CN108303545A - Therapeutic evaluation and prognosis evaluation reagent kit and application thereof after pulmonary tuberculosis intensive treatment - Google Patents
Therapeutic evaluation and prognosis evaluation reagent kit and application thereof after pulmonary tuberculosis intensive treatment Download PDFInfo
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Abstract
The invention discloses therapeutic evaluation and prognosis evaluation reagent kit and application thereof after pulmonary tuberculosis intensive treatment, which includes ELISA Plate, Protein standards composition and biotin labelled antibodies composition；Wherein the ELISA Plate is coated with four kinds of ELISA Plates of anti-ANGT antibody, anti-CO7 antibody, anti-PLMN antibody, anti-APOC2 antibody respectively；The Protein standards composition includes ANGT, CO7, PLMN and APOC2 Protein standards；The biotin labelled antibodies composition includes the anti-APOC2 antibody of the anti-ANGT antibody of biotin labeling, the anti-CO7 antibody of biotin labeling, the anti-PLMN antibody of biotin labeling and biotin labeling.The present invention is 0.779 to the AUC value of pulmonary tuberculosis intensive treatment phase therapeutic evaluation, it is 0.837 to pulmonary tuberculosis intensive treatment prognosis evaluation AUC value, prognosis rate of accuracy reached 84.62%, with higher accuracy, it can be achieved, to consumptive's intensive treatment therapeutic evaluation and prognosis evaluation, new evaluation method to be provided for the treatment of pulmonary tuberculosis.
Description
Technical field
The invention belongs to curative effect of disease evaluation and prognosis evaluation technical fields, more particularly to are treated after pulmonary tuberculosis intensive treatment
Effect evaluation and prognosis evaluation reagent kit and application thereof.
Background technology
Pulmonary tuberculosis is chronic infectious disease caused by being infected by mycobacterium tuberculosis, at present the conventional therapy of pulmonary tuberculosis point
For intensive treatments in 2 months and after treatment in 4 months.In the treatment of pulmonary tuberculosis, the sputum bacteria for just controlling the positive patient of painting is turned out cloudy
Rate is to reflect the core index of therapeutic effect.And for the patient after pulmonary tuberculosis intensive treatment, sputum bacteria transformation situation is to adjust
Whole therapeutic scheme, the most important thing for thoroughly curing disease.
Currently, feelings can only be changed to the sputum bacteria of consumptive by way of traditional Sputum smears or Phlegm incubation
Condition is assessed, so as to adjust therapeutic scheme.But Sputum smears have the characteristics that positive rate is low, poor specificity, and contained by patient
The influence of many factors such as amount of expectoration, detection technique.And the Phlegm incubation method of goldstandard is diagnosed as pulmonary tuberculosis, time-consuming,
2~8 weeks incubation times are needed, and specificity is only 57.8%, has seriously affected the timely adjustment of therapeutic scheme.And some patientss
Have the characteristics that no phlegm, few phlegm, treatment evaluation can not be carried out by the method that phlegm is examined.Therefore, it is clinical there is an urgent need to it is a kind of it is new,
Efficiently, special pulmonary tuberculosis intensive treatment phase therapeutic evaluation and prognostic evaluation methods.
Invention content
In view of this, it is an object of the invention to propose therapeutic evaluation and prognosis evaluation reagent after pulmonary tuberculosis intensive treatment
Box and application thereof, therapeutic evaluation and prognosis evaluation reagent kit can efficiently and accurately Pre-Evaluation after the pulmonary tuberculosis intensive treatment
Curative effect after consumptive's intensive treatment and treatment prognosis situation, have higher specificity and accuracy.
Based on above-mentioned purpose, therapeutic evaluation and prognosis evaluation reagent kit after pulmonary tuberculosis intensive treatment provided by the invention,
Including ELISA Plate, Protein standards composition and biotin labelled antibodies composition；Wherein the ELISA Plate is coated with respectively
There are four kinds of ELISA Plates of anti-ANGT antibody, anti-CO7 antibody, anti-PLMN antibody, anti-APOC2 antibody；The Protein standards group
It includes ANGT, CO7, PLMN and APOC2 Protein standards to close object；The biotin labelled antibodies composition includes biotin mark
Anti- ANGT antibody, the anti-CO7 antibody of biotin labeling, the anti-PLMN antibody of biotin labeling and the biotin labeling remembered resist
APOC2 antibody.
In some embodiments of the invention, ANGT, CO7, PLMN and APOC2 Protein standards concentration difference
For 100~500ng/mL, 20~60ng/mL, 40~120ng/mL, 50~150ng/mL.
In some embodiments of the invention, further include sample and antibody diluent, detergent, horseradish peroxidase mark
Remember Avidin, Chromogenic Substrate Solution, terminate liquid and ELISA Plate note plate.
