CN109521200A - It is a kind of while detecting the kit of Multiple components content, method and its application in blood plasma - Google Patents
It is a kind of while detecting the kit of Multiple components content, method and its application in blood plasma Download PDFInfo
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Abstract
The kit of Multiple components content, method and its application in blood plasma are detected simultaneously the invention discloses a kind of, belong to field of biotechnology, including detection solution R1, detection solution R2, antibiont positive markers quality controlled serum, antibiont marker negative quality controlled serum and concentration washing lotion, pass through the content based on pancreas egg TPS II, SSA, hs-CRP, PCT and phylaxin in latex enhancing immune turbidimetry joint-detection human plasma.The present invention can be realized the investigation of the acute abdomen disease to initiations such as acute pancreatitis, other abdominal viscera Lymph nodes, the perforation of acute digestive tract ulcer, realize multi objective joint-detection to achieve the purpose that identify common acute abdomen disease.
Description
Technical field
The present invention relates to a kind of biotinylation kits, method and its application, detect simultaneously more particularly to one kind more in blood plasma
The kit of kind component content, method and its application, belong to field of biotechnology.
Background technique
Acute abdomen (acuteabdominalpain) is one of most common illness of Emergency Patients, is gone to a doctor whole year
Occur, peak is autumn (9~November) and summer (6~August) respectively, and the medical age concentrates between 15~44 years old (accounting for medical
The 72.7% of sum), often it is related to the disease of inside and outside, youngster, even neural, the spiritual each section of woman's production, Etiological is abdominal viscera
Organ occurs caused by lesion, structure malfunction, the outer adjacent organs damage of abdomen or systemic disease.According to statistics, non-wound impatient
The disease incidence highest of acute pancreatitis, followed by acute gastroenteritis, acute ileus, acute appendicitis, urgency in disease abdominal pain disease
Property cholangitis, ulcer acute perforation etc..Such disease often has the characteristics that morbidity is anxious, variation is fast, the state of an illness is complicated and serious, adds
It, Hospitals at Present emergency department clinician tends to rejuvenation, and experience is insufficient in terms of emergency diagnosis, is easy to diagnose wrong or treatment
Improperly, it may cause adverse consequences in this way, or even dead.Therefore, more demanding to the diagnosis of acute abdomen, it can make in time
Specific diagnosis, is reasonably treated, will directly affect the treatment and prognosis of disease.For this phenomenon, the present invention is proposed
By Trypsinogen-2 in serum (TPS II), serum amyloid A protein (SSA), hs-CRP (hs-CRP), calcitonin
The acute abdomen that former (PCT) and phylaxin carry out joint-detection checks scheme, corresponding detection kit is developed, to acute abdomen
Common acute pancreatitis, acute appendicitis, acute peritonitis, acute gastroenteritis are excluded with exedens acute perforation.
Currently, the external context of detection in clinical acute disease abdominal pain, mainly by combining patient medical history, carry out a medical examination,
Laboratory auxiliary examination selectively carries out blood glucose, pregnancy tests, blood electrolyte, hepatic and renal function, abdominal CT according to conditions of patients
Inspection, abdominal cavity diagnostic puncture and ascites analysis etc. check, and then comprehensively consider, propose corresponding therapeutic scheme.This
Outside, the investigation of acute pancreatitis is also carried out, but for acute disease abdomen using the detection to acute abdomen Urinary Trypsinogen-2
Report of the investigation of bitterly relevant other diseases still without relevant biomarkers object.At home, the clinic of acute abdomen is examined
Survey is similar to external clinical detection, but application development of the TPS II in external clinical diagnosis is more early, and is at home nearly 2 years lands
Continuous just to start to enter into clinical application in Grade III Class A hospital, its clinical value is still in further exploration.This
Invention is by carrying out joint quantitative detection with phylaxin to TPS II, SSA, hs-CRP, PCT in acute abdomen patients serum, to urgency
Disease abdominal pain common disease is checked, and is made reasonable diagnosis in time for clinician and is provided auxiliary tool.
Summary of the invention
The main object of the present invention be solve the related disease of acute abdomen in the prior art investigation sensitivity it is low, accurate
Property difference and cumbersome and inconvenient problem, and provide kit that is a kind of while detecting Multiple components content in blood plasma.
