CN101221171A - Liquid phase chip used for detecting bone metabolism biochemical marker and its preparing method - Google Patents

Abstract

The invention discloses a liquid phase chip kit which is used for the parallel detection of a plurality of bone metabolism biochemical markers, and the invention mainly includes: coated microspheres, detection antibodies which are respectively labelled by biotin and streptavidin phycoerythrin. The liquid phase chip kit which is used for the parallel detection of a plurality of bone metabolism biochemical markers and is provided by the invention has the advantages of high detection efficiency, fewer required samples, strong specificity, high sensitivity, and so on. At the same time, all the bone metabolism biochemical markers can be combined freely, and the usage is convenient. At the same time, as all the reactions are positioned in the liquid phase environment, the invention is more conductive to the maintenance of the native conformation of protein, the reaction of a probe and the object to be detected is faster and more complete, while the detection sensitivity and the linear range are both greatly improved.

Description

Be used for liquid-phase chip of bone metabolism biochemical marker detection and preparation method thereof

Technical field

The invention belongs to the medicine bioengineering class, relate to liquid-phase chip that is used for multinomial bone metabolism biochemical marker parallel detection and preparation method thereof specifically.

Background technology

Osteoporosis (OP) is to destroy with the minimizing of bone amount, bone tissue microstructure to cause bone fragility to increase, and the metabolic disease that easy generation is fractured.Osteoporosis has leapt to common disease, frequently-occurring disease the 7th, and the inland of China total prevalence rate is 1 ten two four, and the ill ratio of elderly population surpasses over half, and the incidence of wherein fracturing is near 1/3rd.Data according to the academic conference of global osteoporosis in 2006 show: osteoporosis has reached the degree that spreads in China, in the period of past 30, the sufferers of osteoporosis face of China has increased by 300%, and the annual medical expense that is caused by 8,800 ten thousand patients needs 15,000,000,000 yuans at least.As a kind of serious chronic disease, osteoporosis not only influences patients ' life quality, and can cause the fracture at positions such as backbone, four limbs, is the common cause that elderly population causes death, disables.Simultaneously, osteoporosis medical expense height causes huge medical resource to take by its fracture that causes, brings heavy burden to society.Because osteoporosis can prevent, and can postpone it and develop, therefore significant to its early diagnosis and prediction.

The detection of bone density measurement (BMD), bone tissue morphometric and stereologic analysis and bone metabolism biochemical marker is three kinds of important detection meanss that the evaluation bone that develops over nearly 30 years changes.X-ray film is the method for detection bone density the earliest.It is to come for Diagnosis of osteoporosis provides foundation by the observation to the sclerotin inner constructional form, but its susceptibility is low, when bone loss reaches 30%-50% often, just can show on the X-ray film, so it can not the early diagnosis osteoporosis.The quantitative measurement technology of bone density comprises single photon absorptiometry (SPA).Two-photon absorption method (DPA), dual energy X-ray absorptiometry (DXA), QCT technology (QCT) and radiation absorption method (RA).Wherein, DEXA is the osteoporotic main surveying instrument of current diagnosis, but its cost an arm and a leg, instrument is bulky, operation inconvenience, it is limited to survey object, is not suitable in the community in urban areas, basic medical unit such as vast rural area and frontier area carries out the diagnosis and the control of senile osteoporosis disease.Bone morphometry method can provide the situation of instant bone structure and bone content, but owing to belong to traumatic inspection, needs to get a bone on one's body patient and analyze, many difficult acceptance of patient.Method with respect to traditional research bone metabolism situation comprises histology, iconography method, the detection of bone metabolism biochemical marker is except that easy, quick, nothing are created, more can in time dynamically reflect ongoing bone reconstruction situation, early diagnosis, prediction bone loss rate and monitoring curative effect of medication etc. to metabolic bone disease all have extremely important clinical meaning.

Think that at present detecting bone metabolism biochemical marker has following meaning: (1) prediction bone loss rate and fracture incidence: studies confirm that the bone metabolism index, especially the mensuration of bone resorption marker is the useful means of fracture risk after the prediction menopause.12 years long-term longitudinal research finds to have high turnover person by detecting blood BGP level, and its vertebral fracture incidence obviously raises.Crosslinked C-end peptide (CTX) of same type i collagen and urine collagen Deoxypyridinoline (DPD) basic value are than the obvious rising person of control group, and its hip fracture rate is obviously increase also.If bone densitometry and bone metabolism mark are measured binding analysis, then separately with the obvious prediction dynamics (predictive power) that improves fracture risk of BMD.Measure multinomial bone metabolism biochemical marker simultaneously and measure a kind of biochemical marker more merely, for judging that the bone loss rate is more meaningful.(2) be used for the monitoring of curative effect of medication: this is a clinical application the most widely at present.It is similar with other chronic diseases of control to prevent and treat osteoporosis, and its curative effect is judged usually needs the long time, and changes of bone mineral density is generally at least more than half a year.And bone metabolism biochemical marker can marked change occur in early stage (1～3 month) of medication.Short-term bone metabolism mark reduces the rising significant correlation with long-term (after 2 years) hip and centrum bone density.The clinical research of most anti-bone resorption treatments shows that changes of bone mineral density is no more than 10% after treating 1 year, and fracture risk can reduce about 50% usually.Show that such as the clinical research to Raloxifene this drug therapy is after 1 year, lumbar spine bmd increases by 2%, and the danger of vertebral fracture reduces by 68%.Changes of bone mineral density only can be explained and reduce 15% of curative effect of fracture, illustrates that the effect of anti-bone resorption treatment is the raising that possible be not limited only to bone density.Lower such as treatment back bone turnover rate, thus changed bones microstructure, mineralization degree, collagen quality and reduced the accumulation of micro-damage, and then improved the quality of bone.The variation of observing treatment back bone metabolism biochemical marker helps to judge the curative effect of this medicine.Allan sodium phosphate treatment back urine NTX and CTX can reduce by 70%, and the corresponding bone metabolism mark in Raloxifene treatment back can reduce about 40%, and hormone replacement therapy can make the bone conversion reduce by 40%～60% according to the difference of dosage.Observe above variation and can provide early stage instructive information for continuing to treat the strategy of taking.

