CN103018465A - Human cystatin C chemiluminescence quantitative detection method - Google Patents

Abstract

The invention provides a human cystatin C quantitative detection method. According to the method, a magnetic micro-particle separation technology and an enzymatic chemiluminescence technology are combined, the principle of a double-antibody sandwich one-step reaction method is adopted, and the method can be used for content determination of human cystatin C in serum, plasma and urine samples and assistance in diagnosis and treatment of various renal dysfunction diseases. The sensitivity of the method is not more than 0.01mu g/L, the quantitative detection range is 0.05-8mu g/L, the precision in analysis is less than 5%, the detection time is 10 minutes, the recovery rate of the added serum and urine is 90-115%, and the correlation analysis R2 with a reagent serum value detected by a latex-enhanced immunoturbidimetric method is 0.9862.

Description

A kind of human cystatin C chemiluminescence quantitative detecting method

Technical field

The invention belongs to biological technical field, provide a kind of accurately, sensitive, fast, quantitatively detect the chemical luminous immune detection method of bladder chalone C in human serum, blood plasma and the urine specimen (CYS-C).

Background technology

Bladder chalone C is a kind of protein of non-saccharification, is a kind of of cystatin, is comprised of 122 amino acid, and relative molecular weight is about 13kDa.It is produced in a kind of constant mode by most of karyocytes, by glomerular filtration, suctions receipts and decomposition fully at proximal convoluted tubule.Its molecular weight is little, and production rate is stable, because its concentration and glomerular filtration rate(GFR (GFR) in serum has close relationship, and more more responsive than creatinine to glomerular filtration rate(GFR, so bladder chalone C has extremely important meaning to measuring glomerular filtration rate(GFR.Bladder chalone C is heavily absorbed by proximal convoluted tubule fully, does not appear in the urine under normal circumstances.If the heavy absorption function of proximal convoluted tubule has infringement, obvious rising can appear in bladder chalone C concentration in the urine.Serum and plasma bladder chalone C reference range, normal male: (0.63-1.25) μ g/ml, normal female: (0.54-1.15) μ g/ml, greater than 50 years old normal person: (0.6-1.55) μ g/ml.Normal person's urine reference range: 0.25-0.317 μ g/ml.

The method that detects at present bladder chalone C mainly contains latex enhancing immune turbidimetry, euzymelinked immunosorbent assay (ELISA), chemoluminescence method etc.The advantage of Immunoturbidimetry is that detection specificity is good, simple to operate, precision is high, sensing range is wide, and shortcoming is that sensitivity is lower.(application number: 201010245894.2) sensitivity is very high, but complicated operation detects length consuming time, and sensing range is narrow, and sample needs pre-dilution process for euzymelinked immunosorbent assay (ELISA) and chemoluminescence method.

Summary of the invention

The object of the present invention is to provide a kind of new chemical luminous immune detection method.The method accuracy is good, precision is high, highly sensitive, sample need not pre-dilution, simple to operate saving time, sensing range is wide.

The reagent of human cystatin C (CYS-C) quantitative determination reagent kit (Magnetism particulate immuno chemistry luminescence method) of the present invention's preparation forms and the reagent principal ingredient comprises:

Reagent 1(R1): the solution that contains the anti-human CYS-C antibody of fluorescein (FITC) mark;

Reagent 2(R2): the solution that contains the anti-human CYS-C antibody of alkaline phosphatase (ALP) mark;

Magnetic separation agent: the suspension that contains the magnetic particle of coated anti-FITC mAb.

Technical solution of the present invention is as follows:

The preparation of magnetic separation agent: (EDC) makes coupling agent with carbodiimide, and then the surface with the covalently bound magnetic particle containing carboxyl (COOH) of anti-FITC mAb is diluted to working concentration with damping fluid, finishes the preparation of magnetic separation agent.

The preparation of reagent 1: with anti-human CYS-C antibody and FITC in molar ratio 1:20-1:200 in the carbonate buffer solution of 0.1-0.2mol/L pH=9.0-10.0, mix, room temperature reaction surpasses 12h.With the G-25 gel column anti-human CYS-C antibody-FITC connector is separated with free FITC, obtain the anti-human CYS-C antibody strong solution that coupling has the FITC molecule, then this solution is diluted to working concentration with damping fluid, finish the preparation of reagent 1.

The preparation of reagent 2: anti-human CYS-C antibody is connected as crosslinking chemical with SMCC, 2IT with ALP.Anti-human CYS-C antibody and ALP mol ratio are 1:1-1:2.Crosslinked rear method purifying with gel chromatography, acquisition connects the anti-human CYS-C antibody strong solution of ALP, and this solution is diluted to the preparation that working concentration is finished reagent 2 with damping fluid.