The purpose of the present invention can reach by using following technical solution:
Kit that is a kind of while detecting Multiple components content in blood plasma, including detection solution R1, detection solution R2, antibiosis
Object positive markers quality controlled serum and antibiont marker negative quality controlled serum.
Detecting solution R1 includes phosphate buffer and Macrogol 6000；Detection solution R2 includes phosphate buffer, coupling
There are the sensitizing latex and stabilizer Proclin300 of biomarker antibody；Biomarker include TPS II, SSA, hs-CRP,
PCT and phylaxin.
Sensitizing latex is obtained with biomarker antibody by physical absorption and/or covalent bond by latex particle.Glue
Newborn particle is that carboxyl polystyrene microsphere, magnetic polystyrene microsphere, aminopolystyrene microballoon, dimethylamino polystyrene are micro-
Ball, sulfonated polystyrene microballoon, polystyrene microsphere, poly (methyl methacrylate) micro-sphere, poly lactide-glycolide acid are micro-
Any one in ball.
Biomarker antibody is monoclonal antibody, and monoclonal antibody passes through monoclonal hybridoma technology and prepares
It obtains or is obtained by phage antibody technology screening.
Sensitizing latex is prepared especially by following method:
Step 1: latex activation
Latex is added in suitable MES buffer, after ultrasonication, is slowly added to thereto under stirring conditions
EDA and NHS, constant-temperature incubation for a period of time after, centrifugation, remove supernatant, then PBS buffer solution is added into precipitating, carry out ultrasonication,
Repeat 1 time from addition PBS to the step of progress ultrasonication, obtains latex particle；
Step 2: antibody linked
PBS is added into latex particle, after carrying out ultrasonication, the monoclonal for being separately added into biomarker thereto is anti-
Body, additional amount is 1.5~6mg/ml, after mixing, is placed in be incubated at room temperature to reaction solution and is evenly distributed state, sensitizing latex；
Step 3: closing
The glycine for being 3.3%mg/ml with PBS buffer solution compound concentration, by glycine solution and sensitizing latex according to body
Product is mixed than 100:5, and after ultrasonication, 40~50min is incubated at 25 DEG C, and pH7.5,50mM are then added thereto and includes
The PBS buffer solution of 0.05wt%BSA after mixing, is incubated overnight at 25 DEG C；After centrifugation, PBS, ultrasonication are added thereto；From
The heart removes supernatant, and 0.05% tween is added, is centrifuged again after ultrasonication, removes supernatant, after 0.05% BSA is added, surpassed
After sound is broken, the stabilizer Proclin300 of 0.05% BSA and 0.1% is continuously added, carries out ultrasonication, ultrasonication
Condition is 100Hz, 0.8s/ times, carries out 2min altogether, is then saved for use for 4 DEG C.
Biomarker positive quality control serum and the biomarker feminine gender quality controlled serum are all normal human serums, biology
Positive markers quality controlled serum is by adding TPS II, SSA, hs-CRP, PCT and phylaxin mark respectively in normal human serum
Quasi- product are made, and additive amount is 3 times of the critical value of TPS II in normal serum, SSA, hs-CRP, PCT and resistance cellulose content.
Kit includes the detection cup of five kinds of different colours label, respectively to TPS II in serum, SSA, hs-CRP, PCT and
The detection of phylaxin, the range of linearity of detection are followed successively by 2.5 μ g/L of μ g/L~1250,0.1ng/ml~300ng/ml, 0.3mg/L
~150mg/L, 0.1ng/ml~250ng/ml, 1ng/ml~200ng/ml.
Method that is a kind of while detecting Multiple components content in blood plasma, includes the following steps:
By the way that the corresponding antibodies of substance to be checked are coupled with latex particle, by the specific bond of antigen and antibody,
So that latex particle is aggregated, changes the turbidity of antigen-antibody reaction system, improve the sensitivity of detection；
By the joint-detection of serum T PS II and hs-CRP, Acute Pancreatitis with Acute is checked, joint-detection serum is passed through
The content of middle PCT and hs-CRP, diagnosis investigation that is timely, effectively completing acute peritonitis, facilitates the observation of clinical efficacy；
By the joint-detection of serum SSA and hs-CRP, acute appendicitis is checked, by being supported in patients serum
Exedens acute perforation is checked in the detection of anti-element and the content of hs-CRP into property；
The serum sample that will be obtained after TPS II, SSA, hs-CRP, PCT and phylaxin standard items or the centrifugation of EDTA anticoagulated blood
This is mixed with the prepared sensitizing latex containing corresponding antibodies respectively, reacts antigen-antibody sufficiently, to antigen-antibody
After reacting a period of time, with the absorbance of full automatic biochemical apparatus measurement reaction solution, reference standard curve can determine detection respectively
The content of TPS II, SSA, hs-CRP, PCT and phylaxin in sample.