Because osteoporotic control is a long-term process, need to improve the compliance of treatment.There are some researches show and have only 50% patient after the osteoporotic treatment of beginning, can adhere to more than 1 year.Transform biochemical marker by results of regular determination bone before and after treatment, observe the variation of the bone conversion after the treatment, can increase doctor and patient's confidence, improve the compliance of treatment.Simultaneously, responsive bone biochemical indicator and the BMD that detects reflection bone loss speed clinically measures the effective ways that use in conjunction is screening risk of fractures crowd.Owing to can monitor the bone metabolism situation in a short time, therefore the detection of the bone biochemical indicator in the treatment is to judge curative effect and adjust the simple and effective method of therapeutic scheme.

At present, the bone metabolism biomarker that generally uses clinically mainly comprises:

(1) reflect osteoplastic biochemical indicator:

1. bone alkaline phosphatase (Bone alkaline phosphatase, BALP): ALP and bone mineralising are closely related, because bone calcification is the most active in alkaline environment, the AL P that Gegenbaur's cell discharges can make the inorganic phosphate salt hydrolysis, thereby reduce pyrophosphate concentration, help the mineralising of bone.The topmost source of the TALP of serum is bone and liver, the key that detects bone ALP is that liver and bone ALP are separated, its method is more, as heat inactivation method, Chemical Inhibition Method, gel electrophoresis, the wheat germ coagulin precipitation method etc., these methods belong to indirect method mostly, and are unsatisfactory for the specificity effect that improves index, and use the BALP monoclonal antibody to carry out the double-antibody sandwich reaction, then can directly detect the BALP in the blood, and the specificity height.

2. (BGP): BGP is a kind of noncollagen protein matter of vitamin k-dependent to BGP for Osteocalcin, OSC or BoneGlaprotein.Present known BGP is synthetic with bone matrix and mineralization rate is relevant.Other experiment in vivo and vitro show that BGP also participates in the adjusting of bone resorption, and it can be raised well and activate the heavy absorptive cell of bone, and when BGP reduced, the osteoclast like cell was assembled minimizing.BGP can be used to monitor osteoporosis and judges other metabolic bone disease result of treatment as the osteoplastic specific index of straight reflection.Detect the method for BGP and use immunization method more. owing to also have the hydrolysis fragment of BGP in the blood, the detection technique of the anti-ox BGP polyclonal antibody that adopts can't be distinguished the hydrolysis fragment usually.And adopt the detection method of the monoclonal antibody of anti-people BGP can discern different fragments.Reflect that osteoplastic specificity obviously improves

3. I procollagen type propetide (PICP, PINP): type i collagen is when Gegenbaur's cell is synthetic, and at first synthetic is procollagen.Respectively have one to prolong peptide at the N-of procollagen end and C end, be called propetide.Behind the synthetic justacrine precollagen of Gegenbaur's cell, the peptide at two ends is cut off under the protease effect, forms ripe collagen.Because collagen is principal ingredient in the bone matrix, the speed of the synthetic type i collagen of bone is faster than its hetero-organization, so can think that PICP in the blood, PINP level can represent the ossein aggregate velocity.

(2) biochemical indicator of reflection bone resorption

1. the anti-tartaic acid phosphatase of blood plasma (Tartrate resistant acid phosphatase, TRAP5b): the acid phosphatase in the blood derives from multiple tissue, as bone, prostate, red blood cell and blood platelet etc., bone source property acid phosphatase can be resisted tartaric inhibition, thereby is referred to as anti-tartaic acid phosphatase.TRAP5b mainly asks the release that comes from osteoclast in the bone resorption process in the blood, and the activity of TRAP5b parallels with the bone resorption situation, is not subjected to meals, motion and age effects.

2. urinate collagen pyridinoline (PYD) and urine collagen Deoxypyridinoline (DPYD): in the ripe collagenous fibres of bone matrix, 3 lysines of α peptide chain end are cross-linked to form the pyridine crosslinking protein, the amino acid that this cross-linking agent is looked on the spiral part crosslink sites is hydroxylysine residue or lysine residue, if the former just is pyridinoline (PYD), the latter then claims Deoxypyridinoline (DPYD).

