CN109596836A - A kind of LBP checkout and diagnosis reagent detection system and method - Google Patents
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Abstract
The invention belongs to Biological Detection technical field, a kind of LBP checkout and diagnosis reagent detection system and method are disclosed, mouse totally 80 is selected, is divided into systemic inflammatory response syndrome group, pyemia survival group and pyemia death group；Separately 10 healthy mice serum are selected as Normal group；ELISA detects each group sample serum LBP, c reactive protein and Procalcitonin concentration；And ROC curve, effect of the evaluation LBP in the diagnosis of pyemia mouse and prognosis prediction are done to sepsis diagnosis and prognosis prediction with the scoring of APACHE II, serum LBP, CRP and PCT concentration.The present invention carries out the work such as product stability, positive cutoff value, Performance Evaluation according to " external diagnosis reagent registration management method ", completes the clinical evaluation of large sample；The present invention provides a kind of new LBP diagnostic reagent registration certificate.
Description
Technical field
The invention belongs to Biological Detection technical field more particularly to a kind of LBP checkout and diagnosis reagent detection system and sides
Method.
Background technique
Currently, the prior art commonly used in the trade is such that
LBP is a kind of glycoprotein that molecular weight is 60kD, can be synthesized in many animals body, gene is all located at No. 20
Between chromosome q11,23 and q12, with the homology of height between different animals kind.LBP is mainly by liver and enteric epithelium
Cell synthesis and secretion, other are as there is also different degrees of tables in coated fibr tissue around lung, kidney, the heart, spleen and inflammation
It reaches.The N-terminal of LBP can be in conjunction with LPS, and C-terminal can be the important carrier that LPS plays biological action in conjunction with CD14.
Mainly there are quantitative approach and qualitative checking method for the detection of LBP, quantitative detecting method has double-antibody method, puts
Radioimmunoassay method, immunochromatographic method etc., wherein radio immunoassay has certain limitation in clinic.
In conclusion problem of the existing technology is:
Preceding usually used ELISA method detection, detection range 0.312-20pg/mL, can detect recombination or it is natural to
Survey substance, and with other GAP-associated protein GAP no cross reactions, the sensitivity of ELISA is higher, but complicated for operation, and time-consuming, quantitative to examine
Survey deviation it is larger, to the bad control of the dilution of sample, immunochromatographic method be mainly used for it is qualitative, can quickly provide as a result, but
It is to be unable to quantitative data, error is larger.
Summary of the invention
In view of the problems of the existing technology, the present invention provides a kind of LBP checkout and diagnosis reagent detection system and methods.
The invention is realized in this way a kind of LBP diagnostic reagent detection method, the LBP diagnostic reagent detection method, packet
It includes:
Mouse totally 80 are selected, it is dead to be divided into systemic inflammatory response syndrome SIRS group, pyemia survival group and pyemia
Group；All mouse are interior for 24 hours after entering ICU to be acquired serum sample and carries out the scoring analysis of APACHE II；
Separately 10 healthy mice serum are selected as Normal group；
ELISA detects each group sample serum LBP, c reactive protein CRP and Procalcitonin PCT concentration；And it is commented with APACHE II
Divide, serum LBP, CRP and PCT concentration does ROC curve to sepsis diagnosis and prognosis prediction.
Further, all mouse are interior for 24 hours after entering ICU acquires serum sample and carries out the scoring analysis of APACHE II
In, comprising:
3 groups of mouse of collection of serum sample are in entering the 1st day blood sample collection of ICU；Whole blood is placed in be aggregated naturally at room temperature
20~30min, with kapok stick along test tube wall separate blood clot, then seal test tube, with the speed of 2000~3000r/min from
Serum is moved in EP pipe by heart 10min with capillary pipet, and EP pipe has marked date, acquisition time, patient's title and case history
Number, it is put in -80 DEG C of low temperature refrigerators and saves.
Further, all mouse are interior for 24 hours after entering ICU acquires serum sample and carries out the scoring analysis of APACHE II
In, further comprise:
Preparation the precise KCl0.2g, KH of PBS buffer solution2PO40.2g, NaCl8.0g, Na2HPO4·2H2O1.56g；
Each ingredient is successively dissolved in 1000mL in volumetric flask 3 are steamed in water, are mixed well, are adjusted pH to 7.4；121 in pressure cooker
DEG C, sterilize 20min.