It is related to be used to prepare investigation acute abdomen for application that is a kind of while detecting the kit of Multiple components content in blood plasma
On the reagent of disease, acute abdomen related disease is acute pancreatitis, acute peritonitis, Peptic Ulcer with Acute Perforation, acute
Any one in ecphyaditis and septicemia.
Advantageous effects of the invention:
(1) screening indexes of the TPS II as acute pancreatitis have sensibility strong, specific high, in acute pancreatitis
There is very high value in clinical diagnosis；
(2) specificity and the sensitivity of acute peritonitis can be improved in the joint-detection of hs-CRP and PCT；
(3) by phylaxin, this emerging Testing index detects the present invention, helps to burst to digestibility in acute abdomen
The investigation of ulcer acute perforation；
(4) by the joint-detection of SSA and hs-CRP in serum, acute appendicitis is checked；
(5) serum T PS II, SSA, hs-CRP, PCT and the multiple indexs of phylaxin are subjected to joint-detections, it is available and
When effective diagnostic result, screening is carried out to the common disease of acute abdominalgia, helps clinician to make and timely and effectively controls
Treatment measure also provides valuable clinical data for the prognosis of disease；
(6) all detection reagent high sensitivities of the present invention detect sample size used and control in 5 μ L, realize clinic
The milligram ammonia of test samples solves the problems, such as sample collection difficulty；
(7) the detection side of multiple indexs has been unified based on the multi objective associated detecting method of latex enhancing immune turbidimetry
Method is detected them in same reaction system, has saved the consumption of sample, while also saving people
Power, material resources and financial resources.
Detailed description of the invention
Fig. 1 is the preparation process flow chart of sensitizing latex；
The testing principle of Fig. 2 latex enhancing immune turbidimetry.
Specific embodiment
To make the more clear and clear technical solution of the present invention of those skilled in the art, the present invention is made below further
Detailed description, embodiments of the present invention are not limited thereto.
Embodiment 1:
The kit provided in this embodiment being used for while measuring serum T PS II, hs-CRP, SSA, PCT and phylaxin, should
Kit includes such as lower component: reagent one (R1), wherein including phosphate buffer and polyethylene glycol；Reagent two (R2), wherein wrapping
Phosphorous acid buffer, coupling have anti-TPS II monoclonal antibody a (hereinafter the sensitizing latex of abbreviation monoclonal antibody a), coupling have it is anti-
Hs-CRP monoclonal antibody b (hereinafter the sensitizing latex of abbreviation monoclonal antibody b), be associated with the monoclonal antibody c of anti-SSA (hereinafter
The sensitizing latex stabilizer of abbreviation monoclonal antibody c), be associated with anti-PCT monoclonal antibody d (hereinafter the sensitizing latex of abbreviation monoclonal antibody d),
It is associated with monoclonal antibody e (the hereinafter sensitizing latex of abbreviation monoclonal antibody e) of anti-phylaxin；It TPS II, hs-CRP, SSA, PCT and supports
Anti- element positive quality control serum；TPS II, hs-CRP, SSA, PCT and phylaxin feminine gender quality controlled serum.Wherein, TPS II, hs-CRP,
SSA, PCT and phylaxin positive quality control serum are to add TPS II, hs-CRP, SSA, PCT in adding respectively in normal human serum
And phylaxin standard items are made, additive amount is TPS II in normal serum, hs-CRP, SSA, PCT and the critical value for resisting cellulose content
3 times；Wherein, the critical value of TPS II, SSA, hs-CRP, PCT and resistance cellulose content are respectively 50 μ g/L in normal serum,
0.5ng/ml, 3mg/L, 0.5ng/ml, 14ng/ml；The biomarker feminine gender quality controlled serum is normal human serum.Wherein
Content of the content of normal human serum TPS II lower than 50 μ g/L, SSA is lower than 0.5ng/ml, and hs-CRP content is lower than 3mg/L, PCT
Content be lower than 0.5ng/ml, the content of phylaxin is lower than 14ng/ml.