In some embodiments of the invention, the sample and antibody diluent be 0.01mol/L pH be 7.2 it is molten
Liquid, including 0.39g/L NaH2PO4·2H2O、1.27g/L Na2HPO4、0.85g/L NaCl、200mg/L NaN3Aqueous solution；
The pH value of the detergent is 7.4, including 2.0g/L NaCl, 0.2g/L KH2PO4、2.9g/L Na2HPO4·
12H2O、0.2g/L KCl、0.2g/L NaN3, 0.5mL/L polysorbas20 aqueous solution；
The Chromogenic Substrate Solution be 0.8~1.2% containing volume ratio 3,3', 5,5'- tetramethyl benzidines are molten
Liquid；The terminate liquid is the H of 1.5~3mol/L2SO4Aqueous solution.
Based on identical inventive concept, the present invention also provides therapeutic evaluatioies after the pulmonary tuberculosis intensive treatment and prognosis to comment
Estimate the application method of kit, in particular to uses therapeutic evaluation after the pulmonary tuberculosis intensive treatment and prognosis evaluation reagent kit
Curative effect after consumptive's intensive treatment and prognostic evaluation, the treatment for instructing consumptive's consolidation.
The present invention provides the users of therapeutic evaluation and prognosis evaluation reagent kit after the pulmonary tuberculosis intensive treatment
Method, using curative effect after therapeutic evaluation after pulmonary tuberculosis intensive treatment and prognosis evaluation reagent kit progress pulmonary tuberculosis intensive treatment
Evaluation, specifically includes following steps：
(1) peripheral blood is acquired, centrifuges, suct clearly；
(2) by the supernatant diluted obtained by step (1), four kinds of ELISA Plates are separately added into, are incubated；
(3) ELISA Plate is patted dry, diluted biotin labelled antibodies are separately added into, is incubated, is washed with the detergent；
(4) diluted Horseradish peroxidase-conjugated avidin is added, is washed with the detergent after incubation；
(5) Chromogenic Substrate Solution is added in each reacting hole, is incubated；
(6) terminate liquid is added in each reacting hole and terminates reaction, each reacting hole OD values are detected in microplate reader；
(7) each sample albumen concentration is calculated, the regression equation of Logistic gradually models is brought into, lung knot is evaluated after being computed
Core disease intensive treatment curative effect.
In some embodiments of the invention, the regression equation of the Logistic gradually models is：
Logit (p)=0.167-0.030 × (APOC2 albumen concentration)+0.064 × (CO7 albumen concentration) -0.011 ×
(PLMN albumen concentration)
Logit (p) value is obtained, P values are obtained by P=exp [Logit (p)]/(1+exp [Logit (p)]) conversions, with
0.5 is boundary, judges pulmonary tuberculosis intensive treatment curative effect.
The present invention also provides the uses of therapeutic evaluation and prognosis evaluation reagent kit after the pulmonary tuberculosis intensive treatment
Method is carried out pre- after pulmonary tuberculosis intensive treatment using therapeutic evaluation after pulmonary tuberculosis intensive treatment and prognosis evaluation reagent kit
After assess, specifically include following steps：
(1) peripheral blood is acquired, centrifuges, suct clearly；
(2) by the supernatant diluted obtained by step (1), four kinds of ELISA Plates are separately added into, are incubated；
(3) ELISA Plate is patted dry, diluted biotin labelled antibodies are separately added into, is incubated, is washed with the detergent；
(4) diluted Horseradish peroxidase-conjugated avidin is added, is washed with the detergent after incubation；
(5) Chromogenic Substrate Solution is added in each reacting hole, is incubated；
(6) terminate liquid is added in each reacting hole and terminates reaction, each reacting hole OD values are detected in microplate reader；
(7) each sample albumen concentration is calculated, each sample albumen concentration is substituted into decision-tree model, judges affiliated Class classes
Not, according to the classification of affiliated Class 0 or Class 1, judge the prognosis situation after pulmonary tuberculosis intensive treatment.
In some embodiments of the invention, ANGT, CO7 protein concentration are substituted into decision-tree model, belonging to judgement
Class classifications are determined as that Class's 1 shows that good therapeutic effect can be reached by continuing after treatment after intensive treatment,
It is determined as that Class's 0 shows that continue after treatment prognosis after intensive treatment poor, needs suitably to adjust therapeutic scheme.
In the present invention, the decision-tree model program of prognosis evaluation can be the prior art after the pulmonary tuberculosis intensive treatment
In program or following programs：
Further, the present invention also provides therapeutic evaluation and prognosis evaluation reagents after the pulmonary tuberculosis intensive treatment
Purposes of the box after preparing pulmonary tuberculosis intensive treatment in therapeutic evaluation reagent.