Further, all mouse are interior for 24 hours after entering ICU acquires serum sample and carries out the scoring analysis of APACHE II
In, further comprise: ELISA serum sample is diluted according to ELISA kit specification with PBS buffer solution；If gauge orifice 8
Hole sequentially adds the standard items of 100 μ L various concentrations；100 μ L of sample to be tested is added in remaining 88 hole, and ELISA Plate adds overlay film,
Liquid is discarded after 37 DEG C of incubation 2h, is dried；Every hole is abandoned after detection solution A working solution 100 μ L, 37 DEG C of incubation 1h is added on ELISA Plate
Remove liquid in hole；Every hole is washed with the cleaning solution of 350 μ L, is repeated board-washing 3 times.
Further, every hole is washed with the cleaning solution of 350 μ L, after repeating board-washing 3 times, also needs to carry out:
After board-washing, cleaning solution in hole is dried completely；100 μ L of detection solution B working solution, 37 DEG C of incubations are added in every hole
Liquid in hole is discarded after 30min, every hole is repeated board-washing 5 times with the cleaning solution of 350 μ L；Every hole is added 90 μ L of substrate solution, and 37 DEG C
After being protected from light colour developing 20min, 50 μ L of stop bath is added in every hole, terminates reaction；The suction in each hole is measured in 450nm wavelength through microplate reader
Luminosity (A) value.
Further, the LBP diagnostic reagent detection method further comprises: statistical procedures: counting soft with SPSS16.0
Part is analyzed；All experimental datas are all made of mean ± standard deviation (mean ± SD) expression, 4 comparison among groups single factor test variance
It analyzes, the comparison between 2 groups is examined with LSD-t；Using true positive rate as ordinate, false positive rate is that abscissa draws ROC curve；With
P < 0.05 is that difference is statistically significant.
Further, the LBP diagnostic reagent detection method further comprises:
The scoring analysis of mouse APACHE II；
The scoring of APACHE II and serum LBP, CRP and PCT concentration analyze the ROC curve of sepsis diagnosis；
The scoring of APACHE II and serum LBP, CRP and PCT concentration analyze the ROC curve of pyemia prognosis prediction；
5) scoring of APACHE II and the scoring of LBP correlation analysis APACHE II and serum LBP concentration are positively correlated analysis.
Another object of the present invention is to provide a kind of LBP diagnostic reagents for implementing the LBP diagnostic reagent detection method
Detection system.
Another object of the present invention is to provide a kind of nephrosis LBP diagnosis using the LBP diagnostic reagent detection method
Kit.
Another object of the present invention is to provide a kind of hepatopathy LBP diagnosis using the LBP diagnostic reagent detection method
Kit.
In conclusion advantages of the present invention and good effect are as follows:
The present invention carries out product stability, positive cutoff value, performance according to " external diagnosis reagent registration management method " and comments
The work such as estimate, completes the clinical evaluation of large sample；The present invention provides a kind of new LBP diagnostic reagent registration certificate.
The present invention selects mouse totally 80, is divided into systemic inflammatory response syndrome (SIRS) group (negative control group), septicopyemia
Disease survival group and pyemia death group；All mouse acquire serum sample in into after ICU for 24 hours and carry out APACHE II and comment
Analysis；Separately 10 healthy mice serum are selected as Normal group；ELISA detects each group sample serum LBP, C and reacts egg
White (CRP) and Procalcitonin (PCT) concentration；And with the scoring of APACHE II, serum LBP, CRP and PCT concentration to sepsis diagnosis
ROC curve, effect of the evaluation LBP in the diagnosis of pyemia mouse and prognosis prediction are done with prognosis prediction.As a result: with SIRS group phase
Than the scoring of APACHE II of pyemia group, serum LBP, CRP, PCT concentration increase (P < 0.05)；With pyemia survival group phase
Than the scoring of APACHE II of death group and serum LBP concentration increase (P < 0.05), and serum CA125 and PCT concentration are in pyemia
No significant difference between survival group and dead group；Diagnosis of sepsis disease is quick when LBP serum-concentration is higher than 26.84mg/L
Perception and specificity are respectively 97.1% and 95.9%；Prediction pyemia prognosis is quick when LBP serum-concentration is higher than 54.16mg/L
Perception and specificity are respectively 85.2% and 80.0%.Conclusion: compared with traditional pyemia biomarker CRP, PCT, LBP
All there is better effect in terms of diagnosis of sepsis and prediction.