Wherein, various pieces are prepared via a method which:
(1) it identifies the preparation of the monoclonal antibody a of TPS II: being prepared and (trained by increment by monoclonal hybridoma technology
The method of supporting expands preservation hybridoma cell strain, and the monoclonal antibody in culture solution is then purified by octanoic acid-saturated ammonium sulfate method) or
Person obtains (passing through built-up expression vector is cloned by the high-affinity obtained after screening by phage antibody technology screening
The monoclonal antibody of high-affinity is obtained after transfection, expression, purification).In the present invention, II monoclonal antibody a of TPS selects anti-human pancreas
Proproteinase -2 (TPS II) monoclonal antibody (article No. FR-A-021 is bought from Shanghai Yan Tuo biotechnology company)；
(2) identify the preparation of the monoclonal antibody b of hs-CRP: the monoclonal antibody can also pass through monoclonal hybridoma technology
It is prepared and (preservation hybridoma cell strain is expanded by increment cultivation, then passes through sad saturated ammonium sulfate method purifying culture
Monoclonal antibody in liquid) or obtain by phage antibody technology screening (to be cloned by the high-affinity obtained after screening
Built-up expression vector obtains the monoclonal antibody of high-affinity after transfection, expression, purification), it selects in the present invention anti-
Hs-CRP monoclonal antibody b selects anti-human hs-CRP (hs-CRP) monoclonal antibody, and (article No. TM011 is bought from grand celebration
Mai Baikang Bioisystech Co., Ltd)；
(3) it identifies the preparation of the monoclonal antibody c of SSA: being prepared by monoclonal hybridoma technology (by increment culture
Method expands preservation hybridoma cell strain, and the monoclonal antibody in culture solution is then purified by octanoic acid-saturated ammonium sulfate method) or
It obtains (built-up expression vector being cloned through turning by the high-affinity obtained after screening by phage antibody technology screening
The monoclonal antibody of high-affinity is obtained after dye, expression, purification), in the present invention, the monoclonal antibody c of anti-SSA selects anti-human serum starch
Sample albumin A (SSA) monoclonal antibody (article No. A90885Hu01 is bought from Shanghai Wu Hao Trade Co., Ltd.)；
(4) it identifies the preparation of the monoclonal antibody d of PCT: being prepared by monoclonal hybridoma technology (by increment culture
Method expands preservation hybridoma cell strain, and the monoclonal antibody in culture solution is then purified by octanoic acid-saturated ammonium sulfate method) or
It obtains (built-up expression vector being cloned through turning by the high-affinity obtained after screening by phage antibody technology screening
The monoclonal antibody of high-affinity is obtained after dye, expression, purification), in the present invention, calcium drops in the monoclonal antibody d of anti-PCT selection anti-human serum
Plain original (PCT) monoclonal antibody (article No. 901010 is bought from Guangzhou Growth hormone secretagogue Biotechnology Co., Ltd).
