Background technology
C reactive protein (C-reactive protein) is the special albumen that liver cell produced in the human body, is the index of inflammatory response; The molecular weight of C-reactive protein is 115KD, is made up of the polypeptide subunit of containing five identical terminal saccharideization, and 187 amino acid are contained in each subunit, connects to the pentamer of ring-type between these subunits through non-covalent bond, and an interchain disulfide bond is arranged.
The C-reactive protein is that people such as Tillett and Francis found in acute lobar pneumonia patient's serum when nineteen thirty the earliest, they find to exist a kind of can be at Ca
2+The material of the C-polysaccharide generation specificity precipitation reaction when existing and in the pneumococcus cell membrane.Nineteen forty-one, it is a kind of protein that Avery etc. predict it, so be called c reactive protein (C-reactive protein).Nineteen forty-four, Jones with it as one of less important index of clinical rheumatic fever diagnostic criteria.Afterwards, people had measured the C-reactive protein in noninfectious disease and infectious diseases patient's acute phase serum, so it is believed that, the C-reactive protein is a kind of nonspecific reaction of tissue damage.Discover that further factors such as virus or bacterial infection, infraction, immune complex deposit all can cause tissue damage.In the acute stage of tissue damage, some synthetic plasma proteinss of liver significantly increase, and these protein are commonly referred to as acute phase protein, and wherein the C-reactive protein is that variation is the most a kind of in the acute phase protein.
The C-reactive protein only contains trace in normal human serum, generally below 1mg/L, its concentration maintains a long-term stability, long half time (about 18-20 hour), and do not receive the influence of sex and food; Sustain damage at tissue, the C-reactive protein can sharply rise in several hours when inflammation, infection or tumor destruction; Can increase several times or hundreds of times, 2-3 days peakings, concentration can reach 1000mg/L during acute inflammation; Treat to descend gradually when the state of an illness is improved, recover normal.The C-reactive protein is widely used in the early diagnosis and the antidiastole of clinical disease; Its rising is found in: 1, tissue damage, infection, tumour, miocardial infarction and a series of active chronic inflammation property disease, like rheumatic arthritis, systemic vasculitis, polymyalgia rheumatism; 2, the index of POI and complication: patients after surgery C-reactive protein raises, and C-reactive protein level should descend in postoperative 7-10 days, does not reduce or rising once more prompting possibility accompanying infection or thromboembolism like the C-reactive protein; 3, can be used as the antidiastole of bacterial infection and viral infection: most of bacterial infections can cause that patients serum C-reactive protein raises, and viral infection then majority does not raise.
Ultra sensitive C-reactive protein (High sensitivity C-reactive protein; Hereinafter to be referred as the hs-C-reactive protein) with the C-reactive protein be not two kinds of albumen; Just from sensitivity, distinguish, ultra sensitive C-reactive protein (hs-C-reactive protein) LDL reaches 0.1mg/l; Thought originally that the C-reactive protein was that normal serum finds that but that angiocardiopathy takes place is closely related with following, a large amount of research datas show, atheromatous thrombosis is except being also to be a chronic inflammation process the process of fat accumulation; The slight rising of hs C-reactive protein is relevant with coronary artery events, apoplexy and peripheral angiopathy, is independently hazards; The hs-C-reactive protein has been proved to be the independent hazard factor that is caused angiocardiopathy by chronic inflammation, detects that its concentration plays an important role to the intervention of angiocardiopathy and prognosis and by clinical attention.Epidemiology survey also shows, the probability that acute apoplexy takes place hs-C-reactive protein level rising person is 2 times of normal healthy people, and the probability that myocardial infarction takes place is 3 times of normal person.European hypertension prevention and control guide (ESH/ESC) formal recommendation in 2003, the hyperpietic need detect hs-C-reactive protein level.