Detailed description of the invention
Fig. 1 is LBP checkout and diagnosis reagent test method flow chart provided in an embodiment of the present invention.
Fig. 2 is the correlation analysis figure of APACHE II scoring and LBP provided in an embodiment of the present invention.
Specific embodiment
In order to make the objectives, technical solutions, and advantages of the present invention clearer, with reference to embodiments, to the present invention
It is further elaborated.It should be appreciated that the specific embodiments described herein are merely illustrative of the present invention, it is not used to
Limit the present invention.
The present invention carries out product stability, positive cutoff value, performance according to " external diagnosis reagent registration management method " and comments
The work such as estimate, completes the clinical evaluation of large sample；The present invention provides a kind of new LBP diagnostic reagent registration certificate.
Application of the invention is further described below with reference to concrete analysis.
Fig. 1, LBP checkout and diagnosis reagent test method provided in an embodiment of the present invention, comprising:
S101: selecting mouse totally 80, is divided into systemic inflammatory response syndrome (SIRS) group (negative control group), pyemia
Survival group and pyemia death group；All mouse are interior for 24 hours after entering ICU to be acquired serum sample and carries out the scoring of APACHE II
Analysis；
S102: 10 healthy mice serum separately are selected as Normal group；
S103:ELISA detects each group sample serum LBP, c reactive protein (CRP) and Procalcitonin (PCT) concentration；And with
The scoring of APACHE II, serum LBP, CRP and PCT concentration do ROC curve to sepsis diagnosis and prognosis prediction, evaluate LBP in purulence
Effect in the diagnosis of toxication mouse and prognosis prediction.
A kind of LBP checkout and diagnosis reagent detection system provided in an embodiment of the present invention.
To application of the invention, the invention will be further described combined with specific embodiments below.
LBP checkout and diagnosis reagent test method provided in an embodiment of the present invention, comprising:
1,3 groups of mouse of analysis of clinical (SIRS group, pyemia survival group and pyemia death group) routine monitoring body
Temperature, breathing, mean arterial pressure, electrocardiogram etc., and interior progress acute physiology function and the chronic health for 24 hours after entering ICU
It scores (acutephysiologyandchronichealthevaluation II, APACHE II).
2,3 groups of mouse of the collection of serum sample are in entering the 1st day blood sample collection of ICU.Whole blood is placed in naturally solidifying at room temperature
Collect 20~30min, lightly separates blood clot along test tube wall with kapok stick, seal test tube, then with 2000~3000r/min
Speed be centrifuged 10min, serum is moved in EP pipe with capillary pipet, EP pipe has marked date, acquisition time, Bing Renming
Title and medical record number are put in -80 DEG C of low temperature refrigerators and save.
3, preparation the precise KCl0.2g, KH of PBS buffer solution2PO40.2g, NaCl8.0g, Na2HPO4·
2H2O1.56g.In volumetric flask by each ingredient be successively dissolved in 1000mL 3 steam water in (attention should be complete to former reagent
A kind of ingredient under being redissolved after dissolution), it mixes well, adjusts pH to 7.4.121 DEG C in pressure cooker, 20min is sterilized.
4, ELISA serum sample is diluted according to ELISA kit specification with PBS buffer solution.If 8 hole of gauge orifice,
Sequentially add the standard items of 100 μ L various concentrations.Remaining 88 hole is added 100 μ L of sample to be tested, and ELISA Plate adds overlay film, and 37 DEG C
Liquid is discarded after being incubated for 2h, is dried.Every hole is added detection 100 μ L of solution A working solution and (says according to ELISA kit on ELISA Plate
Bright book is in prepared before use), liquid in hole is discarded after 37 DEG C of incubation 1h.Every hole is washed with the cleaning solution of 350 μ L, repeats board-washing 3
It is secondary.After last time board-washing, cleaning solution in hole is dried completely.Detection 100 μ L of solution B working solution is added (according to ELISA in every hole
Kit specification is in prepared before use), liquid in hole is discarded after 37 DEG C of incubation 30min, every hole is repeated with the cleaning solution of 350 μ L
Board-washing 5 times.90 μ L of substrate solution is added in every hole, and after 37 DEG C are protected from light colour developing 20min, 50 μ L of stop bath is added in every hole, is terminated anti-
It answers.Absorbance (A) value in each hole is measured in 450nm wavelength through microplate reader.
