CN103033624A - Human myeloperoxidase chemiluminescent immunodetection kit - Google Patents
Human myeloperoxidase chemiluminescent immunodetection kit Download PDFInfo
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- CN103033624A CN103033624A CN2012105432515A CN201210543251A CN103033624A CN 103033624 A CN103033624 A CN 103033624A CN 2012105432515 A CN2012105432515 A CN 2012105432515A CN 201210543251 A CN201210543251 A CN 201210543251A CN 103033624 A CN103033624 A CN 103033624A
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Abstract
The invention discloses a human myeloperoxidase (MPO) chemiluminescent immunodetection kit. The kit comprises the following components: 1) a human MPO standard product; 2) a vector coating a human MPO monoclonal antibody; 3) the enzyme-labeled human MPO monoclonal antibody; 4) a chemiluminescent substrate on which the enzyme acts; and 5) a concentrated cleaning solution. The kit can perform concentrated quantitative detection on the MPO molecules in a serum sample of a patient. The kit effectively utilizes a chemiluminescent technology principle and adopts the enzyme to catalyze the chemiluminescent substrate on the basis of enzyme-linked immunoassay so as to improve the detection sensitivity, has the advantages of high stability, high specificity, excellent accuracy, simplicity in operation and no radioactive pollution, and can provide specific, quick and reliable basis for clinical diagnosis of cardiovascular diseases such as atherosclerosis (AS), acute coronary syndrome (ACS) and coronary heart disease (CHD).
Description
Technical field
The present invention relates to the immunoassay medical domain, concrete, the invention provides a kind of people's myeloperoxidase chemiluminescence immune detection reagent kit.
Background technology
Angiocardiopathy (Cardiovascular disease, CVD) is a class serious harm human life and healthy excessive risk disease.At present, having become the first cause of the death of China's city and country population, also is the highest Non Communicable Diseases (NCD) of the rate of causing death.WHO points out can effectively reduce clinical accident and the too early generation of death to the early diagnosis of CVD disease and prevention.
In recent years, multiple angiocardiopathy mark is come out one after another, and the index of downright bad damage is many just is detected after substantive damage occurs cardiac muscle because great majority can reflecting myocardium, is difficult in early days provide the prevention foundation of science falling ill.Myeloperoxidase (Myeloperoxidase, MPO) be a kind of hemoprotein that is present in a large number neutrophilia multinuclear leucocyte (granulocyte), it is expressed and active increasing promotes atherogenesis, can bring out cardiovascular functional disorder, and then to myocardium injury.The expression of human body MPO and release are everlasting before the myocardial damage, and with other myocarditis marks without obvious correlativity, therefore early diagnosis and the observation of curative effect of patient's angiocardiopathy are had more importantly medical value.MPO is considered at present one of the most promising cardiac marker.The rising that has been proved MPO concentration among the patients serum can be used as the risk indicator of atherosclerotic and coronary heart disease, and is indicating the early stage risk of myocardial infarction.
In recent years, the immune analysis method for the clinical diagnosis field mainly contains: radioimmunology, euzymelinked immunosorbent assay (ELISA), colloidal gold immunization, fluorescent immune method and chemiluminescence immunoassay method etc.The radioimmunology detection sensitivity is relatively not high, has again certain radioactive contamination, and is replaced gradually; Though the euzymelinked immunosorbent assay (ELISA) no radioactivity pollute, influenced factor is many, is easy to cause as a result distortion, and accuracy is relatively poor; The colloidal gold method detection speed is fast, but stability and reliability are undesirable; Fluorescence method also exists the shortcoming of poor stability.
Chemiluminescence immunoassay technology is a new immunoassay of exempting to analyze, growing up after fluoroimmunoassay and the time resolved fluoro-immunoassay continue radioimmunology analysis, enzyme.It combines the high sensitivity of chemiluminescence analysis with the high specific of antigen-antibody reaction, compare with other immunoassays at present commonly used have highly sensitive, high specificity, detection time weak point, measure linear wide ranges, good stability, can realize the plurality of advantages such as robotization, easy to use, safety, no radioactivity pollute, therefore extremely people's favor, this technology have been widely used in the various fields such as the early diagnosis, animals and plants inspection and quarantine of communicable disease, obesity and relevant disease, internal system, hereditary disease, tumour.