(5) it identifies the preparation of the monoclonal antibody e of phylaxin: being prepared by monoclonal hybridoma technology and (pass through increment
Cultivation expands preservation hybridoma cell strain, and the monoclonal antibody in culture solution is then purified by octanoic acid-saturated ammonium sulfate method)
Or it obtains (built-up expression vector being cloned by the high-affinity obtained after screening by phage antibody technology screening
The monoclonal antibody of high-affinity is obtained after transfection, expression, purification), in the present invention, the monoclonal antibody e of anti-phylaxin selection is anti-human
Serum Resistin Levels monoclonal antibody (article No. A90847Hu01 is bought from Shanghai Wu Hao Trade Co., Ltd.)；
(6) preparation of sensitizing latex particle, preparation process such as Fig. 1:
Latex activation (latex article No. PM001 and PM005 are bought from Nanjing Egg-based Biotechnology Co., Ltd.): by latex
It is added in suitable MES buffer, after ultrasonication, is slowly added to EDA and NHS thereto under stirring conditions, constant temperature is incubated
After educating a period of time, centrifugation removes supernatant, then PBS buffer solution is added into precipitating, and carrying out ultrasonication, (operation repeats 1
It is secondary) to get arrive work microballoon；
It is antibody linked: PBS is added into work microballoon, after carrying out ultrasonication, is separately added into a certain amount of list thereto
Anti- a, monoclonal antibody b, monoclonal antibody c, monoclonal antibody d and monoclonal antibody e (amount of addition be respectively 0.65mg/ml, 0.85mg/ml, 1mg/ml, 1mg/ml,
1.2mg/ml), it after mixing, is placed in be incubated at room temperature to reaction solution and is evenly distributed state, this i.e. sensitizing latex；
Closing: after a certain amount of glycine is dissolved in PBS buffer solution, taking and mix in right amount with sensitizing latex, after ultrasonication,
It is incubated for；After being incubated for a period of time, the PBS buffer solution dissolved with BSA is added thereto, after mixing, is incubated overnight；After centrifugation,
PBS, ultrasonication are added thereto；Supernatant is removed in centrifugation, and tween is added, is centrifuged again after ultrasonication, removes supernatant, is added
After BSA, after carrying out ultrasonication, BSA and nitrine trisodium are continuously added, after carrying out ultrasonication, 4 DEG C are saved for use.
Detection method and the course of work: the present invention uses latex enhancing immune turbidimetry, is combined using mentioned reagent box and is examined
The content of TPS II, hs-CRP, SSA, PCT and phylaxin are surveyed, testing principle is as shown in Fig. 2, include the following steps:
1) specific protein analyzer basic parameter: Two point end assay, wavelength 600nm, basic parameter such as Tables 1 and 2 is set
It is shown；2) sample to be tested of 5 μ L is added in 200 μ LR1, is incubated for 300 seconds after mixing, is then added thereto 50 μ LR2, survey the
One absorbance A 1 is incubated for 300 seconds after mixing, surveys the second absorbance A 2, and the corresponding absorbance value of the turbidity of compound is Δ (A2-
A1).Automatic clinical chemistry analyzer carries the program parameter input method of itself, and above-mentioned basic parameter need to be in conjunction with full-automatic biochemical point
The included program parameter input method of analyzer, reagent ability necessary instrument automatically determines after carrying out set factors input；
3) positive, negative quality controlled serum, 37 DEG C of incubation 30min are detected according to the above method simultaneously；
4) after measuring, analyze and draw respectively the standard curve of TPS II, hs-CRP, SSA, PCT and phylaxin.
1 full-automatic specific protein analysis-e/or determining basic parameter of table
| Sample size | Amount of reagent (R1/R2) | Reaction temperature | Reaction time (T1/T2) |
| 5μL | 200μL/50μL | 37℃ | 300 seconds/300 seconds |
2 full-automatic specific protein analysis-e/or determining basic parameter of table
| Unit | The Direction of Reaction | Standard curve simulation equation |
| mg/L | Upwards | Multiple spot calibration, nonlinear computation model |
Embodiment 2:
The standard of serum T PS II, hs-CRP, SSA, PCT and two latex enhancing immune turbidimetry detection kit of phylaxin
Exactness
(1) accuracy and precision
The standard items of high, medium and low three concentration are selected, are respectively containing TPS II, hs-CRP, SSA, PCT and resistin level
500ng/ml, 200ng/ml, 450ng/ml, 450ng/ml and 300ng/ml, 200ng/ml, 150ng/ml, 180ng/ml,
170ng/ml and 140ng/ml, 50ng/ml, 30ng/ml, 100ng/ml, 70ng/ml and 75ng/ml, the glue designed with the present invention
Cream enhancing immunoturbidimetry detection kit carries out above-mentioned sample each Concentration Testing 10 times, and acquired results are compared,
Determine the accuracy and precision of this kit, obtained testing result such as the following table 3:
3 TPS II of table, hs-CRP, SSA, PCT and phylaxin testing result compare
The T for doing paired sample to this group of data is examined, and obtains P > 0.05, obtain difference between detection group acquired results without
Statistical significance, illustrate latex immunoturbidimetry used by this project no matter it is high, in or low concentration TPS II, hs-
CRP, SSA, PCT and phylaxin can obtain the higher result of accuracy in detecting.