3. the crosslinked terminal peptide of type i collagen: crosslinked N-end peptide of type i collagen and C-end peptide (NTX, CTX) by 3-hydroxyl pyridine cross-linking agent with 2 adjacent tropocollagen molecules separately 1 peptide chain of N-end or C-end link to each other with another tropocollagen molecule spiral place that adjoins and form.The multinomial urine NTX and CTX and bone mineral density (BMD) of studies confirm that is remarkable negative correlation, is the special and responsive index of reflection bone resorption.

The laboratory diagnosis technology development in recent years of reflection bone metabolism mark is rapid, accuracy and degree of accuracy improve constantly, from the radioimmunology to the euzymelinked immunosorbent assay (ELISA) again to the electrochemiluminescence immunization that can detect automatically, the bone metabolism index that can detect automatically becomes increasingly abundant, technology is perfect day by day, from be used for research be applied to gradually clinical, for the observation of the diagnosis of bone metabolic disease, monitoring, result of treatment provides many valuable information.Yet the each test of these methods can only obtain the concentration of a mark.

Summary of the invention

The technical issues that need to address of the present invention provide a kind of liquid-phase chip that is used for multinomial bone metabolism biochemical marker parallel detection, this liquid-phase chip is to bone alkaline phosphatase in the serum (BALP), BGP (BGP), I procollagen type propetide (PICP, PINP), the anti-tartaic acid phosphatase of blood plasma (TRAP5b), six indexs of the crosslinked C-end peptide (CTX) of type i collagen, and the detection of running simultaneously of urinating collagen pyridinoline (PYD) in the urine and urinating collagen Deoxypyridinoline (DPYD), the crosslinked N-end peptide of type i collagen (NTX).

The technical scheme that solves the problems of the technologies described above is as follows:

A kind of liquid-phase chip that is used for multinomial bone metabolism biochemical marker parallel detection mainly includes:

1) wraps by microballoon: contain and wrap respectively by the microballoon of bone alkaline phosphatase (BALP) capture antibody, bag is by the microballoon of BGP (BGP) capture antibody, bag is by the microballoon of I procollagen type N-end propetide (PINP) capture antibody, the microballoon of I procollagen type C-end propetide (PICP) capture antibody, bag is by the microballoon of the anti-tartaic acid phosphatase of blood plasma (TRAP5b) capture antibody, bag is by the microballoon of the crosslinked C-end of type i collagen peptide (CTX) capture antibody, and/or contain bag by the microballoon of urine collagen pyridinoline (PYD) capture antibody, bag is urinated the microballoon of collagen Deoxypyridinoline (DPYD) capture antibody and is wrapped by the microballoon of the crosslinked N-end of type i collagen peptide (NTX) capture antibody, and above-mentioned microballoon has the different colours coding respectively;

2) biotin labeling detects antibody: the detection antibody of using biotin labeled bone alkaline phosphatase (BALP), BGP (BGP), I procollagen type N-end propetide (PINP), I procollagen type C-end propetide (PICP), I procollagen type N-end propetide (PINP) the anti-tartaic acid phosphatase of blood plasma (TRAP5b), the crosslinked C-end peptide of type i collagen (CTX) respectively; And/or use biotin labeled urine collagen pyridinoline (PYD), urine collagen Deoxypyridinoline (DPYD) and the crosslinked N-of type i collagen to hold the detection antibody of peptide respectively;

3) streptavidin phycoerythrin (SA-PE).

The capture antibody of bone alkaline phosphatase (BALP), BGP (BGP), I procollagen type propetide (PICP, PINP), the anti-tartaic acid phosphatase of blood plasma (TRAP5b), the crosslinked C-end peptide of type i collagen (CTX) wraps respectively by on the microballoon of different colours coding, uses the detection antibody of biotin labeling bone alkaline phosphatase (BALP), BGP (BGP), I procollagen type propetide (PICP, PINP), the anti-tartaic acid phosphatase of blood plasma (TRAP5b), the crosslinked C-end peptide of type i collagen (CTX) simultaneously respectively.Bag is mixed by six kinds of good microballoons, be suspended in liquid phase, add sample again, in suspension on the microballoon in the capture antibody of mark and the sample some epi-positions of relevant detection thing combine different in naturely, adding biotin labeled detection antibody afterwards combines with another epitope specificity of respective detection thing in the sample, the streptavidin that adds fluorescent material-phycoerythrin mark after reacting completely, because streptavidin phycoerythrin (SA-PE) can combine with the biotin high degree of specificity, therefore form six kinds of compounds of " microballoon-capture antibody+thing to be detected+biotin labeled detection antibody+SA-PE " in the reaction system at last, with the microballoon is carrier, detect by Luminex series liquid-phase chip analytical instrument, read the color numbers of microballoon and the fluorescent value of SA-PE.The microballoon color numbers can be distinguished test item, SA-PE fluorescent value and each detect substrate concentration and are proportionate, by measuring bone alkaline phosphatase (BALP), BGP (BGP), I procollagen type propetide (PICP, PINP), the anti-tartaic acid phosphatase of blood plasma (TRAP5b), the fluorescent value of crosslinked C-end peptide (CTX) standard items of type i collagen under variable concentrations, can obtain each and detect index standard items concentration-fluorescent value typical curve and typical curve equation.Test serum pattern detection gained fluorescent value substitution typical curve equation can be tried to achieve the amount of the crosslinked C-end peptide of bone alkaline phosphatase (BALP), BGP (BGP), I procollagen type propetide (PICP, PINP), the anti-tartaic acid phosphatase of blood plasma (TRAP5b), type i collagen (CTX) in the sample to be tested respectively.