At present, chemiluminescence immunoassay technology is still blank in the application facet of Human megakaryopoietin immunoassay product.Kit of the present invention can be for open chemiluminescent analyzer, and method is simple and efficient, and is with low cost, is easy to promote the use of in clinical diagnosis and research work.
Summary of the invention
The objective of the invention is to overcome the deficiencies in the prior art, a kind of people's myeloperoxidase chemiluminescence immune detection reagent kit is provided.
Technical scheme of the present invention is summarized as follows:
A kind of people's myeloperoxidase chemiluminescence immune detection reagent kit comprises following component:
1) people's myeloperoxidase standard items;
2) be coated with the carrier of people's myeloperoxidase monoclonal antibody;
3) monoclonal antibody of people's myeloperoxidase of enzyme labeling;
4) chemical luminous substrate of above-mentioned enzyme effect;
5) concentrated cleaning solution,
Wherein, the described carrier that is coated with people's myeloperoxidase monoclonal antibody prepares through following steps:
1) employing pH value is that 9.6 50mM carbonate buffer solution is that solvent mixes with people's myeloperoxidase monoclonal antibody, is made into the mixed liquor that concentration is 5 μ g/mL, is carried on the carrier;
2) be the PBS of 7.3-7.5 with the pH value or be that the PBST of 7.3-7.5 cleans carrier with the pH value;
3) the pH value is carried on the carrier after the cleaning for the confining liquid of 7.2-7.5; Described confining liquid is made with following method: get 8gNaCl, 1.43g anhydrous Na
2HPO
4, 0.24g anhydrous K H
2PO
4, 0.2g KCl, 10g BSA, 25g sucrose and 0.5mL Proclin300, add water 1000mL;
4) confining liquid is outwelled, drying obtains being coated with the carrier of people's myeloperoxidase monoclonal antibody.
People's myeloperoxidase standard items are to make with following method: with 0.02mol/L, the pH value be 7.4 phosphate buffer and hyclone by volume the ratio of 4:1 be hybridly prepared into basic damping fluid, people's myeloperoxidase is diluted to that concentration is 0,25,64,160,400,1000ng/mL with basic damping fluid.
Described carrier is microwell plate, magnetic-particle, plastic tube or plastic bead.
The enzyme that step 3) is used for mark is alkaline phosphatase or horseradish peroxidase.
Described chemical luminous substrate is luminol or different luminol, (diamantane)-1,2-two oxidative ethanes, 3-(2 '-spiral diamantane)-4-methoxyl-4-(3 '-phosphorus acyloxy) phenyl-1,2-two oxidative ethanes, CSPD or CDP-Star.
Described concentrated cleaning solution is to make with following method: get 24g trishydroxymethylaminomethane, 160g NaCl, 0.5mLTween-20,0.5mL Proclin300 adds water to 1000mL, and regulating the pH value is 7.2-7.5.
What kit of the present invention adopted is the reaction pattern of " double-antibody sandwich single stage method ", can carry out very single-minded quantitative detection to people's myeloperoxidase (MPO) molecule in patients serum's sample.It had both effectively utilized the chemiluminescence principle, use the enzymatic luminous substrate on the basis of enzyme-linked immuno assay again, improved the sensitivity that detects, have also simultaneously that stability is high, specificity is good, accuracy is good, the advantage of simple to operate and no radioactivity pollute, the clinical diagnosis that can be the angiocardiopathies such as atherosclerotic (AS), acute coronary syndrome (ACS), coronary heart disease (CAD) provides more special, quick, reliable foundation.
Description of drawings
Fig. 1 is the prepared kit Plays product linear graph of embodiment 1.
Fig. 2 has illustrated determining of people's myeloperoxidase chemiluminescence immune detection reagent kit critical value of the present invention.