In addition, the present invention also provides therapeutic evaluatioies after the pulmonary tuberculosis intensive treatment and prognosis evaluation reagent kit to exist
Purposes after preparation pulmonary tuberculosis intensive treatment in prognosis evaluation reagent.
The principle of therapeutic evaluation and prognosis evaluation reagent kit is after pulmonary tuberculosis intensive treatment of the present invention：
Serum tetra- kinds of protein expression levels of ANGT, CO7, PLMN, APOC2 are detected using ELISA method, wherein
ANGT albumen is hypertensinogen (Angiotensinogen), is the important composition portion of hypertensin system
Point, it is a kind of effective blood pressure, body fluid and electrolyte balance conditioning agent；
CO7 albumen, be complement component C7 (Complement component C7), be membrane attack complex at point it
One, can in the plasma membrane of target cell pore-forming, and then play a crucial role in congenital and adaptive immune response；
PLMN albumen is plasminogen (Plasminogen), is the premise substance of plasmin,
By the effect of plasminogen activators, activity becomes plasmin；
APOC2 albumen is Apolipoprotein C2 (Apolipoprotein C-II), is chylomicron, extra-low density fat egg
In vain, the constituent of low-density lipoprotein and high-density lipoprotein.Lipoprotein lipase can be activated, is risen in lipoprotein metabolism
Important function；This in four protein with there are certain functional cohesions with consumptive.
According to the characteristic of protein, above-mentioned APOC2 albumen, PLMN albumen, CO7 albumen these three expressing quantities are brought into
Into Logistic Gradual regression analysis models, the curative effect that is computed after the rear preliminary judgement intensive treatment phase；Again by ANGT, CO7 albumen
Expression quantity substitutes into decision-tree model, and the prognosis situation strengthened after phase treatment to patient by decision-tree model is assessed.
By the above as it can be seen that therapeutic evaluation and prognosis evaluation reagent after pulmonary tuberculosis intensive treatment provided by the invention
Box judges the curative effect after pulmonary tuberculosis intensive treatment, and further assessment patient's prognosis situation using serum proteins composition,
Easy to operate, quick, detection efficiency is good, and has higher sensitivity and specificity, is applied better than phlegm used by clinic, phlegm
The methods of culture and rabat detection, a kind of new side is provided for the treatment evaluation of pulmonary tuberculosis intensive treatment phase and prognostic evaluation
Method.
Description of the drawings
Fig. 1 is normal healthy controls person of the embodiment of the present invention, just controls pulmonary tuberculosis after non-medication consumptive, intensive treatment
The expression comparison schematic diagram of patients serum's APOC2 protein；
Fig. 2 is normal healthy controls person of the embodiment of the present invention, just controls pulmonary tuberculosis after non-medication consumptive, intensive treatment
The expression comparison schematic diagram of patients serum's CO7 protein；
Fig. 3 is normal healthy controls person of the embodiment of the present invention, just controls pulmonary tuberculosis after non-medication consumptive, intensive treatment
The expression comparison schematic diagram of patients serum's PLMN protein；
Fig. 4 be normal healthy controls person of the embodiment of the present invention, just control non-medication consumptive, intensive treatment after sputum bacteria do not turn
Sputum bacteria is turned out cloudy the expression comparison schematic diagram of the serum ANGT protein of patient after cloudy patient, intensive treatment；
Fig. 5 be normal healthy controls person of the embodiment of the present invention, just control non-medication consumptive, intensive treatment after sputum bacteria do not turn
Sputum bacteria is turned out cloudy the expression comparison schematic diagram of the change of serum C O7 protein of patient after cloudy patient, intensive treatment；
Fig. 6 is the ROC curve schematic diagram of serum of embodiment of the present invention APOC2, CO7, PLMN and composition；
Fig. 7 is serum ANGT, CO7 decision-tree model figure of the embodiment of the present invention.
Specific implementation mode
To make the objectives, technical solutions, and advantages of the present invention clearer, below in conjunction with specific embodiment, and reference
Attached drawing, the present invention is described in more detail.
Embodiment 1：The composition of therapeutic evaluation and prognosis evaluation reagent kit after pulmonary tuberculosis intensive treatment
In the present embodiment, therapeutic evaluation and prognosis evaluation reagent kit after the pulmonary tuberculosis intensive treatment, including enzyme mark
Plate, Protein standards composition and biotin labelled antibodies composition, sample and antibody diluent, detergent, horseradish peroxide
Compound enzyme marks Avidin, Chromogenic Substrate Solution, terminate liquid and ELISA Plate note plate, specially：
(1) ELISA Plate includes：It is coated with the ELISA Plate of anti-ANGT antibody, is purchased from ray biotech companies；It is coated with
The ELISA Plate of anti-CO7 antibody, the ELISA Plate for being coated with anti-PLMN antibody are purchased from abcam companies；It is coated with anti-APOC2 antibody
ELISA Plate is purchased from abnova companies.