Description of drawings

Fig. 1. serum sample testing result correlativity

Embodiment

Embodiment 1: anti-FITC mAb and magnetic particle coupling, preparation magnetic separation agent.

Material and instrument

1. magnetic particle: contain carboxyl (COOH) reactive group, every gram (g) magnetic particle (dry weight) carboxyl-content is not less than 0.4 mM (mmol), has superparamagnetism, and diameter is between 0.5-2 μ m.

2. anti-FITC mAb: can be polyclonal antibody, also can be monoclonal antibody, and purity should surpass 90%, and dilution is tired and should be surpassed 1:100 ten thousand.

3.2-morpholino b acid (MES), carbodiimide (EDC), TRIS and other reagent should reach chemical pure.

Operation steps

1. get the 100mg magnetic particle, magnetic divides the supernatant of leaving away, and uses 0.05mol/L, and the MES damping fluid 10ml of pH=4.5-5.0 is resuspended;

2. the anti-FITC mAb that adds 2-4mg, room temperature suspendible 30-60min;

3. add 0.5-1ml, the EDC aqueous solution of freshly prepared 10mg/ml, room temperature suspendible 2-12h;

4. magnetic separates, and removes supernatant, uses the TRIS damping fluid of the 0.1mol/L that contains 0.5% bovine serum albumin(BSA) (BSA) pH=8.0 to be resuspended to 1mg/ml, finishes the preparation of magnetic separation agent.

Embodiment 2:FITC is connected preparation reagent 1 with anti-CYS-C antibody

Material and instrument

1. anti-human CYS-C monoclonal antibody is prepared by the Beijing Leaderman Biochemistry Co., Ltd, and purity surpasses 95%, and concentration surpasses 1mg/ml, preserves with phosphate buffer;

2. fluorescein isothiocynate (FITC), the reagent such as sodium carbonate should reach chemical pure;

3.G-25 the buying of gel-purified post is from GE company.

Operation steps

1. prepare the FITC solution of 0.5mg/ml with the carbonate buffer solution of 0.1-0.2 mol/L pH=9.0-10.0;

2. be that the ratio of 1:20-1:200 adds FITC solution in anti-human CYS-C antibody-solutions according to anti-human CYS-C antibody and FITC mol ratio, mix that room temperature leaves standstill above 12h;

3. with the G-25 gel column anti-human CYS-C antibody-FITC connector is separated with free FITC, obtaining coupling has the anti-human CYS-C antibody strong solution of FITC molecule, use the TRIS damping fluid of the 0.1mol/L that contains 0.5% bovine serum albumin(BSA) (BSA) pH=8.0 to be diluted to 0.5-1 μ g/ml this solution, finish the preparation of reagent 1.

Embodiment 3:ALP is connected preparation reagent 2 with anti-human CYS-C antibody

Material and instrument

1. anti-human CYS-C monoclonal antibody is prepared by the Beijing Leaderman Biochemistry Co., Ltd, and purity surpasses 95%, and concentration surpasses 5mg/ml, preserves with phosphate buffer;

2. alkaline phosphatase purity should surpass 95%, and specific activity should surpass 1000 U/mg, and concentration surpasses 5mg/ml;

3. coupling agent SMCC, 2-IT is available from THERMO company, and the chemical reagent such as TRIS should reach chemical pure;

4.AKTA-purifier protein purification instrument and Supperdex200 gel-purified post are GE company product.

Operation steps

1. get the anti-human CYS-C antibody of 1mg, add the coupling agent 2-IT solution 2-4 μ l of 10mg/ml, room temperature leaves standstill 20min, adds the glycine solution 10 μ l of 0.1mol/L, and room temperature leaves standstill 5min.With G-25 gel column desalination, collect the antibody after activating, 2-8 ℃ saves backup;

2. get the ALP solution of 1.5mg, add the SMCC solution 10-20 μ l of 5mg/ml, room temperature leaves standstill 30min, with G-25 gel column desalination, collects the rear antibody of activation, and 2-8 ℃ saves backup;

3. the anti-human CYS-C antibody with above-mentioned activation mixes with the ALP of activation, leaves standstill 12-24h under the 2-8 ℃ of condition, with Supperdex200 gel-purified post purifying conjugate, obtains anti-human CYS-C antibody-ALP connector strong solution, and 2-8 ℃ saves backup.

4. use the TRIS damping fluid of the 0.1mol/L that contains 0.5% bovine serum albumin(BSA) (BSA) pH=8.0 to be diluted to 0.5-1 μ g/ml anti-human CYS-C antibody-ALP connector strong solution, finish the preparation of reagent 2.

Embodiment 3: the evaluation of the enforcement of detection and detection effect

Material and instrument

1. bladder chalone C reagent 1, and bladder chalone C reagent 2 and magnetic separation agent are by above-described embodiment 1,2,3 preparations.

