indicators, included in the sample were measured using the respective standard concentration curves. The results are shown in the table below.

Bone Specific Alkaline Phosphatase Standard Concentration Curve (FIG. 2) Concentration (ng / ml) 0 4 8 16 32 Intensity (× 10⁴) 540 2586 5862 10468 21578

Y = 660.65X + 279 (r = 0.999)

Bone Specific Alkaline Phosphatase Sample Analysis sample Dilution factor Intensity (× 10⁴) Concentration (ng / ml) result One 1: 2 4044.7 5.7 n / d 1: 3 4309.0 6.1 n / d 2 1: 2 3120.0 4.3 n / d 1: 3 3978.6 5.6 n / d 3 1: 2 19371.8 28.9 p / d 1: 3 23137.5 34.6 p / d

Reference Range: 6.9-26.4 (U / L)

n / d (Normal Detection): means that the result is within the normal range (the same applies below)

p / d (Patient Detection): This means that the result is outside the normal range (the same applies below).

Osteocalcin Standard Concentration Curve (Figure 3) Concentration (ng / ml) 0 4 8 16 32 Intensity (× 10⁴) 540 1567 3154 6852 16850

Y = 519.96X-446.95 (r = 0.982)

Osteocalcin Sample Analysis sample gender Dilution factor Intensity (× 10⁴) Concentration (ng / ml) result One male 1: 2 4180.7 8.9 n / d 1: 3 4724.8 6.1 n / d 2 female 1: 2 4076.7 8.7 n / d 1: 3 4232.7 9.0 n / d 3 female 1: 2 1216.9 3.2 p / d 1: 3 1008.9 2.8 p / d

Reference Range: Female 3.7-10.0 (ng / ml)

Male 3.4-9.1 (ng / ml)

Calcitonin Standard Concentration Curve (Figure 4) Concentration (ng / ml) 0 4 8 16 32 Intensity (× 10⁴) 540 2150 4521 9465 16850

Y = 520.46X + 459.65 (r = 0.998)

Calcitonin Sample Analysis sample Dilution factor Intensity (× 10⁴) Concentration (ng / ml) result One 1: 2 21798.5 41 n / d 1: 3 34289.6 65 n / d 2 1: 2 27523.6 52 n / d 1: 3 41576.0 79 n / d 3 1: 2 9827.9 18 p / d 1: 3 6705.2 12 p / d

Refernce Range:> 20 (pg / ml)

Free deoxypyridinolin standard concentration curve (FIG. 5) Concentration (nmol / l) 0 4 8 16 32 Intensity (× 10⁴) 540 1540 3465 7562 15422

Y = 478.17X-32.225 (r = 0.998)

Free Dioxypyridinoline Sample Analysis sample gender Dilution factor Intensity (× 10⁴) Concentration (nmol / l) result One male 1: 5 8574.8 3.5 n / d 1:10 4749.5 3.6 n / d 2 female 1: 5 3793.1 5.4 n / d 1:10 1880.5 5.1 n / d 3 female 1: 5 13356.5 8.6 p / d 1:10 11443.9 7.9 p / d

Refernce Range: Female 3.0-7.4 (nM / mM)

Male 2.3-5.4 (nM / mM)

Free Pyridinoline Standard Concentration Curve (Figure 6) Concentration (nmol / l) 0 2 4 8 16 Intensity (× 10⁴) 450 2560 5004 10456 24561

Y = 1522.3X-527.83 (r = 0.996)

Free pyridinolin sample analysis sample gender Dilution factor Intensity (× 10⁴) Concentration (nmol / l) result One female 1: 5 26873.6 1.3 n / d 1:10 14695.2 1.39 n / d 2 female 1: 5 11650.6 1.47 n / d 1:10 5561.4 1.8 n / d 3 female 1: 5 42096.6 3.0 p / d 1:10 36007.4 3.5 p / d

Refernce Range: Female 1.29-1.81 (nmol / l)

Male 1.21-1.97 (nmol / l)

Cyclopeptide Standard Concentration Curve (FIG. 7) Concentration (nmol / l) 0 40 80 160 320 Intensity (× 10⁴) 456 984 1951 3897 8123

Y = 24.516 X + 140.33 (r = 0.998)

Cyclopeptide Sample Analysis sample gender Dilution factor Intensity (× 10⁴) Concentration (nmol / l) result One female 1: 2 2052.6 78 n / d 1: 3 1930.0 73 n / d 2 male 1: 2 2469.4 95 n / d 1: 3 3106.8 121 n / d 3 female 1: 2 3989.3 157 p / d 1: 3 3719.7 146 p / d