4, statistical procedures
It is analyzed with SPSS16.0 statistical software.All experimental datas are all made of mean ± standard deviation (mean ± SD) table
Show, 4 comparison among groups one-way analysis of variances, the comparison between 2 groups is examined with LSD-t.It is vertical sit with true positive rate (sensibility)
Mark, false positive rate (1- specificity) is that abscissa draws ROC curve.It is that difference is statistically significant with P < 0.05.
The invention will be further described combined with specific embodiments below.
Analysis of clinical
1) the clinical basic document of mouse is shown in 3 groups of mouse no difference of science of statistics, but mean arterial pressure, APACHE
There is statistical difference (P < 0.05).Pyemia mouse is mostly gram positive bacterial infection, such as Acinetobacter bauamnnii and
The blood culture of pseudomonas aeruginosa, only 3 mouse finds gram-positive bacteria (staphylococcus aureus).
2) scoring of mouse APACHE II is analyzed and serum LBP, CRP and PCT concentration are compared with SIRS group, pyemia group
The scoring of APACHE II, serum LBP, CRP and PCT concentration increase (P < 0.05), and compared with pyemia survival group, dead group
The scoring of APACHE II and serum LBP concentration increase (P < 0.05), and serum CA125 and PCT concentration are in pyemia survival group and dead
Die the no significant difference (P > 0.05) between group.
3) scoring of APACHE II and serum LBP, CRP and PCT concentration analyze each index to the ROC curve of sepsis diagnosis
Area LBP is 0.996, A-PACHEII 0.772, PCT 0.937, CRP 0.782, table 2 under ROC curve.LBP serum is dense
Spend diagnosis of sepsis disease the equal highest of sensibility and specificity, concentration be higher than 26.84mg/L when diagnosis of sepsis disease sensibility and
Specificity is respectively 97.1% and 95.9%.Then, with this it is horizontal to another 30 mouse (SIRS group 11, pyemia group 19
Example) pyemia whether occurs is judged, only 2 mouse misjudgments as the result is shown, all judgement is being just for remaining 28 mouse
Really.The accuracy of diagnosis of sepsis disease is up to 92.8% when LBP serum-concentration is higher than 26.84mg/L.
4) scoring of APACHE II and serum LBP, CRP and PCT concentration analyze the ROC curve of pyemia prognosis prediction:
Area LBP is 0.828, APACHEII 0.897 under the ROC curve of each index, and PCT 0.608, CRP are
The sensibility and specificity of 0.682, LBP serum-concentration prediction pyemia prognosis is slightly below the scoring of APACHE II, and concentration is higher than
Predict that the sensibility and specificity of pyemia prognosis is respectively 85.2% and 80.0% when 54.16mg/L.Then, we use this
Level predicts the final result of another 18 pyemia mouse (survival group 12, death group 6), only 2 small as the result is shown
Mouse misjudgment, remaining 16 mouse whole correct judgment.As shown in table 5, to purulence when LBP serum-concentration is higher than 54.16mg/L
The accuracy of toxication mouse final result prediction is up to 87.5%.
5) scoring of APACHE II and the scoring of LBP correlation analysis APACHE II and serum LBP concentration are positively correlated (P <
0.05), see Fig. 2.
Below with reference to specific effect, the invention will be further described.
LBP is a kind of glycoprotein synthesized by liver, has high affinity with the lipoid A in LPS.As LPS carrier
Albumen, LBP can mediate the combination of LPS and CD14, and the release inflammatory mediator such as stimulation monocyte, endothelial cell causes whole body scorching
Property response syndrome, septic shock, acute lung injury even multiple organ dysfunction syndrome.Opal etc. has found severe septicopyemia
Disease and septic shock patients blood plasma's LBP concentration significantly increase.Zeng etc. is it has also been found that LBP can prompt body acute inflammatory reaction.