The detection commonly used of c reactive protein has immunoturbidimetry and enzyme linked immunosorbent assay etc.Because enzyme linked immunosorbent assay detects consuming time and complicated operation, can not satisfy the requirement that the large hospital fast quantification detects, and immunoturbidimetry develops out multiple tachysynthesis than turbid detection technique along with the popularizing of full automatic biochemical apparatus.Sternberg had founded the rate scattering immunoturbidimetry in 1977; Its ultimate principle is: antigen-antibody forms antigen antibody complex fast in damping fluid; When the light of certain wavelength when transverse axis shines; Run into short grained immune complex and can cause light scattering, scattering strength is directly proportional with the content of immune complex.But immunoturbidimetry is because detection sensitivity still can not reach clinical requirement, so latex enhance immunity turbidimetry occurred.The ultimate principle of latex enhance immunity turbidimetry is at first antibody to be adsorbed on a kind of latex particle, and when running into corresponding antigen, antigen-antibody combines and latex agglutination occurs.The size of single latex particle is within incident wavelength, and light can see through, and when plural latex particle agglutination, can hinder light and see through, and makes transmitted light reduce, and its minimizing degree is directly proportional with the amount of antigen.This method has improved the sensitivity and the accuracy that detect, has obtained using widely.Two kinds (common C-reactive protein detection kit and ultra quick C-reactive protein detection kits) have appearred in the domestic latex enhance immunity turbidimetry C-reactive protein detectable that is used for full automatic biochemical apparatus at present; Higher linearity is arranged common C-reactive protein detection kit but sensitivity is bad; Higher sensitivity is arranged ultra quick C-reactive protein detection kit but linearity is lower; Two kinds of titles of a kind of reagent so just occurred, purposes is difference to some extent also.In recent years; Along with the development of inspection technology, begin on the market a kind of full-range C-reactive protein detection kit to have occurred, but the sensitivity of this full-range C-reactive protein detection kit is still not high; Can only reach 0.5mg/L, not reach clinical detection requirement ultra sensitive C-reactive protein; And upper limit of detection also has only 300mg/L, has also satisfied not the diagnostic requirements of part acute inflammation patient high concentration c reactive protein.
And can produce deposition after the reaction of latex enhance immunity turbidimetry, and be unfavorable for the cleaning of biochemical instruments, and the latex manufacturing cost is higher, cause the latex enhance immunity more on the high side than turbid kit.
Summary of the invention
Technical matters to be solved by this invention provides a kind of highly sensitive full-range C-reactive protein colloid gold immune than turbid detection kit; This kit not only range of linearity is wide, highly sensitive; And cheap for manufacturing cost, do not produce deposition after the reaction and make things convenient for biochemical instruments to clean.
The present invention solves the problems of the technologies described above the technical scheme that is adopted: a kind of full-range C-reactive protein colloid gold immune comprises reagent R1 and reagent R2 than turbid detection kit; The component of said reagent R1 comprises: the BSA of the damping fluid of 10~20mmol/L, the set accelerator of 0.5%~2%w/v, 0.5%~2%w/v, the antiseptic of 0.07%~0.1%w/v; The component of said reagent R2 comprises: 0.1%~0.5%w/v is marked with the colloid gold particle of anti-human C-reactive protein antibodies, the stabilizing agent of 0.2%~2%w/v, the damping fluid of 10~20mmol/L, the antiseptic of 0.07%~0.1%w/v; Said colloid gold particle diameter is 35nm~60nm.
Because the activity of antibody receives influences such as the decomposition of proteinase, high-temperature denatured, osmotic pressure easily, so must add structure and the activity that stabilizing agent comes stabilization of antibodies.Said stabilizing agent is one or more in protein, PEG, sugar and the inorganic salts; Said protein is BSA or gelatin; Said sugar is one or more in sucrose, trehalose, glucosan, mannose and the glucose; Said inorganic salts are one or more in sodium chloride, potassium chloride, the lime chloride.
Described damping fluid is one or more in phosphate buffer, borate buffer solution, glycine buffer, citric acid-phosphate buffer, tris damping fluid and the HEPES damping fluid; Said pH of buffer value is 6~8.5.Said damping fluid can have the damping fluid of similar features for other.
Said set accelerator is PEG20000.It is to increase antigen-antibody reaction speed that set accelerator mainly acts on.The PEG20000 of the preferred 0.05%~2%w/v of set accelerator of the present invention.
In order to prevent that reagent from receiving microbial contamination, also added antiseptic among reagent R1 of the present invention and the reagent R2, antiseptic of the present invention is one or more in sodium azide, thimerosal, the phenol.Preferably, antiseptic of the present invention is the sodium azide of 0.07%~0.1%w/v.
The potpourri that said anti-human C-reactive protein antibodies is polyclonal antibody or polyclonal antibody and monoclonal antibody.
The preparation method of said reagent R2 is: the preparation colloidal gold solution; To join in the said colloidal gold solution with the anti-human C-reactive protein antibodies solution after the damping fluid dilution; Prepare the solution that contains collaurum-anti-people CRP antibody complex, the centrifugal supernatant that removes dissolves collaurum-anti-people CRP antibody complex with solution A; Return to 1/50~1/10 of the said liquor capacity that contains collaurum-anti-people CRP antibody complex, make reagent R2; The component of said solution A comprises: damping fluid, stabilizing agent and antiseptic.