(2) the Protein standards composition includes：ANGT, CO7, PLMN and APOC2 Protein standards.Wherein,
ANGT standard items are purchased from ray biotech companies, and CO7, PLMN standard items are purchased from abcam companies, and APOC2 standard items are purchased from
Abnova companies.
(3) the biotin labelled antibodies composition includes：The anti-ANGT antibody of biotin labeling is purchased from ray
Biotech companies；The anti-CO7 antibody of biotin labeling, the anti-PLMN antibody of biotin labeling are purchased from abcam companies；Biotin
The anti-APOC2 antibody of label is purchased from abnova companies.
(4) Horseradish peroxidase-conjugated avidin divides purchased from ray biotech, abcam and abnova companies
It Yong Yu not corresponding protease target.
(5) sample and antibody diluent：
By 0.39g NaH2PO4·2H2O、1.27g Na2HPO4、0.85g NaCl、200mg NaN3, with 1000mL H2O
Dissolving adjusts pH to 7.2, dilutes 10 times with distilled water using preceding, is used as sample and antibody diluent.
(6) detergent：
2.0g NaCl、0.2g KH2PO4、2.9g Na2HPO4·12H2O、0.2g KCl、0.2g NaN3, use 1000mL
H2O dissolves, and adds 0.5mL tweens (Tween) 20, adjusts pH to 7.4.
(7) Chromogenic Substrate Solution：
With absolute ethyl alcohol or dimethylformamide by 3,3', it is 0.8 that 5,5'- tetramethyl benzidines (TMB), which are made into volume ratio,
~1.2% solution can be kept in dark place in 4 DEG C.
As a preferred embodiment, the chromogenic substrate is with absolute ethyl alcohol by 3,3', 5,5'- tetramethyl benzidines
(TMB) it is made into the solution that volume ratio is 1%, can be kept in dark place in 4 DEG C, is used in half a year.
(8) terminate liquid：
1.5~3mol/L H2SO4Aqueous solution.
As a preferred embodiment, terminate liquid is 2mol/L H2SO4Aqueous solution.
Embodiment 2：Therapeutic evaluation and prognosis evaluation reagent kit Logistic successive Regression moulds after pulmonary tuberculosis intensive treatment
Type is established
Normal healthy controls person is detected using therapeutic evaluation after pulmonary tuberculosis intensive treatment and prognosis evaluation reagent kit respectively, is just controlled
ANGT, CO7, PLMN, APOC2 protein in consumptive's serum after non-medication consumptive, intensive treatment
Content.
(1) serum sample collection：
According to the pulmonary tuberculosis diagnostic criteria that ministry of Health of China is issued, acquisition just controls non-medication consumptive 48,
Consumptive 81 after intensive treatment, normal healthy controls person 40.Subject excludes tumour, lung outer tuberculosis, diabetes, second
The diseases such as liver, AIDS, non-tuberculous mycobacteria, drug Resistant Pulmonary Tuberculosis disease, and exclude immunosuppressor user.Between each group patient
Age, gender are without significant difference.
All subjects are to extract on an empty stomach from morning, using disposal vacuum not anticoagulant blood-collecting pipe, acquire peripheral blood
5.0mL, within 4 hours, 3000g, 10min, 4 DEG C, centrifugation.Clear rear packing is sucted, is preserved in -80 DEG C of refrigerators.All patients,
Normal healthy controls person's age, the equal no difference of science of statistics of gender.
(2) protein expression level measures：
The serum sample for taking normal healthy controls person, just controlling consumptive after non-medication consumptive, intensive treatment,
Using dilution, by 1：100 dilution proportions, for measuring ANGT protein contents；
The serum sample for taking normal healthy controls person, just controlling consumptive after non-medication consumptive, intensive treatment,
Using dilution, by 1：8000 dilution proportions, for measuring CO7 protein contents；
The serum sample for taking normal healthy controls person, just controlling consumptive after non-medication consumptive, intensive treatment,
Using dilution, by 1：20000 dilution proportions, for measuring PLMN protein contents；
The serum sample for taking normal healthy controls person, just controlling consumptive after non-medication consumptive, intensive treatment,
Using dilution, by 1：20000 dilution proportions, for measuring APOC2 protein contents.
ANGT, CO7, PLMN, APOC2 Protein standards concentration is respectively 100~500ng/mL, 20~60ng/
ML, 40~120ng/mL, 50~150ng/mL.