LBP horizontal lower (< 2mg/L) in normal person's blood, when having microorganism infection, inflammation to occur, LBP is generated simultaneously by rapid induction
Significantly (200~800mg/L) increases.Therefore, LBP level can be used as the important mark of certain disease early diagnosis and prognosis prediction
Will.But, also studies have found that LBP is only capable of reflection body acute inflammatory reaction, and cannot function as judging disease severity and
The index of prognosis.Meaning of the LBP in pyemia report it is different may with patient context, to enter the treatment done before ICU etc. different
It is related.There are the LBP of certain level in normal serum, but in patients serum, and LBP concentration significantly increases.Compared with SIRS group,
The serum LBP level of pyemia group increases；Compared with pyemia survival group, death group serum LBP is horizontal further to be increased,
Prompt LBP level increases the generation that not only can reflect infection, but also it is related to the prognosis of pyemia mouse.Though
Area ratio APACHE II scores slightly lower under the ROC curve of right LBP serum-concentration prediction pyemia prognosis, but its sensibility and special
Property has been more than 80.0%.Further analysis serum LBP concentration finds that the two is in positive with the correlation that APACHE II scores
It closes, LBP is prompted to can be used as the index of reflection disease severity and prognosis prediction.When the concentration of LBP in serum is higher than
The sensibility and specificity of diagnosis of sepsis disease is more than 95.0% when 26.84mg/L, and serum LBP concentration is higher than 54.16mg/L
When prediction pyemia prognosis sensibility and specificity more than 80.0%.For the reliability of further confirmatory experiment, in addition with
Machine picks a batch, takes its serum as verifying sample data.The results show that LBP serum-concentration diagnoses when being higher than 26.84mg/L
Pyemic accuracy is up to 92.8%, and its concentration is higher than the accuracy predicted when 54.16mg/L sepsis patient final result
Up to 87.5%.
Current clinically common pyemia biomarker has CRP and PCT.CRP has clinically used many years
, but its specificity is not high.Although the specificity of PCT ratio CRP is more preferable, it still cannot preferably judge pyemic prognosis,
Therefore its value for clinical application is also constantly subjected to query.This research also shows traditional pyemia biomarker PCT's and CRP
Serum levels have in auxiliary diagnosis pyemia to have certain effect, but they prediction pyemia mouse prognosis in terms of then
Effect is poor.Although result of study shows APACHE II, scoring also contributes to pyemic prognosis prediction, its diagnosis of sepsis disease
Sensibility and specificity be far inferior to LBP.LBP is pyemic compared with traditional pyemia biomarker PCT and CRP
Diagnosis and prediction aspect all have better effect, and sensibility and specificity is higher.The above results suggest that LBP is septicopyemia
Disease diagnosis and one of prognosis prediction new, potential biomarker, clinical value need further to be assessed.
The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, all in essence of the invention
Made any modifications, equivalent replacements, and improvements etc., should all be included in the protection scope of the present invention within mind and principle.
Claims (10)
1. a kind of LBP diagnostic reagent detection method, which is characterized in that the LBP diagnostic reagent detection method, comprising:
Mouse totally 80 are selected, systemic inflammatory response syndrome SIRS group, pyemia survival group and pyemia death group are divided into；Institute
There is mouse is interior for 24 hours after entering ICU to acquire serum sample and carry out the scoring analysis of APACHE II；
Separately 10 healthy mice serum are selected as Normal group；
ELISA detects each group sample serum LBP, c reactive protein CRP and Procalcitonin PCT concentration；And scored with APACHE II,
Serum LBP, CRP and PCT concentration does ROC curve to sepsis diagnosis and prognosis prediction.
2. LBP diagnostic reagent detection method as described in claim 1, which is characterized in that all mouse are after entering ICU
Serum sample is acquired in for 24 hours and is carried out in the scoring analysis of APACHE II, comprising:
3 groups of mouse of collection of serum sample are in entering the 1st day blood sample collection of ICU；Whole blood be placed at room temperature naturally agglutination 20~
30min is separated blood clot along test tube wall with kapok stick, then seals test tube, be centrifuged with the speed of 2000~3000r/min
Serum is moved in EP pipe by 10min with capillary pipet, and EP pipe has marked date, acquisition time, patient's title and case history
Number, it is put in -80 DEG C of low temperature refrigerators and saves.