A kind of full-range C of the present invention-reactive protein colloid gold immune is used for measuring the C-reactive protein content of human serum or blood plasma than turbid detection kit.
The preparation method of kit of the present invention:
(1) reagent R1 preparation: add PEG20000, sodium azide etc. in the damping fluid, mixing makes R1 reagent;
(2) reagent R2 preparation: preparation colloidal gold solution; To join in the said colloidal gold solution with the anti-human C-reactive protein antibodies solution after the damping fluid dilution; Prepare the solution that contains collaurum-anti-people CRP antibody complex; Centrifugally remove the deposition that supernatant obtains containing collaurum-anti-people CRP antibody complex; With the said deposition that contains collaurum-anti-people CRP antibody complex of solution A dissolving, return to 1/50~1/10 of the said liquor capacity that contains collaurum-anti-people CRP antibody complex.
Principle of the present invention: the present invention utilizes antigen-antibody selectivity specific reaction principle and gold grain to assemble the principle that causes color change; To resist colloid gold particle on the human C-reactive protein antibodies mark; Human C-reactive albumen and a plurality of antibody in the testing sample react, and produce antigen antibody complex, make colloid gold particle be gathered into bulky grain; Thereby cause solution colour to take place obviously to change; Light absorption changes, and the variable quantity of light absorption is directly proportional with the amount of antigen antibody complex, thereby is directly proportional with C-reactive protein antigen amount.Supporting full automatic biochemical apparatus can realize that then automatic ration detects.
In sum, the present invention compared with prior art has following advantage:
(1), kit of the present invention have highly sensitive, high specificity, the characteristics of good stability can be used for detecting C-reactive protein content in serum or the blood plasma, are applicable to clinical automatic clinical chemistry analyzer.The sensitivity that kit of the present invention detects the C-reactive protein can reach 0.01mg/L, and upper limit of detection reaches 500mg/L.
(2), do not produce deposition after the kit of the present invention reaction, be convenient to the cleaning of biochemical instruments, prolonged the serviceable life of biochemical instruments.
(3), kit of the present invention adopts colloid gold label antibody, reduced the manufacturing cost of C-reactive protein immunoturbidimetry detection kit, has the bigger market competitiveness.
Embodiment
Following embodiment can make those skilled in the art more fully understand the present invention, but does not limit the present invention in any way.
Embodiment one
Kit preparation 1:
(1) preparation of reagent R1 as follows: in 20mmol/L Tris damping fluid (pH7.5), adding final concentration is the BSA of 0.5% (w/v), and adding final concentration is the PEG20000 of 2% (w/v), and adding final concentration is the NaN3 of 0.09% (w/v).Make reagent R1 thus.
(2) preparation of collaurum is as follows:
A) cleaning of glass container: rinse well with tap water flowing water, use 1%~2% hydrochloric acid soaked overnight then, take out, rinse well with the abluent washing and with flowing water then, spend the night with distilled water immersion again, carry out the collaurum rinse then with a large amount of tap water flushings.
B) prepare 1% gold chloride and 1% sodium citrate solution, 0.2um membrane filtration.
C) get 1 of 1000ml round-bottomed flask, put into rotor, add the 500ml ultrapure water.
D) add 5ml 1% gold chloride, 600rpm, heat to the solution boiling, is drawn 1% sodium citrate solution 5ml immediately and is joined in the flask fast approximately.Continue the about 10min of heated and stirred, solution turns black earlier, and purpling is red gradually then.
E) cooling: close heater switch and continue and stir 10min, stop stirring then and be cooled to room temperature, return to original volume with ultrapure water.
F) the colloid gold particle diameter that makes of electron-microscope scanning is 40nm, and the collaurum that makes is subsequent use.