Fig. 3 has shown that people's myeloperoxidase chemiluminescence immune detection reagent kit of the present invention and the German IBL MPO of company ELISA measuring reagent kit detect the result of 103 routine cardiovascular patient correlativitys.
Wherein, the value of IBL kit measurement is X-axis, and the measured value of kit of the present invention is Y-axis, and the result learns by statistics and processes to get correlation coefficient r=0.9866, y=0.9775x+11.633.
Embodiment
The present invention is further illustrated below in conjunction with specific embodiment.
Embodiment 1
A kind of people's myeloperoxidase chemiluminescence immune detection reagent kit comprises following component:
1) people's myeloperoxidase standard items;
2) be coated with the microwell plate of people's myeloperoxidase monoclonal antibody;
3) monoclonal antibody of people's myeloperoxidase of alkali phosphatase enzyme mark;
4) the chemical luminous substrate AMPPD of above-mentioned enzyme effect;
5) concentrated cleaning solution;
6) semi-manufacture and finished product form;
Wherein:
1. people's myeloperoxidase standard items are made with following method: with 0.02mol/L, the pH value be 7.4 phosphate buffer and hyclone by volume the ratio of 4:1 be hybridly prepared into basic damping fluid, people's myeloperoxidase is diluted to respectively that concentration is 0,25,64,160,400,1000ng/mL with basic damping fluid.
2. the preparation process that is coated with the microwell plate of people's myeloperoxidase monoclonal antibody is:
1) employing pH value is that 9.6 50mM carbonate buffer solution is that solvent mixes with people's myeloperoxidase monoclonal antibody, is made into the mixed liquor that concentration is 5 μ g/mL, adds in each hole of microwell plate, and every hole 120 μ L place 24h for 4 ℃;
2) be that 7.4 PBS cleans microwell plate with the pH value, clean 3 times that every hole is each with 300 μ L;
3) be that 7.3 300 μ L confining liquids add in each microwell plate after cleaning with the pH value, 4 ℃ of placements are spent the night, and get rid of confining liquid, pat dry at thieving paper, and Vacuum Package immediately behind the room temperature removal moisture drying 24h is stored in 4 ℃;
Described confining liquid is to make with following method: get 8g NaCl, 1.43g anhydrous Na
2HPO
4, 0.24g anhydrous K H
2PO
4, 0.2gKCl, 10g BSA, 25g sucrose and 0.5mL Proclin300, add deionized water to 1000mL;
3. the monoclonal antibody of people's myeloperoxidase of alkali phosphatase enzyme mark
Monoclonal anti body and function glutaraldehyde cross-linking method and the alkaline phosphatase coupling of people's myeloperoxidase, be that 7.3 PBS damping fluid is fully dialysed with the pH value, add isopyknic glycerine in the bond solution after dialysis, the concentration that is diluted to enzyme labelled antibody with 20% cow's serum enzyme dilution is 1:10000, preserves below-20 ℃;
The prescription of described 20% cow's serum enzyme dilution is:
4. the chemical luminous substrate 3-(2 ' of above-mentioned enzyme effect-spiral diamantane)-and 4-methoxyl-4-(3 '-phosphorus acyloxy) phenyl-1,2-two oxidative ethanes (AMPPD)
The prescription of described AMPPD chemical luminous substrate is:
5. concentrated cleaning solution
The prescription of described concentrated cleaning solution is:
Dissolving and mixing is adjusted pH to 7.3.
6. semi-manufacture and finished product form
The packing of above-mentioned steps products obtained therefrom is semi-manufacture.Randomly draw three parts and carry out specificity, precision, sensitivity and stability test, qualified rear assembling is people's myeloperoxidase chemiluminescence immune detection reagent kit.
Experimental results show that: with the microwell plate of plastic tube alternate embodiment 1 step 2, the other the same as in Example 1 can be prepared corresponding people's myeloperoxidase chemiluminescence immune detection reagent kit.