3. LBP diagnostic reagent detection method as claimed in claim 2, which is characterized in that all mouse are after entering ICU
For 24 hours in acquisition serum sample and carry out APACHE II scoring analysis in, further comprise:
Preparation the precise KCl0.2g, KH of PBS buffer solution2PO40.2g, NaCl8.0g, Na2HPO4·2H2O1.56g；Holding
Each ingredient is successively dissolved in 1000mL in measuring bottle 3 are steamed in water, are mixed well, are adjusted pH to 7.4；121 DEG C in pressure cooker, disappear
Malicious 20min.
4. LBP diagnostic reagent detection method as claimed in claim 2, which is characterized in that all mouse are after entering ICU
Serum sample is acquired in for 24 hours and is carried out in the scoring analysis of APACHE II, and further comprise: ELISA serum sample is tried according to ELISA
Agent box specification is diluted with PBS buffer solution；If 8 hole of gauge orifice, the standard items of 100 μ L various concentrations are sequentially added；It is remaining
88 holes 100 μ L of sample to be tested is added, ELISA Plate adds overlay film, discards liquid after 37 DEG C of incubations 2h, drying；Every hole on ELISA Plate
Liquid in hole is discarded after detection solution A working solution 100 μ L, 37 DEG C of incubation 1h is added；Every hole is washed with the cleaning solution of 350 μ L, weight
After backwashing plate 3 times.
5. LBP diagnostic reagent detection method as claimed in claim 4, which is characterized in that every hole is washed with the cleaning solution of 350 μ L
It washs, after repeating board-washing 3 times, also needs to carry out:
After board-washing, cleaning solution in hole is dried completely；100 μ L of detection solution B working solution, 37 DEG C of incubations are added in every hole
Liquid in hole is discarded after 30min, every hole is repeated board-washing 5 times with the cleaning solution of 350 μ L；Every hole is added 90 μ L of substrate solution, and 37 DEG C
After being protected from light colour developing 20min, 50 μ L of stop bath is added in every hole, terminates reaction；The suction in each hole is measured in 450nm wavelength through microplate reader
Luminosity (A) value.
6. LBP diagnostic reagent detection method as claimed in claim 2, which is characterized in that the LBP diagnostic reagent detection method
Further comprise: statistical procedures: being analyzed with SPSS16.0 statistical software；All experimental datas are all made of mean ± standard
Poor (mean ± SD) is indicated, 4 comparison among groups one-way analysis of variances, and the comparison between 2 groups is examined with LSD-t；With true positive rate
For ordinate, false positive rate is that abscissa draws ROC curve；It is that difference is statistically significant with P < 0.05.
7. LBP diagnostic reagent detection method as described in claim 1, which is characterized in that the LBP diagnostic reagent detection method
Further comprise:
The scoring analysis of mouse APACHE II；
The scoring of APACHE II and serum LBP, CRP and PCT concentration analyze the ROC curve of sepsis diagnosis；
The scoring of APACHE II and serum LBP, CRP and PCT concentration analyze the ROC curve of pyemia prognosis prediction；
5) scoring of APACHE II and the scoring of LBP correlation analysis APACHE II and serum LBP concentration are positively correlated analysis.
8. a kind of LBP diagnostic reagent detection system for implementing LBP diagnostic reagent detection method described in claim 1.
9. a kind of nephrosis LBP diagnostic kit using LBP diagnostic reagent detection method described in claim 1.
10. a kind of hepatopathy LBP diagnostic kit using LBP diagnostic reagent detection method described in claim 1.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201811497824.9A CN109596836A (en) | 2018-12-07 | 2018-12-07 | A kind of LBP checkout and diagnosis reagent detection system and method |
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| CN117647645A (en) * | 2024-01-29 | 2024-03-05 | 中国人民解放军总医院第一医学中心 | Application of LBP, ATF6 and M-CSFR combination in preparation of product for diagnosing autoimmune liver disease and kit |
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| CN117647645A (en) * | 2024-01-29 | 2024-03-05 | 中国人民解放军总医院第一医学中心 | Application of LBP, ATF6 and M-CSFR combination in preparation of product for diagnosing autoimmune liver disease and kit |
| CN117647645B (en) * | 2024-01-29 | 2024-04-12 | 中国人民解放军总医院第一医学中心 | Application of LBP, ATF6 and M-CSFR combination in preparation of product for diagnosing autoimmune liver disease and kit |
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