Use the detection of running simultaneously that same principle can carry out urinating collagen pyridinoline (PYD) in the urine and urine collagen Deoxypyridinoline (DPYD), the crosslinked N-of type i collagen hold peptide (NTX).

Another technical issues that need to address of the present invention provide the above-mentioned preparation method who is used for the liquid-phase chip of multinomial bone metabolism biochemical marker parallel detection.

A kind of preparation is used for the method for the liquid-phase chip of multinomial bone metabolism biochemical marker parallel detection, mainly may further comprise the steps:

(1) corresponding capture antibody bag is by microballoon:

-with after the activation of 50 μ L microballoons, 〉=8000g is centrifugal;

-inhale and to abandon supernatant, microballoon is resuspended in the solution of MES of 230-280 μ L 50mM of pH5.0, the about 20s of vortex vibration, ultrasonic about 20s, after with microballoon in 〉=8000g, centrifugal 1-2min; Repeat this step;

-inhale and to abandon supernatant, microballoon is resuspended in the solution of MES of 100 μ L 50mM pH5.0 the about 20s of vortex vibration, ultrasonic about 20s;

-in the microballoon that suspends, add corresponding 0.8-1.2 μ g antibody, with the MES solution of the pH5.0 of 50mM cumulative volume is mended the L to 450-550 μ;

-usefulness vortex oscillator mixing, room temperature lucifuge vibration 1.5-2.5hr;

-coupling the speed of the microballoon behind the antibody with 〉=8000g, centrifugal 1-2min;

-inhale and abandon supernatant, microballoon is resuspended in the 500 μ L PBS-TBN solution the about 20s of vortex vibration, the about 20s of sonic oscillation;

-room temperature lucifuge vibration 30min; Again in 〉=8000g, centrifugal 1-2min;

-inhale and abandon supernatant, microballoon is resuspended in the 1mL PBS-TBN solution the about 20s of vortex vibration, ultrasonic about 20s; Microballoon 〉=8000g, centrifugal 1-2min; Repeat this step once;

-inhale and abandon supernatant, microballoon is resuspended in the 500-1000 μ L PBS-TBN solution;

-every kind bag is placed on 2-8 ℃ of lucifuge by good microballoon by luminex instrument counting and preserves separately, during use, needs equal proportion to mix according to detecting;

(2) every kind of biotin labeling that detects antibody:

-calculate the volume that the detection antibody-solutions is diluted to 1mg/ml according to detecting antibody concentration, be considered as target volume;

The NHS-Biotin reactant liquor of-configuration 10mg/ml;

In reaction tube, added respectively in-1: 100 in molar ratio and detect antibody and NHS-Biotin reactant liquor;

-add volume and be 1/10 the NaHCO of the pH8.9 of target volume ₃Solution;

The PBS of-adding pH7.4 mends to target volume;

-wrap aluminium foil, reaction tube is placed on the oscillator, lucifuge was hatched 3-5 hour in 25 ℃ of constant temperature ovens, and rotating speed is 800rpm-1000rpm;

-reactant liquor is gone in the dialysis cassette, dialysed overnight in the PBS damping fluid is to remove unreacted NHS-Biotin;

-every kind of as required that mark is good when using detection antibody equal proportion is mixed.

The step of described activation microballoon is as follows:

-usefulness vortex oscillator or ultrasound wave suspension microballoon;

-get the centrifugal 1-2min of 50 μ L microballoons;

-remove supernatant, microballoon is resuspended in the distilled water of 100 μ L, with vortex oscillator or the about 20s of ultrasound wave suspension microballoon, the centrifugal 1-2min of 〉=8000g;

-inhale and abandon supernatant, add the phosphate buffer (pH6.2) of 80 μ L, the about 20s of vortex vibration, the about 20s of ultrasound wave suspension microballoon;

-add 10 μ L 50mg/mL Sulfo-NHS, use vortex oscillator mixing lightly;

-add 10 μ L 50mg/mL EDC, use vortex oscillator mixing lightly;

-room temperature lucifuge leaves standstill 20min, every 10min vortex oscillator mixing lightly.

But of the present invention be used for multinomial bone metabolism biochemical marker parallel detection liquid-phase chip then primary first-order equation finish the detection by quantitative of multiple bone metabolism biochemical marker simultaneously, thereby to the bone metabolism state, comprise bone resorption and bone the conversion comprehensively estimate, reach accurate quick diagnosis osteoporosis, prediction risk of bone fracture degree and instruct the purpose of drug therapy.Multinomial bone metabolism biochemical marker parallel detection liquid-phase chip provided by the present invention has the detection efficiency height, and required sample size is few, high specificity, advantages such as sensitivity height.Simultaneously, but every bone metabolism biochemical marker independent assortment is easy to use.And because institute responds and all is in liquid phase environment, more help keeping the native conformation of protein, make the reaction of probe and detected material faster more complete, so detection sensitivity and the range of linearity all are greatly improved.In addition, preparation method of the present invention is simple, good stability, and the various technological parameters in its technical scheme such as the amount of microballoon and antibody, course of reaction etc. all draw on a large amount of experimental basis, are the parameter value of preparation process the best.