With the microwell plate in magnetic-particle or plastic bead alternate embodiment 1 step 2, and put into blank microwell plate or plastic tube in described kit, the other the same as in Example 1 can be prepared corresponding people's myeloperoxidase chemiluminescence immune detection reagent kit;
Experimental results show that: be 7.3 or 7.5 PBS with the pH value, or the pH value of 7.3,7.4 or 7.5 PBST alternate embodiment 1 step 2 is 7.4 PBS, the other the same as in Example 1 can be prepared corresponding people's myeloperoxidase chemiluminescence immune detection reagent kit;
Experimental results show that: the pH value that with the pH value is 7.2,7.4 or 7.5 confining liquid alternate embodiment 1 step 2 is 7.3 confining liquid, and the other the same as in Example 1 can be prepared corresponding people's myeloperoxidase chemiluminescence immune detection reagent kit;
Experimental results show that: with (diamantane)-1, the 3-(2 ' of 2-two oxidative ethanes, CSPD or CDP-Star alternate embodiment 1 step 4-spiral diamantane)-and 4-methoxyl-4-(3 '-phosphorus acyloxy) phenyl-1,2-two oxidative ethanes, the other the same as in Example 1 can be prepared corresponding people's myeloperoxidase chemiluminescence immune detection reagent kit;
Experimental results show that: the pH value that with the pH value is 7.2,7.4 or 7.5 concentrated cleaning solution alternate embodiment 1 step 5 is 7.3 concentrated cleaning solution, and the other the same as in Example 1 can be prepared corresponding people's myeloperoxidase chemiluminescence immune detection reagent kit.
Embodiment 2
A kind of people's myeloperoxidase chemiluminescence immune detection reagent kit comprises following component:
1. people's myeloperoxidase standard items (with embodiment 1)
2. be coated with the microwell plate (with embodiment 1) of people's myeloperoxidase monoclonal antibody
3. the monoclonal antibody of people's myeloperoxidase of horseradish peroxidase-labeled
Dissolving 4mg horseradish peroxidase (HRP) is in the 1mL deionized water, adding 0.4mL concentration is the sodium periodate aqueous solution of 50mM, room temperature is shaken 30min, through 1mM sodium-acetate buffer (the pH value is 4.4) dialysed overnight, the monoclonal antibody that adds 6mg MPO, 2-8 ℃ of concussion spent the night, be that the NaBH4 of 200mM reduces with 400 μ L concentration, it is 7.4 0.02M PBS dialysed overnight by the pH value, secondarily purified with HPLC, collect protein peak and add equal-volume glycerine, under-20 ℃ of conditions, preserve.
4. the chemical luminous substrate luminol of above-mentioned enzyme effect
The prescription of described luminol chemiluminescence substrate is:
1) chemical luminous substrate A liquid:
Dissolving and mixing is adjusted pH to 8.0.
2) chemical luminous substrate B liquid:
Dissolving and mixing is adjusted pH to 7.6.
5. concentrated cleaning solution (with embodiment 1)
6. semi-manufacture and finished product form (with embodiment 1)
Experimental results show that: with the microwell plate in plastic tube alternate embodiment 2 steps 2, the other the same as in Example 1 can be prepared corresponding people's myeloperoxidase chemiluminescence immune detection reagent kit.
With the microwell plate in magnetic-particle or plastic bead alternate embodiment 2 steps 2, in described kit, put into blank microwell plate or plastic tube, the other the same as in Example 1 can be prepared corresponding people's myeloperoxidase chemiluminescence immune detection reagent kit;
Experimental results show that: be 7.3 or 7.5 PBS with the pH value, or the pH value of 7.3,7.4 or 7.5 PBST alternate embodiment 2 steps 2 is 7.4 PBS, the other the same as in Example 1 can be prepared corresponding people's myeloperoxidase chemiluminescence immune detection reagent kit;
Experimental results show that: the pH value that with the pH value is 7.2,7.4 or 7.5 confining liquid alternate embodiment 2 steps 2 is 7.3 confining liquid, and the other the same as in Example 1 can be prepared corresponding people's myeloperoxidase chemiluminescence immune detection reagent kit;
Experimental results show that: with the luminol of different luminol alternate embodiment 2 steps 4, the other the same as in Example 1, can prepare corresponding people's myeloperoxidase chemiluminescence immune detection reagent kit;
Experimental results show that: the pH value that with the pH value is 7.2,7.4 or 7.5 concentrated cleaning solution alternate embodiment 2 steps 5 is 7.3 concentrated cleaning solution, and the other the same as in Example 1 can be prepared corresponding people's myeloperoxidase chemiluminescence immune detection reagent kit.