(3) preparation of reagent R2 is as follows:
A) will resist the human C-reactive protein antibodies to be diluted to 0.1mg/ml with phosphate buffer (pH7.2);
B) use K
2CO
3Regulate the pH to 8.0 of colloidal gold solution;
C) antibody-solutions is joined in the collaurum, and stirring reaction 30 minutes, wherein the volume ratio of antibody-solutions and colloidal gold solution is 1:10;
D) be that the BSA of 2% (w/v) joins in the above-mentioned colloidal gold solution with final concentration, and stirring reaction 30 minutes;
E) 10000rpm is centrifugal 30 minutes, redissolves to 1/10 of original volume with solution A then, makes reagent R2; The concentration that the reagent R2 for preparing is marked with the colloid gold particle of anti-human C-reactive protein antibodies is 0.1%w/v.The composition of said solution A comprises: the NaN3 of the stabilizing agent, 0.1% (w/v) of 20mmol/L Tris pH of buffer 8.0,0.2% (w/v).The stabilizing agent of said 0.2% (w/v) is meant that the quality of stabilizing agent in the 100ml solution A is 0.2g.The constituent of said stabilizing agent comprises 0.7%NaCl, 2%BSA and 0.5% sucrose.Said 0.7%NaCl is meant that the quality of NaCl accounts for 0.7% of stabilizing agent gross mass.
Embodiment two
Kit preparation 2:
(1) preparation of reagent R1
In 10mmol/L citrate buffer (pH6.5), adding final concentration is the BSA of 2% (w/v), and adding final concentration is the PEG20000 of 0.5% (w/v), and adding final concentration is the NaN3 of 0.09% (w/v).Make R1 reagent thus.
(2) preparation of collaurum:
A) cleaning of glass container: rinse well with tap water flowing water, use 1%~2% hydrochloric acid soaked overnight then, take out, rinse well with the abluent washing and with flowing water then, spend the night with distilled water immersion again, carry out the collaurum rinse then with a large amount of tap water flushings.
B) prepare 1% gold chloride and 1% sodium citrate solution, 0.2um membrane filtration.
C) get 1 of 1000ml round-bottomed flask, put into rotor, add the 500ml ultrapure water.
D) add 5ml 1% gold chloride, 600rpm, heat to the solution boiling, is drawn 1% sodium citrate solution 4ml immediately and is joined in the flask fast approximately.Continue the about 10min of heated and stirred, solution turns black earlier, and purpling is red gradually then.
E) cooling: close heater switch and continue and stir 10min, stop stirring then and be cooled to room temperature, return to original volume with ultrapure water.
F) the colloid gold particle diameter that makes of electron-microscope scanning is 50nm, and the collaurum that makes is subsequent use.
(3) preparation of reagent R2:
A) will resist the human C-reactive protein antibodies to be diluted to 0.1mg/ml with phosphate buffer (pH7.2);
B) regulate the pH to 8.0 of colloidal gold solution with K2CO3
C) antibody-solutions is joined in the collaurum, and stirring reaction 30 minutes, wherein the volume ratio of antibody-solutions and colloidal gold solution is 1:10;
D) be that the PEG20000 of 0.1% (w/v) joins in the above-mentioned colloidal gold solution with final concentration, and stirring reaction 30 minutes;
E) 10000rpm is centrifugal 30 minutes, redissolves to 1/25 of original volume with solution A then, makes reagent R2.The concentration that reagent R2 is marked with the colloid gold particle of anti-human C-reactive protein antibodies is 0.25%w/v.Said solution A comprises: the thimerosal of the stabilizing agent, 0.07% (w/v) of 10mmol/LTris pH of buffer 8.0,2% (w/v).Said stabilizing agent comprises: 0.9%NaCl, 0.1%PEG20000 and 1% trehalose.
Embodiment three
Kit preparation 3
(1) preparation of reagent R1
In 10mmol/L phosphate buffer (pH6.5), adding final concentration is the BSA of 1% (w/v), and adding final concentration is the PEG20000 of 1% (w/v), and adding final concentration is the NaN3 of 0.09% (w/v).Make R1 reagent thus.
(2) preparation of collaurum:
A) cleaning of glass container: rinse well with tap water flowing water, use 1%~2% hydrochloric acid soaked overnight then, take out, rinse well with the abluent washing and with flowing water then, spend the night with distilled water immersion again, carry out the collaurum rinse then with a large amount of tap water flushings.
B) prepare 1% gold chloride and 1% sodium citrate solution, 0.2um membrane filtration.
C) get 1 of 1000ml round-bottomed flask, put into rotor, add the 500ml ultrapure water.
D) add 5ml 1% gold chloride, 600rpm, heat to the solution boiling, is drawn 1% sodium citrate solution 3ml immediately and is joined in the flask fast approximately.Continue the about 10min of heated and stirred, solution turns black earlier, and purpling is red gradually then.
E) cooling: close heater switch and continue and stir 10min, stop stirring then and be cooled to room temperature, return to original volume with ultrapure water.
