CN104730252A - Method, reagent and kit for quantitative determination of procalcitonin in human serum - Google Patents
Method, reagent and kit for quantitative determination of procalcitonin in human serum Download PDFInfo
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- CN104730252A CN104730252A CN201510070009.4A CN201510070009A CN104730252A CN 104730252 A CN104730252 A CN 104730252A CN 201510070009 A CN201510070009 A CN 201510070009A CN 104730252 A CN104730252 A CN 104730252A
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- procalcitonin
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/575—Hormones
- G01N2333/5753—Calcitonin gene related peptide
Abstract
The invention relates to a reagent for quantitative determination of procalcitonin in human serum, the reagent is composed of a reagent I and a reagent II which are placed individually, wherein the reagent I contains sodium dihydrogen phosphate, disodium hydrogen phosphate, polyethylene glycol 8000 and sodium chloride; and the reagent II contains sodium dihydrogen phosphate, disodium hydrogen phosphate, goat anti-human procalcitonin antibody, Twain-20, bovine albumin and latex. The kit and the detection method adopted by the invention only need few tens of microlitres of serum, are free from separation treatment, such as centrifugation or electrophoresis, are simple and convenient to operate, can achieve the requirement of full-automatic analysis, and are suitable for large-scale in-time and accurate detection on samples.
Description
Technical field
The application relates to Procalcitonin method for quantitatively determining, reagent and kit in a kind of human serum.
Background technology
Procalcitonin is a kind of protein, and when serious bacterial, fungi, parasitic infection and pyemia and MOFE, its level in blood plasma raises.When autoimmunity, allergy and virus infections, Procalcitonin can not raise.The bacteriological infection of local finite, slight infection and chronic inflammation can not cause it to raise.Bacterial endotoxin has served as vital effect in Induction Process.
Procalcitonin reflects the active degree of systemic inflammatory response.The factor affecting Procalcitonin Concentrations comprises size and type, the kind of bacterium, the degree of inflammation and the immunoreactive situation of infected organ.In addition, Procalcitonin just can measure at the large-scale surgical postoperative 1 ~ 4d of small number of patients.
The rising of Procalcitonin Concentrations appears at serious shock, systemic inflammatory response syndrome (SIRS) and multiple organ dysfunction derangement syndrome (MODS), even without bacteriological infection or bacillary focus.But Procalcitonin Concentrations is usually less than the patient that those have bacillary focus in these cases.Induction may be caused from the enteron aisle release cells factor or bacterial translocation.
Procalcitonin is the parameter that diagnosis and monitoring bacterium inflammatory disease infect.The mensuration of Procalcitonin can indicate: come antidiastole bacillary and non-bacterial infection and inflammation as an acute parameter.Monitoring have the patient of risk of infection and need intensive care patient, be used for detect bacteriological infection systemic effects or detect septic complications; Evaluate serious inflammatory Disease Clinical process and prognosis.
At present, the method detecting Procalcitonin has a lot, not only can be qualitative, can also be quantitative, and conventional method has: gel chromatography and efficient liquid phase chromatographic analysis, enzyme linked immunosorbent assay (ELISA), immunoluminescence method and colloidal gold chromatography.These methods have the shortcoming of complex operation, length consuming time, expensive, excess waste resource, are unsuitable for routine inspection, detect while especially extensive epidemic investigation or all entries of clinical sample in enormous quantities.
Summary of the invention
The object of the application is Procalcitonin method for quantitatively determining, reagent and kit in a kind of human serum.
The application have employed following technical scheme:
The one side of the application discloses the reagent of Procalcitonin content in a kind of quantitatively determining human serum, this reagent is made up of the reagent I placed respectively and reagent II, wherein, described reagent I contains sodium dihydrogen phosphate, sodium hydrogen phosphate, PEG 8000, sodium chloride; Described reagent II contains sodium dihydrogen phosphate, sodium hydrogen phosphate, goat-anti HCT original antibody, Tween-20, bovine albumin, latex.
Further, in described reagent I, biphosphate sodium content is 50-150mmol/L, and sodium hydrogen phosphate content is 50-150mmol/L, and the concentration of PEG 8000 is 1.5-2.8%, and sodium chloride content is 0.1-0.5mol/L.