Description of drawings

Fig. 1 is the typical curve synoptic diagram that detects BGP;

Fig. 2 is the typical curve synoptic diagram that detects BALP;

Fig. 3 is the typical curve synoptic diagram that detects PINP;

Fig. 4 is the typical curve synoptic diagram that detects PICP;

Fig. 5 is the typical curve synoptic diagram that detects TRACP5b;

Fig. 6 is the typical curve synoptic diagram that detects CTX;

Fig. 7 is the typical curve synoptic diagram that detects PYD;

Fig. 8 is the typical curve synoptic diagram that detects DPYD;

Fig. 9 is the typical curve synoptic diagram that detects NTX.

Embodiment

Embodiment 1

The monoclonal antibody of capture antibody of the present invention for combining with bone alkaline phosphatase (BALP), BGP (BGP), I procollagen type N-end propetide (PINP), I procollagen type C-end propetide (PICP), the anti-tartaic acid phosphatase of blood plasma (TRAP5b), the crosslinked C-end of type i collagen peptide (CTX), urine collagen pyridinoline (PYD), urine collagen Deoxypyridinoline (DPYD) and the crosslinked N-end of type i collagen peptide (NTX) specificity respectively; Monoclonal antibody or the polyclonal antibody of described detection antibody for combining with bone alkaline phosphatase (BALP), BGP (BGP), I procollagen type N-end propetide (PINP), I procollagen type C-end propetide (PICP), the anti-tartaic acid phosphatase of blood plasma (TRAP5b), the crosslinked C-end of type i collagen peptide (CTX), urine collagen pyridinoline (PYD), urine collagen Deoxypyridinoline (DPYD) and the crosslinked N-end of type i collagen peptide (NTX) specificity respectively.

The capture antibody of " BALP, BGP, TRAP5b, PINP, PICP/NTX, CTX " that present embodiment is used and detection antibody are available from Santa Cruz Biotechnology company, and the capture antibody of PYD and DPYD and detection antibody are available from Quidel company.

In the present embodiment, the prescription of described various solution is as follows:

1.50mM MES damping fluid (pH5.0) prescription (250ml):

Reagent	The source	Final concentration	The consumption of every 250ml
				MES (2[N-Morpholino] ethanesulfonic acid)	Sigma M-2933	0.05M	2.44g
5M NaOH	Fisher SS256-500	-	5

2.PBS prescription:

Reagent	Catalog number (Cat.No.)	Final concentration	The consumption of every 1L
				PBS	Sigma-3813	138mM NaCl 2.7mM KCl	1 bag

3.PBS-TBN prescription (be to contain 0.1%BSA among the PBS, 0.02%Tween-20,0.05%Na3N, pH7.4)

Reagent	Catalog number (Cat.No.)	Final concentration	The consumption of every 1L
				PBS pH7.4	Sigma P-3813	138mM NaCl 2.7mM KCl	1 bag
BSA	Sigma A-9647	0.1％	1g
				Tween-20	Sigma P-9416	0.02％	0.2ml

Sodium Azide

Sigma S-8032

0.05％Azide

500mg

1. be used for the liquid phase chip reagent box of multinomial bone metabolism biochemical marker parallel detection, include:

1) 9-plex wraps by microballoon: contain and wrap respectively by No. 18 microballoons of bone alkaline phosphatase (BALP) capture antibody, bag is by No. 20 microballoons of BGP (BGP) capture antibody, bag is by No. 36 microballoons of I procollagen type N-end propetide (PINP) capture antibody, No. 34 microballoons of I procollagen type C-end propetide (PICP) capture antibody, bag is by No. 38 microballoons of the anti-tartaic acid phosphatase of blood plasma (TRAP5b) capture antibody, bag by No. 72 microballoons of the crosslinked C-of type i collagen end peptide (CTX) capture antibody and bag quilt No. 51 microballoons of urine collagen pyridinoline (PYD) capture antibody, bag is urinated No. 53 microballoons of collagen Deoxypyridinoline (DPYD) capture antibody and is wrapped by No. 56 microballoons of the crosslinked N-end of type i collagen peptide (NTX) capture antibody.

2) the 9-plex biotin labeling detects antibody: contain the detection antibody of using biotin labeled bone alkaline phosphatase (BALP), BGP (BGP), I procollagen type N-end propetide (PINP), I procollagen type C-end propetide (PICP), I procollagen type N-end propetide (PICP, PINP) the anti-tartaic acid phosphatase of blood plasma (TRAP5b), the crosslinked C-end peptide of type i collagen (CTX) respectively; Use the detection antibody of the crosslinked N-end of biotin labeled collagen pyridinoline (PYD), urine collagen Deoxypyridinoline (DPYD) and type i collagen peptide respectively;

3) the streptavidin phycoerythrin (SA-PE, 10ug/ml); Also include according to prior art is supporting

4) analysis buffer;

5) 9-plex standard items;

6) control liquid I;

7) control liquid II;

8) serum matrix liquid;

9) seal film;

10) filter plate.