Refernce Range: Female 69-147 (ng / ml)

Male 76-163 (ng / ml)

Entelopeptide standard concentration curve (FIG. 8) Concentration (ng / ml) 0 100 200 400 800 Intensity (× 10⁴) 251 543 1045 2547 5412

Y = 6.6808X-44.625 (r = 0.995)

Entelopeptide Sample Analysis sample Dilution factor Intensity (× 10⁴) Concentration (ng / ml) result One 1: 2 1498.6 231 n / d 1: 3 1598.9 246 n / d 2 1: 2 1578.8 243 n / d 1: 3 1512.0 233 n / d 3 1: 2 2935.0 446 p / d 1: 3 3448.8 478 p / d

Refernce Range: 215-415 (ug / mol)

TGF β3 standard concentration curve (FIG. 9) Concentration (ng / ml) 0 2 4 8 16 Intensity (× 10⁴) 251 456 912 1799 3568

Y = 213.28X + 117.55 (r = 0.998)

TGF β3 Sample Analysis sample Dilution factor Intensity (× 10⁴) Concentration (ng / ml) result One 1: 2 565.4 2.1 n / d 1: 3 189.4 2.0 n / d 2 1: 2 177.1 1.5 n / d 1: 3 209.0 2.8 n / d 3 1: 2 262.9 5.0 p / d 1: 3 361.0 9.0 p / d

Refernce Range: 1-3 (ng / ml)

Osteopontin standard concentration curve (Fig. 10) Concentration (ng / ml) 0 5 10 20 40 Intensity (× 10⁴) 660 1500 2344 4678 8962

Y = 210.82X + 466.5 (r = 0.999)

Osteopontin Sample Analysis sample Dilution factor Intensity (× 10⁴) Concentration (ng / ml) result One 1: 2 327.1 0 n / d 1: 3 207.5 0 n / d 2 1: 2 431.4 0 n / d 1: 3 421.9 0 n / d 3 1: 2 3656.6 12.3 p / d 1: 3 2933.1 1.7 p / d

Refernce Range: No Detection

Osteoclestogenesis Inhibitor (Osteoffertegerin) Standard Concentration Curve (FIG. 11) Concentration (ng / ml) 0 2 4 8 20 Intensity (× 10⁴) 1100 2450 5862 10468 21578

Y = 1035.4X + 1251.2 (r = 0.996)

Osteoclestogenesis Inhibitor (Osteoffotegerin) Sample Analysis sample Dilution factor Intensity (× 10⁴) Concentration (ng / ml) result One 1: 2 2597.2 1.3 n / d 1: 3 2390.1 1.1 n / d 2 1: 2 2079.5 0.8 n / d 1: 3 2390.1 1.1 n / d 3 1: 2 3632.6 2.3 p / d 1: 3 4253.9 2.9 p / d

Refernce Range:> 1.8 ng / ml

As described above, the protein chip of the present invention attaches monoclonal antibodies to various kinds of bone metabolism indices related to the bone turnover process including bone formation and bone resorption on the same substrate to determine the presence, progression, and causes of osteoporosis. Comprehensive information on the ability to accurately diagnose osteoporosis is effective.

In addition, the protein chip of the present invention is more accurate by minimizing the error from the experiment by arranging monoclonal antibodies to simultaneously prepare a standard concentration curve and measure the concentration of various kinds of bone metabolism indicator included in the sample on the same substrate. Has the effect of diagnosing osteoporosis quickly.

Claims (5)

Standard concentration curve of monoclonal antibody and specific antigen for concentration measurement of different bone metabolism indicators included in the sample on the same substrate. Osteoporosis diagnostic protein chip characterized in that the monoclonal antibody attached together. The method of claim 1, wherein the monoclonal antibody is a bone specific alkaline phosphatase, osteopontin, osteocalcin, calcitonin, osteocytogenesis inhibitor, TGF β3, calvindine, enteropeptide, cetopeptide, free pyridinolin or Protein chip, characterized in that the monoclonal antibody against free deoxypyridinoline. 3. The protein chip according to claim 2, wherein the substrate is made of polystyrene, polyvinyl chloride, polypropylene, or glass. A standard concentration curve is prepared by reacting antigens for each monoclonal antibody with concentration using a protein chip according to claim 1 to 3, and each bone metabolism index included in the sample using the standard concentration curve. How to calculate the concentration of. Method for detecting bone metabolism index using the protein chip of claim 1 to claim 3.