Further, in described reagent I, biphosphate sodium content is 100mmol/L, and sodium hydrogen phosphate content is 100mmol/L, and the concentration of PEG 8000 is 2.5%, and sodium chloride content is 0.22mol/L.
Further, in described reagent II, biphosphate sodium content is 50-150mmol/L, sodium hydrogen phosphate content is 50-150mmol/L, goat-anti HCT original antibody content is 0.1-0.5g/L, Tween-20 content is 0.05-0.5% (V/V), bovine albumin content is 0.1-0.5%, and content of latex is 1-2g/L.
Further, in described reagent II, biphosphate sodium content is 100mmol/L, and sodium hydrogen phosphate content is 100mmol/L, goat-anti HCT original antibody content is 0.2g/L, Tween-20 content is 0.1%, and bovine albumin content is 0.4% (V/V), and content of latex is 1.4g/L.
The another aspect of the application provides the kit of Procalcitonin content in a kind of quantitatively determining human serum, and the reagent of Procalcitonin content in above-mentioned quantitatively determining human serum is wherein housed, and this reagent is made up of the reagent I placed respectively and reagent II.
The another aspect of the application provides the method for Procalcitonin content in a kind of quantitatively determining human serum, the method to comprise in blood serum sample, to add described reagent I, hatch 1-5 minute for 37 DEG C, and blank tube returns to zero, working sample absorbance A 1 under certain wavelength; Then in sample, add reagent II, after continuing to hatch 5 minutes, under above-mentioned wavelength, measure absorbance A 2, calculate △ A sample by following formula (1); Use the same method and measure the absorbance △ A standard of calibration solution; The Procalcitonin content of blood serum sample is calculated again by following formula (2):
△A＝A2-A1 (1)
Procalcitonin content (ng/mL) in serum=(△ A sample-△ A is blank)/(△ A standard-△ A is blank) × concentration of standard solution (2)
We's ratio juris is: reacted by the sensitization latex particle of goat-anti HCT original antibody in the Procalcitonin in sample and reagent, there is agglutinating reaction, in the change of its absorbance of 510nm wavelength detecting, its intensity of variation is directly proportional to the Procalcitonin content in sample.
Direct quantitative of the present invention measures the kit of Procalcitonin content in human serum sample, is to be loaded in kit package with different specifications by the mentioned reagent I placed respectively and reagent II.This kit has multiple different specification, can be applicable to the automated chemical analyser of the various domestic and international brand generally used in clinical labororatory at present respectively.
The kit that the present invention adopts and detection method only need tens HL serum, without the need to separating treatment such as centrifugal or electrophoresis, easy and simple to handle, can meet the full-automatic requirement analyzed, be applicable to the promptly and accurately detection of extensive sample.
Embodiment
Embodiment one
According to following compositions and the following reagent I of the present invention and reagent II of proportional arrangement:
Reagent I:
Reagent II:
By 240 μ l reagent I and the mixing of 30 μ l blood serum samples in sample hose, 1-5 minute is hatched at 37 DEG C, use Hitachi 7180 type automatic clinical chemistry analyzer, blank tube returns to zero, measure absorbance A 1 at wavelength 510nm place, in sample, then add 60 μ l reagent II, mixing, at 37 DEG C, hatch 5 minutes, measure the absorbance A 2 under phase co-wavelength.△ A sample is calculated according to following formula (1).Use the same method and the absorbance of condition bioassay standard liquid, and calculate △ A standard.Then the Procalcitonin content of blood serum sample is calculated according to formula (2):
△A＝A2-A1 (1)
Procalcitonin content (ng/mL) in serum=(△ A sample-△ A is blank)/(△ A standard-△ A is blank) × concentration of standard solution (2)
The application's standard items used are Landau standard items.