As a preferred embodiment, ANGT, CO7, PLMN, APOC2 Protein standards concentration is respectively
300ng/mL, 40ng/mL, 80ng/mL, 100ng/mL.ANGT standard items are diluted with dilution when use, establish 300ng/
Eight concentration of mL, 120ng/mL, 48ng/mL, 19.2ng/mL, 7.68ng/mL, 3.072ng/mL, 1.229ng/mL, 0ng/mL
Standard curve；CO7 standard items dilute, and establish 40ng/mL, 20ng/mL, 10ng/mL, 5ng/mL, 2.5ng/mL, 1.25ng/
The standard curve of eight concentration of mL, 0.625ng/mL, 0ng/mL；PLMN standard items dilute, establish 80ng/mL, 40ng/mL,
The standard curve of eight concentration of 20ng/mL, 10ng/mL, 5ng/mL, 2.5ng/mL, 1.25ng/mL, 0ng/mL；APOC2 standards
Product dilute, and establish 100ng/mL, 50ng/mL, 25ng/mL, 12.5ng/mL, 6.25ng/mL, 3.125ng/mL, 1.563ng/
The standard curve of eight concentration of mL, 0ng/mL.
Serum to be checked after dilution is separately added into and is coated with anti-ANGT antibody, CO7 antibody, PLMN antibody, APOC2 antibody
Four kinds of ELISA Plates in, stick ELISA Plate pasting board, in incubation at room temperature 1~2.5 hour.
Liquid is abandoned, ELISA Plate is patted dry, detergent is used in combination to wash 4~5 times.By 1：50 ratio, with diluted biotin
The antibody of label, and the antibody of corresponding biotin labeling is added in each reacting hole, ELISA Plate pasting board is sticked, in incubation at room temperature
1 hour.
Liquid is abandoned, ELISA Plate is patted dry, detergent is used in combination to wash 4~5 times.By 1：100 ratio, with diluted horseradish mistake
Oxide enzyme marks Avidin, and the Horseradish peroxidase-conjugated avidin of releasing of diluted fresh, patch are added in each reacting hole
Upper ELISA Plate pasting board, in incubation at room temperature 30~45 minutes.
Liquid is abandoned, ELISA Plate is patted dry, detergent is used in combination to wash 4~5 times.Phosphoric acid-citric acid the substrate for being 5.0 by 9.9mL pH
Liquid (0.2mol/L Na2HPO410.3mL and 0.1mol/L citric acids 9.7mL is mixed with acquisition) colour developings of 0.1mL 1% are added
In substrate solution, then toward every milliliter of Chromogenic Substrate Solution the H that mass fraction is 30% is added2O21 μ L of solution, after mixing immediately plus
Enter into each reacting hole, stick ELISA Plate pasting board, in incubation at room temperature 15~30 minutes.
50 μ L of terminate liquid are added in each reacting hole and terminate reaction, and detect each reacting hole with microplate reader in 5 minutes
OD values at 450nm, and returned to zero with blank control wells.
3. data processing and statistical analysis：
It according to the OD values of ELISA Plate reacting hole, is converted by standard curve, obtains the corresponding protein of all reaction samples
Content is right to health using Nonparametric Mann-Whitney U test statistical methods by 16.0 softwares of SPSS
According to person, just controls the protein expression level of consumptive after non-medication consumptive, intensive treatment and analyze.It is logical
Cross 5 Software on Drawing protein expression level scatter plots of GraphPad Prism (referring to Fig. 1-Fig. 5).
Logistic Gradual regression analysis model analyses are carried out by 16.0 softwares of SPSS, are built by CO7, PLMN, APOC2 egg
The pulmonary tuberculosis of white matter composition treats the logistic regression equation of therapeutic evaluation after strengthening：
Logit (p)=0.167-0.030 × (APOC2 albumen concentration)+0.064 × (CO7 albumen concentration) -0.011 ×
(PLMN albumen concentration)
The ROC curve and area under the curve of each single protein and the protein model are calculated by MedCalc softwares again
(AUC), as a result show that the AUC value of CO7, PLMN, APOC2 are 0.706,0.688,0.692 respectively, accuracy is relatively low, and the egg
The AUC value of white matter model is 0.779 (referring to Fig. 6), has higher accuracy.
Again by the consumptive after intensive treatment be divided into treatment after phlegm painting turn out cloudy patient and treatment after phlegm painting do not turn out cloudy
Patient analyzes two classes by 16.0 softwares of SPSS using Nonparametric Mann-Whitney U test statistical methods
The protein expression difference of patient, by 5 Software on Drawing protein expression level scatter plots of GraphPad Prism (referring to figure
4- Fig. 5).