2. bladder chalone C calibration object, bladder chalone C quality-control product, luminous substrate, cleaning fluid, bladder chalone C determining reagent kit (immunoturbidimetry) are produced by the Beijing Leaderman Biochemistry Co., Ltd.

3.CI1000 the Full-automatic chemiluminescence analyser is produced by the Beijing Leaderman Biochemistry Co., Ltd.

4.AU400 Biochemical Analyzer is by Olympus production.

Detect and implement

Following detecting step is finished automatically by the Full-automatic chemiluminescence analyser, also manually actuated finishing.

1. add successively 5 μ l samples, 100 μ l reagent, 1,100 μ l reagent, 2,50 μ l magnetic separation agents in the detector tube, mix, react 5min under 37 ± 0.5 ℃ of conditions;

2. make magnetic particle sedimentation in magnetic field, remove supernatant, add the cleaning fluid of 600 μ l, remove magnetic field, concussion makes the abundant suspendible of magnetic particle.Repeat this operation, operate altogether 3 times.

3. with magnetic particle sedimentation in magnetic field, remove supernatant, add the luminous substrate of 100 μ l, remove magnetic field, fully detect 1 second behind the suspendible in relative luminous intensity value (RLU).

Sensitivity is estimated

Detect " 0 " concentration samples, duplicate detection 20 times, calculate mean value (M) and the standard deviation (SD) of relative luminous intensity (RLU), and calculating M+2SD value, carry out 2 regression fits according to the concentration-RLU between zero-dose calibration object and the adjacent calibration object and draw linear function, the M+2SD value is brought in the above-mentioned equation, obtain corresponding concentration value, be lowest detectable limit.This method sensitivity≤0.01 μ g/ml.

Linear evaluation

Be 9 concentration levels with 2 times of dilutions of sample of (8.0 ± 1.0) μ g/ml.With the sample duplicate detection of each concentration 2 times, calculate testing result mean value, as a result mean value and dilution ratio are carried out fitting a straight line, calculate linearly dependent coefficient r.This method in 0.05-8 μ g/ml sensing range, correlation coefficient r 〉=0.9990.

Precision is estimated

With each duplicate detection of sample of (1.0 ± 0.2) μ g/ml and (3.0 ± 0.6) μ g/ml 10 times, calculate mean value M and the standard deviation SD of 10 measurement results, calculate coefficient of variation CV according to formula CV=SD/M * 100%.This method CV≤5%.

Accuracy estimating

Mix the different amount people CYS-C international standard substances of interpolation in the urine specimen at 1 routine pooled serum sample and 1 example, the serum or the urine that form 3 concentration levels are added sample, and the additive volume is less than 10% of cumulative volume.Detect concentration of specimens, by following formula calculate recovery rate.This method urine and the serum matrix recovery are between 90-115%.Data are referring to table 1.

$R=\frac{C\&times;({\mathrm{<figure-callout\; id="0"\; label="V"\; filenames="2012105457175100003DEST\_PATH\_IMAGE001.png"\; state="\{\{state\}\}">V</figure-callout>}}_0+V)-{\mathrm{<figure-callout\; id="0"\; label="C"\; filenames="2012105457175100003DEST\_PATH\_IMAGE001.png"\; state="\{\{state\}\}">C</figure-callout>}}_0\&times;V_0}{V\&times;C_S}\&times;100\%$

R: the recovery;

V: the volume that adds standard solution;

V ₀: the volume of people source sample;

C: people source sample adds the detectable concentration behind the standard solution;

C ₀: the detectable concentration of people source sample;

C _s: the concentration of standard solution.

Table 1. accuracy estimating---add the recovery experiment data

Annotate: people CYS-C concentration of standard solution=100 μ g/ml; Sample volume=1.0ml before adding.Correlativity is estimated

Detect simultaneously 100 routine serum samples with method of production and bladder chalone C determining reagent kit (immunoturbidimetry).Take latex intensified turbidimetry measured value as horizontal ordinate, the method for production measured value is that ordinate is done correlation analysis, coefficient R ²=0.9862, slope=1.01, intercept=0.0581.Referring to Fig. 1.

Evaluation of Thermal Stability

Get the sample in the term of validity, after 7 days, detection accuracy, lowest detectable limit, linearity, repeatability still meet above-mentioned requirements 37 ℃ of placements.

List of references

[1].Abrahamson?M,?Olafsson?I,?Palsdottir?A,?et?al.?Structure?and?expression?of?the?human?cystatin?C?gene.?Biochem?J,?268:287–94。

[2]. bend the first month of spring, Hu Rongrong, LI XUEMEI, etc. the Clinical advances of bladder chalone C. CHINESE JOURNAL OF INTERNAL MEDICINE, 10 phases of 48 volumes in 2009.