Embodiment two
According to following compositions and the following reagent I of the present invention and reagent II of proportional arrangement:
Reagent I:
Reagent II:
By 240 μ l reagent I and the mixing of 30 μ l blood serum samples in sample hose, 1-5 minute is hatched at 37 DEG C, use Abbott Laboratories C16000 type automatic clinical chemistry analyzer, blank tube returns to zero, measure absorbance A 1 at wavelength 510nm place, in sample, then add 60 μ l reagent II, mixing, at 37 DEG C, hatch 5 minutes, measure the absorbance A 2 under phase co-wavelength.△ A sample is calculated according to following formula (1).Use the same method and the absorbance of condition bioassay standard liquid, and calculate △ A standard.Then the Procalcitonin content of blood serum sample is calculated according to formula (2):
△A＝A2-A1 (1)
Procalcitonin content (ng/mL) in serum=(△ A sample-△ A is blank)/(△ A standard-△ A is blank) × concentration of standard solution (2)
Embodiment three
According to following compositions and the following reagent I of the present invention and reagent II of proportional arrangement:
Reagent I:
Reagent II:
By 240 μ l reagent I and the mixing of 30 μ l blood serum samples in sample hose, 1-5 minute is hatched at 37 DEG C, use Olympus AU400 type automatic clinical chemistry analyzer, blank tube returns to zero, measure absorbance A 1 at wavelength 510nm place, in sample, then add 60 μ l reagent II, mixing, at 37 DEG C, hatch 5 minutes, measure the absorbance A 2 under phase co-wavelength.△ A sample is calculated according to following formula (1).Use the same method and the absorbance of condition bioassay standard liquid, and calculate △ A standard.Then the Procalcitonin content of blood serum sample is calculated according to formula (2):
△A＝A2-A1 (1)
Procalcitonin content (ng/mL) in serum=(△ A sample-△ A is blank)/(△ A standard-△ A is blank) × concentration of standard solution (2)
Embodiment four
Use reagent listed in the present embodiment 1, measure Procalcitonin content in the serum of 100 routine blood serum samples according to the method described in embodiment 1 and condition, every part of blood serum sample carries out contrastive mensuration with Procalcitonin assay kit in commercially available serum (norman bio tech ltd, Nanjing) simultaneously.
1 SPSS quantitative statistical analysis
1.1 statistics describe
Table 1 descriptive statistics amount
Table 2 descriptive statistics amount
1.2 paired-samples T-test
Table 3 paired samples related coefficient
| N | Related coefficient | Sig. | |
| To 1 data & contrasting data of the present invention | 100 | .998 | .000 |
Table 4 paired samples is checked
Table 5 paired samples is checked
| t | df | Sig. (bilateral) | |
| To 1 data-contrasting data of the present invention | 5.121 | 99 | .102 |
Wherein, P=0.102, > 0.05, showing that two groups of data are separate, is the result of two different kits
1.3 bivariate correlativitys
Table 6 correlativity
*. at the upper significant correlation of .01 level (bilateral).
R＝0.998
1.4 novel regretional analysis and scatter diagrams
Table 7 model gathers
| Model | R | R side | Adjustment R side | The error that standard is estimated |
| 1 | .998 a | .996 | .996 | 2.74789 |
A. predictive variable: (constant), contrasting data
B. dependent variable: data of the present invention.
Table 8 coefficient
a
A. dependent variable: VAR00127
Equation: Y=1.00*X+1.409, r=0.998, X-axis: contrasting data, Y-axis: data of the present invention
The qualitative statistical study of 2 clinical effectiveness
Clinical research Procalcitonin Measurement kit and reference product measurement result are analyzed:
Cross tabulating is analyzed
Table 9 clinical definite and control group reagent
Take reference product as reference group:
Positive coincidence rate=[29/ (29+2)] × 100%=93.55%
Negative match-rate=[67/ (67+2)] × 100%=97.10%
Total consistance=[(67+31)/100] × 100%=98.00%
3 Performance Analysis
3.1 accuracy
Carry out three horizontal surveies with known Procalcitonin definite value serum to measure, get average, draw:
Table 10
3.2 repeated
Same batch of reagent carries out 10 replications to same sample, calculates sample measurement result mean value, draws batch interior imprecision: CV≤6.0%.
Table 11
3.3 the range of linearity
Choose the serum of Procalcitonin 80ng/ml, doubling dilution is carried out with physiological saline, get five gradient concentrations, each concentration replication is averaged for 3 times, calculate theoretical concentration and corresponding mensuration concentration average carries out linear regression analysis, calculate correlation coefficient r, theoretical value is horizontal ordinate, measured value is that ordinate makes scatter diagram, both discoveries have good linear relation, carry out straight-line regression statistical study, regression equation is Y=125.477X-2.235, and has statistical significance (p<0.0001).