Decision-tree model is built by BPS biomarker prototype softwares, phlegm applies trouble of turning out cloudy after intensive treatment to differentiate
Phlegm painting is not turned out cloudy patient (referring to Fig. 7) after person and intensive treatment, after the intensive treatment which differentiates phlegm painting turn out cloudy patient, i.e., should
1 patients of Class of model, AUC value 0.837, sensitivity 90.90%, specificity 74.07%, it is good accurate to have
Property.Therefore, prognosis evaluation quickly and accurately can be carried out to the consumptive after intensive treatment by the model, it is especially right
It is of great significance in few phlegm, without the difficult consumptive of the phlegm such as phlegm inspection.
Phlegm after patient, intensive treatment of turning out cloudy applied to phlegm after intensive treatment apply 1 year cure rate of the patient that do not turn out cloudy count,
It was found that it is 100% that phlegm, which applies patient's cure rate of turning out cloudy, after intensive treatment, 1 year cure rate that phlegm applies the patient that do not turn out cloudy after intensive treatment is
43.33%, phlegm applies the patient that turns out cloudy substantially less than after intensive treatment, has good treatment prognostic value.Illustrate by certainly
The case where whether the phlegm painting of patient is turned out cloudy after the discriminating intensive treatment of plan tree-model, the prognosis that can be used for after pulmonary tuberculosis intensive treatment
Assessment is particularly suitable for few phlegm, without the difficult patient of the phlegm such as phlegm inspection.
Embodiment 3：It therapeutic evaluation and the verification of prognosis evaluation reagent kit detection result and is answered after pulmonary tuberculosis intensive treatment
With
Holdout is verified：It takes normal healthy controls person, just control pulmonary tuberculosis sufferer after non-medication consumptive, intensive treatment
The serum sample of person, sample collection and dilution mode are the same as described in embodiment 2.
Sample to be checked after dilution is added to being coated with anti-ANGT antibody, CO7 antibody, PLMN antibody, APOC2 antibody
In four kinds of ELISA Plates, ELISA Plate pasting board is sticked, in incubation at room temperature 1~2.5 hour.
Waste liquid is discarded, ELISA Plate is patted dry, detergent is used in combination to wash 4~5 times.And it is added in each reacting hole corresponding fresh
The antibody of the dilution biotin labeling of preparation, sticks ELISA Plate pasting board, in incubation at room temperature 1 hour.
Waste liquid is discarded, ELISA Plate is patted dry, detergent is used in combination to wash 4~5 times.And Fresh is added in each reacting hole
It is diluted to release Horseradish peroxidase-conjugated avidin, ELISA Plate pasting board is sticked, in incubation at room temperature 30~45 minutes.
Waste liquid is discarded, ELISA Plate is patted dry, detergent is used in combination to wash 4~-5 times.And chromogenic substrate is added in each reacting hole
Solution sticked ELISA Plate pasting board, in incubation at room temperature 15~30 minutes.
50 μ L of terminate liquid are added in each reacting hole and terminate reaction, and detect each reacting hole with microplate reader in 5 minutes
OD values at 450nm, are returned to zero with blank control wells, and protein concentration is conversed by standard curve.
After detection, brings each protein concentration into Logistic successive Regression formula, obtain Logit (p) value, pass through P=
Exp [Logit (p)]/(1+exp [Logit (p)]) conversions obtain P values, are boundary with 0.5, P values close to 1 judgement be the reinforcing phase
Treatment is effective, and P values are poor for reinforcing phase therapeutic effect close to 0 judgement.
Bring patient ANGT, CO7 protein concentrations after intensive treatment into decision-tree model, the affiliated Class of judgement patient again
Classification is determined as that Class's 1 shows that good therapeutic effect can be reached by continuing after treatment after intensive treatment, be determined as
Class 0 shows to continue after treatment prognosis after intensive treatment poor, needs suitably to adjust therapeutic scheme.
It is verified by Holdout, finds the difficult consumptive of 15 phlegm inspections, 14 patient's judgements after intensive treatment
It is determined as Class 0 for 1,1 patient of Class.It is determined as that the patient of Class 1,1 patient fall off, 11 patients are after 1 year
It cures；Patient is determined as that the patient of Class 0 does not cure after 1 year.Show the prognosis rate of accuracy reached 84.62% of the model, explanation
The specific protein composition model to patient after pulmonary tuberculosis intensive treatment carry out therapeutic evaluation and prognosis evaluation have compared with
High accuracy.
As it can be seen that therapeutic evaluation and prognosis evaluation reagent kit have the following advantages after the pulmonary tuberculosis intensive treatment of the present invention：
1. using specific protein composition, in conjunction with Logistic successive Regressions formula and decision-tree model, lung is carried out
The evaluation of tuberculosis intensive treatment curative effect, then the prognosis situation after intensive treatment is assessed, detection technique is advanced, quick,
The method design of protein compositions structure is accurate, effective.