2. prepare above-mentioned liquid phase chip reagent box, comprise the steps:

(1) by the composition of mentioned reagent box, every kind of capture antibody bag is by corresponding microballoon, and the preparation method is identical:

-usefulness vortex oscillator or ultrasound wave suspension microballoon, approximately 20s;

-get 50 μ L microballoons (U.S. Luminex company) in the centrifuge tube of 1.5ml, 8, the centrifugal 2min of the above speed of 000g;

-remove supernatant, microballoon is resuspended in the distilled water of 100 μ L, with vortex oscillator or the about 20s of ultrasound wave suspension microballoon, 8000g (or more than) the centrifugal 2min of speed;

-inhale and abandon supernatant, add the phosphate buffer (pH6.2) of 80 μ L, the about 20s of vortex vibration, the about 20s of ultrasound wave suspension microballoon;

-add 10 μ L 50mg/mL Sulfo-NHS, use vortex oscillator mixing lightly;

-add 10 μ L 50mg/mL EDC, use vortex oscillator mixing lightly;

-room temperature lucifuge leaves standstill 20min, every 10min vortex oscillator mixing lightly;

Microballoon after the-activation, 〉=8000g, centrifugal 2min;

-inhale and to abandon supernatant, microballoon is resuspended in the solution of MES (pH5.0) of 250 μ L 50mM the about 20s of vortex vibration, ultrasonic about 20s; Microballoon 〉=8000g, centrifugal 1-2min; Repeat this step once;

-inhale and to abandon supernatant, microballoon is resuspended in the solution of MES (pH5.0) of 100 μ L 50mM the about 20s of vortex vibration, ultrasonic about 20s;

-in the microballoon that suspends, add 1 μ g antibody, with MES (pH5.0) solution of 50mM cumulative volume is mended to 500 μ L;

-usefulness vortex oscillator mixing, room temperature lucifuge vibration 2hr;

Microballoon behind the-coupling antibody is with the speed of 〉=8000g, centrifugal 1-2min;

-inhale and abandon supernatant, microballoon is resuspended in the 500 μ L PBS-TBN solution the about 20s of vortex vibration, ultrasonic about 20s;

-room temperature lucifuge vibration 30min;

-microballoon 〉=8000g, centrifugal 1-2min;

-inhale and abandon supernatant, microballoon is resuspended in the 1mL PBS-TBN solution the about 20s of vortex vibration, ultrasonic about 20s; Microballoon 〉=8000g, centrifugal 1-2min; Repeat this step once;

-inhale and abandon supernatant, microballoon is resuspended in the 600 μ L PBS-TBN solution;

-Bao is counted by the luminex instrument by good microballoon;

-Bao is placed 2-8 ℃ to keep in Dark Place by good microballoon, and the microballoon of every kind of antibody coupling is preserved separately, during use, selects equal proportion to mix according to test item.

(2) by the composition of mentioned reagent box, every kind of biotin labeling that detects antibody:

-calculate reaction system according to protein concentration, fill up a form;

-according to the volume of protein concentration calculating antibody solution dilution, be considered as target volume (V) to 1mg/ml;

-configuration NHS-Biotin reactant liquor;

In reaction tube, added respectively in-1: 100 in molar ratio and detect antibody and NHS-Biotin (10mg/ml) reactant liquor;

The NaHCO of-adding 1/10 target volume ₃(pH8.9) solution;

-add PBS (pH7.4) to mend to target volume;

-wrap aluminium foil, reaction tube is placed on the oscillator, lucifuge is hatched 4h in 25 ℃ of constant temperature ovens, and rotating speed is 900rpm;

-reactant liquor is gone in the dialysis cassette, dialysed overnight in the PBS damping fluid is to remove unreacted NHS-Biotin;

-mensuration protein concentration.

3. be used for detecting, comprise the steps:

1) uses preceding all reagent that take out earlier, place balance to room temperature.

2) dilution of standard items: respectively according to the serology range of concentrations of nine kinds of bone metabolism marks, prepare the standard items of various blood serum designated objects, each standard items is established 6 dilutabilitys, and equal proportion is mixed then, and making each contented final concentration is required concentration.

Each manages dilution process and theoretical concentration (with the expection concentration in the following table):

Attention: all must be with the dilution that just can be used for next concentration behind the thorough mixing of vortex mixed instrument after each concentration standard product has diluted.Blending process avoids producing foam.

3) 96 orifice plate layouts are set, determine the position of standard items, quality-control product, testing sample and blank well on 96 orifice plates.The order of considering instrument readings be according to from the 1st be listed as the 12nd row, capable from A to the vertical reading of the capable order of H, when 96 orifice plate layouts are set, should follow from the 1st be listed as the 12nd row, capable from A to the capable series arrangement of H.

4) according to the 96 orifice plate layouts that set, every hole adds 25 μ l analysis buffer, adds 25ul standard items, quality-control product more respectively in hole separately, and blank well adds the 25ul analysis buffer.