Meanwhile this method coefficient of variation of the repetition testing result of multi objective under high, medium and low concentration is respectively less than 10%,
With preferable precision.
(2) stabilization of kit is tested:
Kit preservation condition is 2-8 DEG C, and after saving 6 months, the indices of assay kit are found in normal model
Within enclosing.Consider in transport and use process, has improper preservation condition and occur, the condition that kit is saved at 37 DEG C
It is lower to place 6 days, carry out accelerated aging tests, the results showed that the kit indices comply fully with requirement, it is contemplated that kit
Freezing happens, and kit is put into -20 DEG C of refrigerator freezings 5 days, and measurement result also indicates that kit indices completely just
Often.It can show that kit can at least save 6 months or more at 2~8 DEG C from result above.
In the present embodiment, the logical one kind provided of the present invention detects TPS II, SSA, hs-CRP, PCT in blood plasma and supports simultaneously
The kit of anti-cellulose content is the related disease of acute abdomen by establishing a kind of immunoassay method of multi objective joint-detection
The investigation of disease provides that a kind of high sensitivity, accuracy be good and kit assay method easily and fast, help clinician couple
Acute abdomen related disease makes timely diagnosis, also carries out auxiliary judgment for condition assessment or Index for diagnosis.
The above, further embodiment only of the present invention, but scope of protection of the present invention is not limited thereto, and it is any
Within the scope of the present disclosure, according to the technique and scheme of the present invention and its design adds those familiar with the art
With equivalent substitution or change, protection scope of the present invention is belonged to.
Claims (10)
1. a kind of kit for detecting Multiple components content in blood plasma simultaneously, which is characterized in that molten including detection solution R1, detection
Liquid R2, antibiont positive markers quality controlled serum and antibiont marker negative quality controlled serum.
2. a kind of kit for detecting Multiple components content in blood plasma simultaneously as described in claim 1, which is characterized in that detection
Solution R1 includes phosphate buffer and Macrogol 6000；Detection solution R2 include phosphate buffer, coupling have biomarker
The sensitizing latex and stabilizer Proclin300 of antibody；Biomarker includes TPS II, SSA, hs-CRP, PCT and phylaxin.
3. a kind of kit for detecting Multiple components content in blood plasma simultaneously as claimed in claim 2, which is characterized in that sensitization
Latex is obtained with biomarker antibody by physical absorption and/or covalent bond by latex particle.
4. a kind of kit for detecting Multiple components content in blood plasma simultaneously as claimed in claim 2, which is characterized in that latex
Particle is that carboxyl polystyrene microsphere, magnetic polystyrene microsphere, aminopolystyrene microballoon, dimethylamino polystyrene are micro-
Ball, sulfonated polystyrene microballoon, polystyrene microsphere, poly (methyl methacrylate) micro-sphere, poly lactide-glycolide acid are micro-
Any one in ball.
5. a kind of kit for detecting Multiple components content in blood plasma simultaneously as claimed in claim 2, which is characterized in that biology
Marker antibody is monoclonal antibody, and monoclonal antibody passes through monoclonal hybridoma technology and is prepared or passes through
Phage antibody technology screening obtains.
6. a kind of kit for detecting Multiple components content in blood plasma simultaneously as claimed in claim 2, which is characterized in that sensitization
Latex is prepared via a method which to obtain:
Step 1: latex activation
Latex is added in suitable MES buffer, after ultrasonication, be slowly added to thereto under stirring conditions EDA and
NHS, constant-temperature incubation for a period of time after, centrifugation removes supernatant, then PBS buffer solution is added into precipitating, ultrasonication is carried out, from adding
Enter PBS to carry out ultrasonication the step of repeat 1 time, obtain latex particle；
Step 2: antibody linked
PBS is added into latex particle, after carrying out ultrasonication, is separately added into the monoclonal antibody of biomarker thereto,
Additional amount is 1.5~6mg/ml, after mixing, is placed in be incubated at room temperature to reaction solution and is evenly distributed state, sensitizing latex；
Step 3: closing
The glycine for being 3.3%mg/ml with PBS buffer solution compound concentration, by glycine solution and sensitizing latex according to volume ratio
100:5 is mixed, and after ultrasonication, 40~50min is incubated at 25 DEG C, and pH7.5,50mM are then added thereto and includes
The PBS buffer solution of 0.05wt%BSA after mixing, is incubated overnight at 25 DEG C；After centrifugation, PBS, ultrasonication are added thereto；From
The heart removes supernatant, and 0.05% tween is added, is centrifuged again after ultrasonication, removes supernatant, after 0.05% BSA is added, surpassed
After sound is broken, the stabilizer Proclin300 of 0.05% BSA and 0.1% is continuously added, carries out ultrasonication, ultrasonication
Condition is 100Hz, 0.8s/ times, carries out 2min altogether, is then saved for use for 4 DEG C.