F) the colloid gold particle diameter that makes of electron-microscope scanning is 60nm, and the collaurum that makes is subsequent use.
(3) preparation of reagent R2:
A) will resist the human C-reactive protein antibodies to be diluted to 0.1mg/ml with phosphate buffer (pH7.2);
B) regulate the pH to 8.0 of colloidal gold solution with K2CO3
C) antibody-solutions is joined in the collaurum, and stirring reaction 30 minutes, wherein the volume ratio of antibody-solutions and colloidal gold solution is 1:10;
D) final concentration is that 1% (w/v) BSA and final concentration are that 0.05% PEG20000 joins in the above-mentioned colloidal gold solution, and stirring reaction 30 minutes;
E) 10000rpm is centrifugal 30 minutes, redissolves to 1/20 of original volume with solution A then, makes reagent R2; The concentration that reagent R2 is marked with the colloid gold particle of anti-human C-reactive protein antibodies is 0.2%w/v.Said solution A comprises: the phenol of the stabilizing agent, 1% (w/v) of 15mmol/LTris pH of buffer 8.0,1% (w/v).Said stabilizing agent comprises: 0.9%NaCl, 1%BSA, 0.05%PEG20000 and 0.2% gelatin.
Embodiment four
Full-range C-reactive protein colloid gold immune is than the clinical use of turbid detection kit:
(1) some test conditions and the parameter of kit of the present invention are following
A) analytical approach: 2 end-point methods; Wavelength 660nm; Sample size: 3ul; R1:200ul; R2:50ul; Calibrating mode: splines Spline; Reactive mode: rise; Measure temperature: 37 ℃.
B) determination step: add earlier reagent R1 200ul, add sample 3ul then, hatch 37 ℃ after 5 minutes, add 50ul reagent R2, read first point immediately, react and read another point after 5 minutes, obtain the difference of absorbance.
C) computing method: 6 calibrations are computation schema with the splines.Value according to absorbance and reference serum is done dosage/response curve, and sample size can be calculated on dosage/response curve according to its absorbance.
(2) examination criteria curve
Through method of the present invention, adopt Hitachi's 7020 type automatic analyzers to obtain the typical curve of the C-reactive protein reference serum of 6 kinds of different contents.Each point is represented the reference serum of a kind of content.Wherein the X axle is represented the content of C-reactive protein, and the Y axle is represented absorbance.
Embodiment five
The detection kit performance test:
(1) sensitivity test
Measure the sample of 5 kinds of different C-reactive protein content, each sample replication 10 times, computation of mean values and standard deviation, the result sees table 1, can know from table 1, the sensitivity of kit of the present invention is 0.01mg/L.
Table 1
?CRP(mg/L) |
Average |
SD |
M±2SD |
?0 |
0.0001 |
0.0028 |
-0.0055~0.0057 |
?0.005 |
0.0006 |
0.0056 |
-0.0086~0.0098 |
?0.01 |
0.0102 |
0.0016 |
0.0070~0.0134 |
?0.02 |
0.0198 |
0.0019 |
0.0160~0.0236 |
?0.03 |
0.0307 |
0.0027 |
0.0253~0.0361 |
(2) high value linear determination
Measure the sample of 7 kinds of different CRP content, every kind of sample is surveyed 3 times, and the result sees table 2, and through linear regretional analysis, regression equation is y=0.9707x+3.5991, R2=0.9991, and the highest detection scope of kit of the present invention reaches 500mg/L.
?CRP(mg/L) |
Average |
?0 |
0.001 |
?50 |
50.4 |
?100 |
100.1 |
[0093]?
200 |
201.1 |
300 |
301.6 |
400 |
396.7 |
500 |
480.0 |
(3) precision test
Use the human serum sample of two parts of different C-reactive protein content, test with kit of the present invention.Withinrun precision (Intra-Assay) and betweenrun precision (Inter-Assay).The result sees table 3, kit of the present invention batch in and batch between CV% all less than 10.
Table 3
(4) interference test
Prepare a duplicate samples (obtaining from hospital laboratory), sample CRP content is 7.78mg/L, gets sample segment, makes enriched sample to wherein adding the CRP standard items, is 41.26mg/L through measuring CRP concentration.In the CRP of above-mentioned two kinds of concentration blood serum sample, add the chaff interference that concentration is the maximum concentration of pathology sample respectively, adopt kit of the present invention to measure then respectively, each concentration determination 20 times.The result sees table 4, chaff interference to the interference of this kit all in 2%.
Table 4