Table 12
Show that this law is in 0.2-80ng/ml Levels of Serum Procalcitonin concentration range, measured value and theoretical value related coefficient are r:0.999.
4. discuss and conclusion
In above-mentioned 100 routine samples, Procalcitonin monstrosity 30 example, Procalcitonin normal specimen 70 example is by cross tabulating analysis, SPSS software statistics, data statistics result shows: accuracy of the present invention is high, reproducible, the range of linearity is wide, meets better with reference product in positive coincidence rate, negative match-rate, total consistance and correlativity etc.Therefore, Procalcitonin Measurement kit of the present invention can meet clinical performance requirement, and validity is strong, and security is high, can meet the full-automatic requirement analyzed, be applicable to the promptly and accurately detection of extensive sample.
Above content is the further description done the application in conjunction with concrete embodiment, can not assert that the concrete enforcement of the application is confined to these explanations.For the application person of ordinary skill in the field, under the prerequisite not departing from the application's design, some simple deduction or replace can also be made, all should be considered as the protection domain belonging to the application.
Claims (7)
1. the reagent of Procalcitonin content in quantitatively determining human serum, this reagent is made up of the reagent I placed respectively and reagent II, and wherein, described reagent I contains sodium dihydrogen phosphate, sodium hydrogen phosphate, PEG 8000, sodium chloride; Described reagent II contains sodium dihydrogen phosphate, sodium hydrogen phosphate, goat-anti HCT original antibody, Tween-20, bovine albumin, latex.
2. reagent according to claim 1, it is characterized in that, in described reagent I, biphosphate sodium content is 50-150mmol/L, and sodium hydrogen phosphate content is 50-150mmol/L, the concentration of PEG 8000 is 1.5-2.8%, and sodium chloride content is 0.1-0.5mol/L.
3. reagent according to claim 2, is characterized in that, in described reagent I, biphosphate sodium content is 100mmol/L, and sodium hydrogen phosphate content is 100mmol/L, and the concentration of PEG 8000 is 2.5%, and sodium chloride content is 0.22mol/L.
4. reagent according to claim 1, it is characterized in that, in described reagent II, biphosphate sodium content is 50-150mmol/L, sodium hydrogen phosphate content is 50-150mmol/L, goat-anti HCT original antibody content is 0.1-0.5g/L, Tween-20 content is 0.05-0.5%, and bovine albumin content is 0.1-0.5%, and content of latex is 1-2g/L.
5. reagent according to claim 4, it is characterized in that, in described reagent II, biphosphate sodium content is 100mmol/L, sodium hydrogen phosphate content is 100mmol/L, goat-anti HCT original antibody content is 0.2g/L, Tween-20 content is 0.1%, and bovine albumin content is 0.4%, and content of latex is 1.4g/L.
6. the kit of Procalcitonin content in a quantitatively determining human serum, it is characterized in that, the reagent of Procalcitonin content in arbitrary described quantitatively determining human serum in claim 1 to 5 is wherein housed, and this reagent is made up of the reagent I placed respectively and reagent II.
7. the method for Procalcitonin content in quantitatively determining human serum, the method comprises and add described reagent I in blood serum sample, hatches 1-5 minute for 37 DEG C, and blank tube returns to zero, working sample absorbance A 1 under certain wavelength; Then in sample, add reagent II, after continuing to hatch 5 minutes, under above-mentioned wavelength, measure absorbance A 2, calculate △ A sample by following formula (1); Use the same method and measure the absorbance △ A standard of calibration solution; The Procalcitonin content of blood serum sample is calculated again by following formula (2):
△A＝A2-A1 (1)
Procalcitonin content (ng/mL) in serum=(△ A sample-△ A is blank)/(△ A standard-△ A is blank) × concentration of standard solution (2).
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106198993A (en) * | 2016-06-22 | 2016-12-07 | 浙江达美生物技术有限公司 | A kind of mensuration reagent of Procalcitonin. albumen and preparation method thereof |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106198993A (en) * | 2016-06-22 | 2016-12-07 | 浙江达美生物技术有限公司 | A kind of mensuration reagent of Procalcitonin. albumen and preparation method thereof |
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Application publication date: 20150624 |