7. a kind of kit for detecting Multiple components content in blood plasma simultaneously as described in claim 1, which is characterized in that biology
Positive markers quality controlled serum and the biomarker feminine gender quality controlled serum are all normal human serum, biomarker positive matter
Control serum is made by adding TPS II, SSA, hs-CRP, PCT and phylaxin standard items respectively in normal human serum, addition
Amount is 3 times of the critical value of TPS II in normal serum, SSA, hs-CRP, PCT and resistance cellulose content.
8. a kind of kit for detecting Multiple components content in blood plasma simultaneously as described in claim 1, which is characterized in that reagent
Box includes the detection cup of five kinds of different colours label, respectively to the inspection of TPS II, SSA, hs-CRP, PCT and phylaxin in serum
Survey, the range of linearity of detection be followed successively by 2.5 μ g/L of μ g/L~1250,0.1ng/ml~300ng/ml, 0.3mg/L~150mg/L,
0.1ng/ml~250ng/ml, 1ng/ml~200ng/ml.
9. it is a kind of as described in claim 1~8 any one while detection blood plasma in Multiple components content method, feature
It is, includes the following steps:
By the way that the corresponding antibodies of substance to be checked are coupled with latex particle, by the specific bond of antigen and antibody, so that
Latex particle is aggregated, and is changed the turbidity of antigen-antibody reaction system, is improved the sensitivity of detection；
By the joint-detection of serum T PS II and hs-CRP, Acute Pancreatitis with Acute is checked, by joint-detection serum
The content of PCT and hs-CRP, diagnosis investigation that is timely, effectively completing acute peritonitis, facilitates the observation of clinical efficacy；
By the joint-detection of serum SSA and hs-CRP, acute appendicitis is checked, by phylaxin in patients serum
With the detection of the content of hs-CRP, exedens acute perforation is checked into property；
The serum sample one's duty that will be obtained after TPS II, SSA, hs-CRP, PCT and phylaxin standard items or the centrifugation of EDTA anticoagulated blood
It is not mixed with the prepared sensitizing latex containing corresponding antibodies, reacts antigen-antibody sufficiently, to antigen-antibody reaction
After a period of time, with the absorbance of full automatic biochemical apparatus measurement reaction solution, reference standard curve can determine test sample respectively
The content of middle TPS II, SSA, hs-CRP, PCT and phylaxin.
10. it is a kind of as described in claim 1~8 any one while detection blood plasma in the kit of Multiple components contents answer
With, which is characterized in that it is used to prepare on the reagent of investigation acute abdomen related disease, acute abdomen related disease is acute pancreas
Any one in inflammation, acute peritonitis, Peptic Ulcer with Acute Perforation, acute appendicitis and septicemia.
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| CN201811640110.9A CN109521200A (en) | 2018-12-29 | 2018-12-29 | It is a kind of while detecting the kit of Multiple components content, method and its application in blood plasma |
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| CN201811640110.9A CN109521200A (en) | 2018-12-29 | 2018-12-29 | It is a kind of while detecting the kit of Multiple components content, method and its application in blood plasma |
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Cited By (3)
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| CN109917063A (en) * | 2019-04-15 | 2019-06-21 | 德阳市人民医院 | A kind of metabolic markers relevant to acute pancreatitis |
| CN111830260A (en) * | 2020-07-27 | 2020-10-27 | 北京安图生物工程有限公司 | Process for reducing batch-to-batch difference of latex enhanced immunoturbidimetric reagent |
| WO2022052012A1 (en) * | 2020-09-10 | 2022-03-17 | 北京肿瘤医院(北京大学肿瘤医院) | Marker and method for early diagnosis of infectious complications in abdominal cavity |
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