5) take out the capture antibody coupling microballoon that detects at BALP, BGP, PINP, PICP, TRAP5b, CTX of above-mentioned preparation respectively, with vortex mixed instrument mixing 30 paper money, sonicated 30 seconds is mixed by equal proportion, makes the microballoon concentration of every kind of capture antibody coupling in the mixed liquor be 40/μ l.Add mixing suspension 25 μ l in every hole at the capture antibody coupling microballoon of BALP, BGP, PINP, PICP, TRAP5b, CTX.Bag should be used preceding mixing facing by microballoon, and should use immediately behind the mixing, can precipitate otherwise place microballoon of a specified duration again.

6) seal all application of sample apertures with sealing film, and encase 96 orifice plates with lucifuge with masking foil, 25 ℃ are placed on the microwell plate oscillator with the concussion of 600 rotary speeds, hatched 60 minutes.

7) hatching every hole after finishing adds the mixed liquor of the biotin labeled detection antibody of the above-mentioned preparation of 25ul (wherein said six kinds of BALP, BGP, PINP, PICP, the biotin labeled detection antibody of TRAP5b, CTX mixes by equal proportion in advance, shake up), seal all application of sample apertures with sealing film, and encase 96 orifice plates with lucifuge with masking foil, 25 ℃ are placed on the microwell plate oscillator with the concussion of 600 rotary speeds, hatched 60 minutes again.

8) hatch that every hole adds 25ul streptavidin phycoerythrin after finishing, seal all application of sample apertures with sealing film, and encase 96 orifice plates with lucifuge with masking foil, 25 ℃ are placed on the microwell plate oscillator and shake with 600 rotary speeds, hatch 30 minutes again.

9) on Luminex series liquid-phase chip analyser, read the result.Instrument is the drawing standard curve automatically, and calculates the measured value of testing sample.

The detection of running simultaneously that fortune uses the same method and can carry out urinating collagen pyridinoline (PYD) in the urine and urinate collagen Deoxypyridinoline (DPYD), the crosslinked N-end peptide of type i collagen (NTX).

From Fig. 1-9 and following table as can be seen, the linearity of each index typical curve of BALP, BGP, PICP, PINP, TRAP5b, CTX, NTX, PYD and DPYD is R2 〉=0.99 in the concentration range that detects, standard items with each index of kit measurement, its relative error＜± 10%, by the joint-detection of nine indexs, can be more convenient, quick, predict bone loss speed, risk of bone fracture degree exactly, the monitoring drug response.Detect required sample size few (25 microlitre), and can in two hours time, finish detection.

Table 1

Table 1	BGP			BALP
							MFI	Expection concentration (pg/ml)	Measured concentration (pg/ml)	MFI	Expection concentration (mIU/L)	Measured concentration (mIU/L)
	Std1	15.5	97.7	92.5	79	97.7							98
Std2							30	390.6	421	382	390.6	402
	Std3	78	1562.5	1490	1366	1562.5							1450
Std4							375	6,250	6,410	5124	6,250	6570
	Std5	1584	25,000	24,600	11720	25,000							26,000
Std6							3820	100,000	101,000	15429	100,000	91,300

Table 2

Table 2	PINP			PICP
							MFI	Expection concentration (pg/ml)	Measured concentration (pg/ml)	MFI	Expection concentration (pg/ml)	Measured concentration (pg/ml)
	Std1	77	97.7	99.7	45	97.7							98.1
Std2							171	390.6	383	202.5	390.6	379
	Std3	658	1562.5	1590	800	1562.5							1580
Std4							2054	6,250	6,480	2627.5	6,250	6230

Std5	4195	25,000	21,900	6867	25,000	25000
							Std6	7953	100,000	112,000	12052	100,000	100,000

Table 3

Table 3	PYD			DPYD
								MFI	Expection concentration (nmol/L)	Measured concentration (nmol/L)	MFI	Expection concentration (nmol/L)	Measured concentration (nmol/L)
Std1	23.5	0.39	0.375	51.5	0.39	0.39
							Std2	48	1.56	1.67	124.5	1.56	1.57
Std3	121	6.25	5.87	381.5	6.25	6.13
							Std4	481	25	25.9	1187	25	26.1
Std5	1777	100	98.5	2748	100	95.2
							Std6	5517.5	400	402	5726	400	409

Table 4

	TRACP5b			CTX
							MFI	Expection concentration (mIU/L)	Measured concentration (mIU/L)	MFI	Expection concentration (pg/ml)	Measured concentration (pg/ml)
	Std1	22	9.77	9.56	21.5	9.77							9.66
Std2							69	39.06	41.5	85.5	39.06	40.1
	Std3	199	156.25	144	448.5	156.25							152
Std4							777	625	672	3175	625	637
	Std5	2228	2,500	2,390	10248	2,500							2410
Std6							5645	10,000	10,100	14165.5	10,000	11,300

Table 5

Table 5	NTX
				MFI	Expection concentration (nmol/L)	Measured concentration (nmol/L)
	Std1	56.5	0.39				0.391
Std2				117.5	1.56	1.55
	Std3	342	6.25				6.35
Std4				1034.5	25	24.6
	Std5	3059	100				101
Std6				7578.5	400	399