2. clinical most common intensive treatment efficacy determination method is Sputum smears and Phlegm incubation, Sputum smears method positive rate
It is low, and influenced containing factors such as amounts of expectoration by patient；And time-consuming for Sputum culturing method, positive rate and specificity are low.And this reagent
Box simple operation, take it is short, accuracy is high, Serum protein levels carry out pulmonary tuberculosis intensive treatment after curative effect evaluation and
Prognosis evaluation has larger advantage to the assessment of the therapeutic scheme of pulmonary tuberculosis.
3. the present invention is 0.779 to the AUC value of pulmonary tuberculosis intensive treatment phase therapeutic evaluation, to pulmonary tuberculosis intensive treatment
Prognosis evaluation AUC value is 0.837, and prognosis rate of accuracy reached 84.62% has higher accuracy, it can be achieved that pulmonary tuberculosis sufferer
Person's intensive treatment therapeutic evaluation and prognosis evaluation provide new evaluation method for the treatment of pulmonary tuberculosis.
Those of ordinary skills in the art should understand that：The discussion of any of the above embodiment is exemplary only, not
It is intended to imply that the scope of the present disclosure (including claim) is limited to these examples；Under the thinking of the present invention, above example
Or can also be combined between the technical characteristic in different embodiments, and there are different aspects present invention as described above
Many other variations, in order to it is concise they do not provided in details.Therefore, all within the spirits and principles of the present invention,
Any omission, modification, equivalent replacement, improvement for being made etc., should all be included in the protection scope of the present invention.
Claims (10)
1. therapeutic evaluation and prognosis evaluation reagent kit after pulmonary tuberculosis intensive treatment, which is characterized in that including ELISA Plate, protein
Standard items composition and biotin labelled antibodies composition；Wherein the ELISA Plate is coated with anti-ANGT antibody, anti-CO7 respectively
Four kinds of ELISA Plates of antibody, anti-PLMN antibody, anti-APOC2 antibody；The Protein standards composition include ANGT, CO7,
PLMN and APOC2 Protein standards；The biotin labelled antibodies composition includes the anti-ANGT antibody of biotin labeling, life
Anti- CO7 antibody, the anti-PLMN antibody of biotin labeling and the anti-APOC2 antibody of biotin labeling of object element label.
2. therapeutic evaluation and prognosis evaluation reagent kit after pulmonary tuberculosis intensive treatment according to claim 1, feature exist
In, ANGT, CO7, PLMN and APOC2 Protein standards concentration be respectively 100~500ng/mL, 20~60ng/mL,
40~120ng/mL, 50~150ng/mL.
3. therapeutic evaluation and prognosis evaluation reagent kit after pulmonary tuberculosis intensive treatment according to claim 1, feature exist
In further including sample and antibody diluent, detergent, Horseradish peroxidase-conjugated avidin, Chromogenic Substrate Solution, terminate liquid
With ELISA Plate note plate.
4. therapeutic evaluation and prognosis evaluation reagent kit after pulmonary tuberculosis intensive treatment according to claim 1, feature exist
In, the sample and antibody diluent be the solution that 0.01mol/L pH are 7.2, including 0.39g/L NaH2PO4·2H2O、
1.27g/L Na2HPO4、0.85g/L NaCl、200mg/L NaN3Aqueous solution；
The pH value of the detergent is 7.4, including 2.0g/L NaCl, 0.2g/L KH2PO4、2.9g/L Na2HPO4·
12H2O、0.2g/L KCl、0.2g/L NaN3, 0.5mL/L polysorbas20 aqueous solution；
The Chromogenic Substrate Solution is the 3,3' for being 0.8~1.2% containing volume ratio, 5,5'- tetramethyl biphenyl amine aqueous solutions；
The terminate liquid is the H of 1.5~3mol/L2SO4Aqueous solution.
5. the user of therapeutic evaluation and prognosis evaluation reagent kit after a kind of pulmonary tuberculosis intensive treatment as described in claim 1
Method, which is characterized in that pulmonary tuberculosis reinforcing is carried out using therapeutic evaluation after pulmonary tuberculosis intensive treatment and prognosis evaluation reagent kit
The evaluation of curative effect, specifically includes following steps after treatment：
(1) peripheral blood is acquired, centrifuges, suct clearly；
(2) by the supernatant diluted obtained by step (1), four kinds of ELISA Plates are separately added into, are incubated；
(3) ELISA Plate is patted dry, diluted biotin labelled antibodies are separately added into, is incubated, is washed with the detergent；
(4) diluted Horseradish peroxidase-conjugated avidin is added, is washed with the detergent after incubation；
(5) Chromogenic Substrate Solution is added in each reacting hole, is incubated；
(6) terminate liquid is added in each reacting hole and terminates reaction, each reacting hole OD values are detected in microplate reader；
(7) each sample albumen concentration is calculated, the regression equation of Logistic gradually models is brought into, pulmonary tuberculosis is evaluated after being computed
Intensive treatment curative effect.