The using method of embodiment 3 kits of the present invention
With the use of the Human megakaryopoietin chemiluminescence immune detection reagent kit of embodiment 1 preparation, concrete operations are as follows:
1) take out kit of the present invention in 4 ℃ of refrigerators, balance is to room temperature;
2) microwell plate of getting the preparation of embodiment 1 step 2 is numbered, and all experiments are all done diplopore and repeated;
3) add respectively in the hole of described microwell plate that concentration is 0,25,64,160,400, the calibration object of 1000ng/mL and each 25 μ L of sample to be tested, a blank hole is established in each experiment, and every hole adds the monoclonal antibody 100 μ L of people's myeloperoxidase of the enzyme labeling that embodiment 1 step 3 prepares except blank well subsequently;
4) vibration 30s mixing on the oscillator;
5) use the sealed membrane shrouding, hatched 45 minutes in 37 ℃;
6) get rid of dereaction liquid, automatic washer or manual wash plate 5 times, each good cleansing solution 300 μ L of dilution that add pat dry at thieving paper;
The dilution process of described concentrated cleaning solution is: during use and deionized water dilute by the 1:19 volume ratio.
7) every hole adds the Chemoluminescent substrate of 100 μ L embodiment, 1 step 4 preparation, the mixing that fully vibrates on oscillator, room temperature (20-27 ℃) lucifuge reaction 20 minutes;
8) measure successively the luminous intensity (RLU) in each hole on the chemiluminescence measuring instrument, Measuring Time is 1 second/hole;
9) take the standard items concentration value as horizontal ordinate, the RLU value is ordinate drawing standard curve (referring to accompanying drawing 1).Find the MPO concentration of this sample at the double logarithmic curve of setting up with the RLU value of each sample to be tested.
The methodology of embodiment 4 kits of the present invention is identified
According to manufacturing conventional in this area and vertification regulation the kit of preparation among the embodiment 1 is examined and determine, be the results are shown in Table 1:
Table 1
| Interventions Requested | Test stone | Assay |
| Sensitivity | ≤1.63ng/mL | Meet standard |
| Precision CV (%) | In batch＜10%, between batch＜15% | Meet standard |
| Accuracy | Average recovery rate 101.83% ± 1.32% | Meet standard |
| Specificity | With the cross reacting rate of analog≤0.01% | Meet standard |
| Stability | Each reagent component place 37 ℃ at least 3 days, still keep stable | Meet standard |
Illustrate that Human megakaryopoietin chemiluminescence immune detection reagent kit Specifications of the present invention meets national external diagnosis reagent standard.
Determining of MPO content in the embodiment 5 normal human serum samples and critical value
Collect 200 routine Healthy Human Serums from domestic certain hospital, be divided at random two groups, measure with " the Human megakaryopoietin chemiluminescence immune detection reagent kit " of preparation among the embodiment 1,2 respectively, the result is as follows:
200 routine Healthy Human Serums, the mean value of trying to achieve out the Healthy People measured value is 193.6ng/mL, standard deviation is 59.7ng/mL, and the mean value that calculates adds the corresponding concentration value of 2 times standard deviation as critical value, and its result is that 313.0ng/mL(is referring to accompanying drawing 2).
The clinical practice of embodiment 6 Human megakaryopoietin chemiluminescence immune detection reagent kits of the present invention and IBL kit are relatively
Use kit and the IBL kit of preparation in the embodiment of the invention 1 to detect simultaneously 103 parts of cardiovascular patients, compare the correlativity between two kinds of kits.