Claims (3)

1. liquid-phase chip that is used for multinomial bone metabolism biochemical marker parallel detection, it is characterized in that, mainly include: 1) bag is by microballoon: contain bag respectively by the microballoon of capture antibody of bone alkaline phosphatase, bag is by the microballoon of BGP capture antibody, bag is by the microballoon of I procollagen type N-end propetide capture antibody, the microballoon of I procollagen type C-end propetide capture antibody, bag is by the microballoon of the anti-tartaic acid phosphatase of blood plasma capture antibody, bag is by the microballoon of the crosslinked C-end of type i collagen peptide capture antibody, and/or contain bag by the microballoon of urine collagen pyridinoline capture antibody, bag is urinated the microballoon of collagen Deoxypyridinoline capture antibody and is wrapped by the microballoon of the crosslinked N-end of type i collagen peptide capture antibody, and above-mentioned microballoon has the different colours coding respectively;

2) biotin labeling detects antibody: the detection antibody of holding peptide respectively with biotin labeled bone alkaline phosphatase, BGP, I procollagen type N-end propetide, I procollagen type C-end propetide, the anti-tartaic acid phosphatase of blood plasma, the crosslinked C-of type i collagen; And/or respectively with biotin labeled urine collagen pyridinoline, urine collagen Deoxypyridinoline) and the detection antibody of the crosslinked N-end of type i collagen peptide; With

3) streptavidin phycoerythrin.

2. one kind prepares the described method that is used for the liquid-phase chip of multinomial bone metabolism biochemical marker parallel detection of claim 1, mainly may further comprise the steps:

(1) corresponding capture antibody bag is by microballoon:

-with after the activation of 50 μ L microballoons, 〉=8000g is centrifugal;

-inhale and to abandon supernatant, microballoon is resuspended in the solution of MES of 230-280 μ L 50mM of pH5.0, the about 20s of vortex vibration, ultrasonic about 20s, after with microballoon in 〉=8000g, centrifugal 1-2min; Repeat this step;

-inhale and to abandon supernatant, microballoon is resuspended in the solution of MES of 100 μ L 50mM pH5.0 the about 20s of vortex vibration, ultrasonic about 20s;

-in the microballoon that suspends, add corresponding 0.8-1.2 μ g antibody, with the MES solution of the pH5.0 of 50mM cumulative volume is mended the L to 450-550 μ;

-usefulness vortex oscillator mixing, room temperature lucifuge vibration 1.5-2.5hr;

-coupling the speed of the microballoon behind the antibody with 〉=8000g, centrifugal 1-2min;

-inhale and abandon supernatant, microballoon is resuspended in the 500 μ L PBS-TBN solution the about 20s of vortex vibration, the about 20s of sonic oscillation;

-room temperature lucifuge vibration 30min; Again in 〉=8000g, centrifugal 1-2min;

-inhale and abandon supernatant, microballoon is resuspended in the 1mL PBS-TBN solution the about 20s of vortex vibration, ultrasonic about 20s; Microballoon 〉=8000g, centrifugal 1-2min; Repeat this step once;

-inhale and abandon supernatant, microballoon is resuspended in the 500-1000 μ L PBS-TBN solution;

-every kind bag is placed on 2-8 ℃ of lucifuge by good microballoon by luminex instrument counting and preserves separately, during use, needs equal proportion to mix according to detecting;

(2) every kind of biotin labeling that detects antibody:

-calculate the volume that the detection antibody-solutions is diluted to 1mg/ml according to detecting antibody concentration, be considered as target volume;

The NHS-Biotin reactant liquor of-configuration 10mg/ml;

In reaction tube, added respectively in-1: 100 in molar ratio and detect antibody and NHS-Biotin reactant liquor;

-add volume and be 1/10 the NaHCO of the pH8.9 of target volume ₃Solution;

The PBS of-adding pH7.4 mends to target volume;

-wrap aluminium foil, reaction tube is placed on the oscillator, lucifuge was hatched 3-5 hour in 25 ℃ of constant temperature ovens, and rotating speed is 800rpm-1000rpm;

-reactant liquor is gone in the dialysis cassette, dialysed overnight in the PBS damping fluid is to remove unreacted NHS-Biotin;

-every kind of as required that mark is good when using detection antibody equal proportion is mixed.

3. preparation method according to claim 2 is characterized in that: the step of described activation microballoon is as follows:

-usefulness vortex oscillator or ultrasound wave suspension microballoon;

-get the centrifugal 1-2min of 50 μ L microballoons;

-remove supernatant, microballoon is resuspended in the distilled water of 100 μ L, with vortex oscillator or the about 20s of ultrasound wave suspension microballoon, the centrifugal 1-2min of 〉=8000g;

-inhale and abandon supernatant, add the phosphate buffer (pH6.2) of 80 μ L, the about 20s of vortex vibration, the about 20s of ultrasound wave suspension microballoon;

-add 10 μ L 50mg/mL Sulfo-NHS, use vortex oscillator mixing lightly;

-add 10 μ L 50mg/mL EDC, use vortex oscillator mixing lightly;

-room temperature lucifuge leaves standstill 20min, every 10min vortex oscillator mixing lightly.

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