[3].Ying?Ma,?Qi?Li,?Jiuping?Wang,?Zhuwei?Xu,?et?al.?Cystatin?C,?a?novel?urinary?biomarker?for?sensitive?detection?of?acute?kidney?injury?during?haemorrhagic?fever?with?renal?syndrome.?Biomarkers,?2010,?15(5):?410–417。

Claims (6)

1. the chemiluminescence quantitative detecting method of a human cystatin C, it is characterized in that it is a kind of double-antibody sandwich single step reaction method immunologic detection method based on magnetic particle isolation technics and enzyme-catalyzed chemical luminescence technology, this detection method has comprised immune response, wash and add the step of substrate solution detection luminous intensity, the kit that uses is mainly by reagent 1, reagent 2 and magnetic separation agent form, wherein reagent 1 is the damping fluid that contains the anti-human bladder chalone C antibody of fluorescein (FITC) mark, reagent 2 is the damping fluid that contains the anti-human bladder chalone C antibody of alkaline phosphatase (ALP) mark, and the magnetic separation agent is the damping fluid that contains the magnetic particle that is coated with anti-FITC mAb.

2. detection method according to claim 1 is characterized in that its detecting step comprises:

1) immune response: with sample to be tested stoste and reagent 1, reagent 2 and magnetic separation agent mixing, incubation;

2) washing: make magnetic particle sedimentation in magnetic field, remove supernatant, add cleaning fluid, remove magnetic field, concussion, magnetic separates, and removes supernatant again, and this step repeats 2-3 time;

3) add substrate solution and detect luminous intensity: add the chemical luminous substrate of alkaline phosphatase enzymatic in detector tube, concussion detects the unit interval inner glow intensity.

3. according to claims 1 or 2 described detection methods, it is characterized in that its detecting step comprises:

1) immune response: in detector tube, add 2-10 μ l sample to be tested stoste, then add 100-200 μ l reagent 1,100-200 μ l reagent 2,20-100 μ l magnetic separation agent, mixing, incubation 2-15min under 37 ± 1 ℃ of conditions;

2) washing: make magnetic particle sedimentation in magnetic field, remove supernatant, add the cleaning fluid of 300-1000 μ l, remove magnetic field, concussion makes the abundant suspendible of magnetic particle, and then magnetic separates, and removes supernatant; This step repeats 2-3 time;

3) add substrate solution and detect luminous intensity: add the chemical luminous substrate of 100-300 μ l alkaline phosphatase enzymatic in detector tube, concussion makes the abundant suspendible of magnetic particle, detects the unit interval inner glow intensity.

4. each described kit according to claim 1-3, it is characterized in that mainly being formed by reagent 1, reagent 2 and magnetic separation agent, wherein reagent 1 is the damping fluid that contains the anti-human bladder chalone C antibody of fluorescein (FITC) mark, reagent 2 is the damping fluid that contains the anti-human bladder chalone C antibody of alkaline phosphatase (ALP) mark, and the magnetic separation agent is the damping fluid that contains the magnetic particle that is coated with anti-FITC mAb.

5. kit according to claim 4, it is characterized in that, the magnetic separation agent is prepared by following method: (EDC) makes coupling agent with carbodiimide, and anti-FITC mAb is covalently bound on the surface of carboxylic magnetic particle, then is diluted to working concentration with damping fluid; Reagent 1 is prepared by following method: anti-human CYS-C antibody is mixed with FITC, and room temperature reaction separates anti-human CYS-C antibody-FITC connector with free FITC, and dilution separates the solution that obtains; Reagent 2 is prepared by following method: anti-human CYS-C antibody and ALP are carried out crosslinked, purifying is with the anti-human CYS-C antibody strong solution dilution of the connection ALP that obtains.

6. according to claim 4 or 5 described kits, it is characterized in that, the magnetic separation agent is prepared by following method: (EDC) makes coupling agent with carbodiimide, and anti-FITC mAb is covalently bound on the surface of carboxylic magnetic particle, then is diluted to working concentration with damping fluid; Reagent 1 is prepared by following method: with anti-human CYS-C antibody and FITC in molar ratio 1:20-1:200 in the carbonate buffer solution of 0.1-0.2mol/L pH=9.0-10.0, mix, room temperature reaction surpasses 12h, with the G-25 gel column anti-human CYS-C antibody-FITC connector is separated with free FITC, obtaining coupling has the anti-human CYS-C antibody strong solution of FITC molecule, then this solution is diluted to working concentration with damping fluid; Reagent 2 is prepared by following method: anti-human CYS-C antibody is connected as crosslinking chemical with SMCC, 2IT with ALP, anti-human CYS-C antibody and ALP mol ratio are 1:1-1:2, crosslinked rear method purifying with gel chromatography, obtain to connect the anti-human CYS-C antibody strong solution of ALP, this solution is diluted to working concentration with damping fluid.

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