One, the source of clinical serum specimen
Collect 103 parts of clinical cardiovascular patients serum samples from Some Domestic hospital, atherosclerotic's 42 examples wherein, Acute Coronary Syndrome Patients 38 examples, patients with coronary heart disease 23 examples.300 parts of normal human serum samples.
Two, kit (by embodiment 5 operations) and the IBL kit (by its instructions operation) with the embodiment of the invention 1 detects 103 parts of serum specimens respectively, result by statistics Epidemiological Analysis processes, show the as a result height correlation that two kinds of methods detect, as shown in Figure 3.
Three, result
Use " people's myeloperoxidase chemiluminescence immune detection reagent kit " to show with the correlation data that the IBL kit detects identical serum specimen, two kinds of method height correlations, correlation coefficient r＞0.98, the P value is less than 0.01.Illustrate that two kinds of methods have equal use value.But a kind of people's myeloperoxidase chemiluminescence immune detection reagent kit of method preparation of the present invention is simple and efficient to handle, with low cost, is easier to promote the use of in clinical diagnosis and research work.
The stability of kit of the present invention stores experiment by 37 ℃ of senile experiments and 2-8 ℃ of deepfreeze and determines that the result shows, can effectively place 6 months for 2-8 ℃, can effectively place 3 days for 37 ℃, and the kit indices all meets stability requirement.
Claims (6)
1. people's myeloperoxidase chemiluminescence immune detection reagent kit, its feature comprises following component:
1) people's myeloperoxidase standard items;
2) be coated with the carrier of people's myeloperoxidase monoclonal antibody;
3) monoclonal antibody of people's myeloperoxidase of enzyme labeling;
4) chemical luminous substrate of above-mentioned enzyme effect;
5) concentrated cleaning solution,
Wherein, the described carrier that is coated with people's myeloperoxidase monoclonal antibody prepares through following steps:
1) employing pH value is that 9.6 50mM carbonate buffer solution is that solvent mixes with people's myeloperoxidase monoclonal antibody, is made into the mixed liquor that concentration is 5 μ g/mL, is carried on the carrier;
2) be the PBS of 7.3-7.5 with the pH value or be that the PBST of 7.3-7.5 cleans carrier with the pH value;
3) the pH value is carried on the carrier after the cleaning for the confining liquid of 7.2-7.5; Described confining liquid is made with following method: get 8gNaCl, 1.43g anhydrous Na
2HPO
4, 0.24g anhydrous K H
2PO
4, 0.2g KCl, 10g BSA, 25g sucrose and 0.5mL Proclin300, add water to 1000mL;
4) confining liquid is outwelled, drying obtains being coated with the carrier of people's myeloperoxidase monoclonal antibody.
2. kit as claimed in claim 1, it is characterized in that people's myeloperoxidase standard items are to make with following method: with 0.02mol/L, the pH value be 7.4 phosphate buffer and hyclone by volume the ratio of 4:1 be hybridly prepared into basic damping fluid, people's myeloperoxidase is diluted to that concentration is 0,25,64,160,400,1000ng/mL with basic damping fluid.
3. kit as claimed in claim 1 is characterized in that described carrier is microwell plate, magnetic-particle, plastic tube or plastic bead.
4. kit as claimed in claim 1 is characterized in that the enzyme that step 3) is used for mark is alkaline phosphatase or horseradish peroxidase.
5. kit as claimed in claim 1, it is characterized in that described chemical luminous substrate is luminol or different luminol, (diamantane)-1,2-two oxidative ethanes, 3-(2 '-spiral diamantane)-4-methoxyl-4-(3 '-phosphorus acyloxy) phenyl-1,2-two oxidative ethanes, CSPD or CDP-Star.
6. kit as claimed in claim 1, it is characterized in that described concentrated cleaning solution is to make with following method: get 24g trishydroxymethylaminomethane, 160g NaCl, 0.5mL Tween-20,0.5mL Proclin300 adds water to 1000mL, regulating the pH value is 7.2-7.5.
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