6. the user of therapeutic evaluation and prognosis evaluation reagent kit after pulmonary tuberculosis intensive treatment according to claim 5
Method, which is characterized in that the regression equation of the Logistic gradually models is：
Logit (p)=0.167-0.030 × (APOC2 albumen concentration)+0.064 × (CO7 albumen concentration) -0.011 × (PLMN
Albumen concentration)
Logit (p) value is obtained, P values are obtained by P=exp [Logit (p)]/(1+exp [Logit (p)]) conversions, are with 0.5
Boundary judges pulmonary tuberculosis intensive treatment curative effect.
7. the user of therapeutic evaluation and prognosis evaluation reagent kit after a kind of pulmonary tuberculosis intensive treatment as described in claim 1
Method, which is characterized in that pulmonary tuberculosis reinforcing is carried out using therapeutic evaluation after pulmonary tuberculosis intensive treatment and prognosis evaluation reagent kit
Prognosis evaluation after treatment, specifically includes following steps：
(1) peripheral blood is acquired, centrifuges, suct clearly；
(2) by the supernatant diluted obtained by step (1), four kinds of ELISA Plates are separately added into, are incubated；
(3) ELISA Plate is patted dry, diluted biotin labelled antibodies are separately added into, is incubated, is washed with the detergent；
(4) diluted Horseradish peroxidase-conjugated avidin is added, is washed with the detergent after incubation；
(5) Chromogenic Substrate Solution is added in each reacting hole, is incubated；
(6) terminate liquid is added in each reacting hole and terminates reaction, each reacting hole OD values are detected in microplate reader；
(7) each sample albumen concentration is calculated, each sample albumen concentration is substituted into decision-tree model, judges affiliated Class classifications, root
According to the classification of affiliated Class 0 or Class 1, the prognosis situation after pulmonary tuberculosis intensive treatment is judged.
8. the user of therapeutic evaluation and prognosis evaluation reagent kit after pulmonary tuberculosis intensive treatment according to claim 7
Method, which is characterized in that ANGT, CO7 protein concentration are substituted into decision-tree model, affiliated Class classifications is judged, is determined as
Class's 1 shows that good therapeutic effect can be reached by continuing after treatment after intensive treatment, be determined as the table of Class 0
It is poor to continue after treatment prognosis after bright intensive treatment, needs suitably to adjust therapeutic scheme.
9. after claim 1-4 any one of them pulmonary tuberculosis intensive treatments prepared by therapeutic evaluation and prognosis evaluation reagent kit
Purposes after pulmonary tuberculosis intensive treatment in therapeutic evaluation reagent.
10. therapeutic evaluation and prognosis evaluation reagent kit are being made after claim 1-4 any one of them pulmonary tuberculosis intensive treatments
Purposes after standby pulmonary tuberculosis intensive treatment in prognosis evaluation reagent.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
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| CN201711397573.2A CN108303545B (en) | 2017-12-21 | 2017-12-21 | Kit for evaluating curative effect and evaluating prognosis after intensive treatment of pulmonary tuberculosis and application of kit |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201711397573.2A CN108303545B (en) | 2017-12-21 | 2017-12-21 | Kit for evaluating curative effect and evaluating prognosis after intensive treatment of pulmonary tuberculosis and application of kit |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109655613A (en) * | 2019-02-15 | 2019-04-19 | 安徽理工大学 | A kind of kit and its detection method for detection sensitivity mycobacterium tuberculosis |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104808003A (en) * | 2015-04-30 | 2015-07-29 | 李继承 | Curative effect evaluation reagent kit of phthisis and application of reagent kit |
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2017
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104808003A (en) * | 2015-04-30 | 2015-07-29 | 李继承 | Curative effect evaluation reagent kit of phthisis and application of reagent kit |
Non-Patent Citations (2)
| Title |
|---|
| CUIPING LI ET AL.: "Discovery and verification of serum differential expression proteins for pulmonary tuberculosis", 《TUBERCULOSIS》 * |
| YURIY SKIBA ET AL.: "Molecular snapshot of Mycobacterium tuberculosis population in Kazakhstan: A country-wide study", 《TUBERCULOSIS》 * |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109655613A (en) * | 2019-02-15 | 2019-04-19 | 安徽理工大学 | A kind of kit and its detection method for detection sensitivity mycobacterium tuberculosis |
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