CN116519941A - Screening kit for acute fatty liver diseases in gestation period - Google Patents
Screening kit for acute fatty liver diseases in gestation period Download PDFInfo
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Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
- G01N33/535—Production of labelled immunochemicals with enzyme label or co-enzymes, co-factors, enzyme inhibitors or enzyme substrates
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
- G01N33/54326—Magnetic particles
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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- Life Sciences & Earth Sciences (AREA)
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- Urology & Nephrology (AREA)
- Biomedical Technology (AREA)
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- Medicinal Chemistry (AREA)
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- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
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- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention relates to the field of medical screening, in particular to a screening kit for acute fatty liver diseases in gestation period. The invention provides a screening kit for acute fatty liver diseases in gestation period, which comprises magnetic particle suspension coated with a recognition total PlGF antibody, a horseradish peroxidase-labeled recognition total PlGF antibody solution, a sample diluent and a calibrator. Compared with the traditional kit for obtaining serum total PlGF by identifying sFlt-1 and free PlGF, the kit can directly obtain the serum total PlGF level, reduces medical cost, has accurate and stable test result, high sensitivity, short reaction time, simple operation and high anti-interference performance, can more intuitively and rapidly predict the risk of pregnant women suffering from gestational acute fatty liver, provides basis for medical decision of doctors, and has good clinical application prospect.
Description
Technical Field
The invention relates to the field of medical screening, in particular to a screening kit for acute fatty liver diseases in gestation period.
Background
Acute fatty liver during pregnancy (AFLP) is a severe liver disease characteristic of pregnancy, and usually occurs 30 weeks after pregnancy. Although unusual, the global incidence rate is estimated to be 1:7000-15000, AFLP is a life-threatening disease for both pregnant women and fetuses, and the fatality rate of the mother reaches 75% -85% once. The exact pathogenesis of AFLP is not known, but it is generally believed that defects in mitochondrial oxidation of the fetus or placenta lead to the accumulation of free fatty acids in maternal blood and hepatocytes, which in turn lead to the detrimental manifestation of the disease. The most notable feature of AFLP is the presence of liver dysfunction, manifested by complications such as hypoglycemia, coagulation dysfunction and renal failure. Up to 20% of AFLP pregnant women are also diagnosed with preeclampsia, which is characterized by the second half of pregnancy with signs of proteinuria or maternal organ damage, including HELLP syndrome. During preeclampsia, placental failure triggers the excessive release of sVEGFR (soluble vascular endothelial growth factor receptor; also known as sFlt-1[ soluble fms-like tyrosine kinase-1 ]), which binds its free circulating ligands VEGF (vascular endothelial growth factor) and PlGF (placental growth factor).
In pre-eclampsia and HELLP syndrome pregnant women patients, total PlGF was substantially consistent with normal gestation levels, whereas in AFLP pregnant women, total PlGF was significantly higher than normal levels prior to delivery, and post-partum total PlGF was rapidly decreased, indicating that in AFLP, plGF was predominantly derived from placenta. The suspected AFLP pregnant women have no clinical specificity in early stage, and for the suspected AFLP pregnant women, the blood coagulation function and the liver function are recommended to be rechecked at intervals of 24 hours. Liver tissue biopsies are the "gold standard" for diagnostic AFLP, but are rarely used in clinical practice due to the invasive nature of penetration. Thus, the effects of placental abnormalities before AFLP occurs can be more intuitively predicted by detecting total pigf levels.
The methods for detecting PlGF in clinic at present mainly comprise enzyme-linked immunosorbent assay (ELISA), time-resolved fluorescence immunoassay (TRFIA), fluorescence Immunochromatography (FICA), electrochemiluminescence (ECLA) and chemiluminescence immunoassay (CLIA). PlGF is a highly specific marker, and in gestational disorders, there is a distinct advantage in detecting total levels of PlGF in pregnant women, which can be used to assess AFLP pregnancy disorders, only in which the total PlGF content in the pregnant woman is significantly elevated prior to the onset of AFLP.
The specific form of the detection marker is not explicitly indicated in the detection method, so that a plurality of uncertain factors are brought to the prediction accuracy of gestational diseases, the clinical diagnosis and treatment efficiency is greatly reduced, and the related medical cost is increased. The literature reports that serum total PlGF (n=42; average calculated KD is 50 pmol/L) can be accurately predicted from sFlt-1 and free PlGF levels using well-known mathematical formulas describing drug-receptor interactions, resulting in a similar thermal dissociation method to previously published sFlt-1-PlGF complexes (r=0.94, P < 0.0001). This procedure requires the measurement of sFlt-1 and free PlGF levels in the same pregnant woman sample and calculation of the total PlGF level by the formula shown in FIG. 1. Since two markers need to be detected simultaneously, this increases the medical cost; furthermore, the mechanism of receptor binding in vivo is complex, and it is doubtful whether the final calculated total PlGF is close to the true result.
Disclosure of Invention
In view of the above, the technical problem to be solved by the invention is to provide a screening kit for acute fatty liver diseases in gestation period.
The invention provides a kit for screening gestational acute fatty liver diseases, which comprises a marker solution and a magnetic particle suspension:
the marker solution comprises a buffer solution and an enzyme-labeled PlGF antibody;
the PlGF antibody is a monoclonal antibody, and the recognition site of the monoclonal antibody does not exist between the sFlt-1 receptor and the PlGF binding site;
the magnetic particle suspension comprises a buffer solution and magnetic beads coated with PlGF antibodies;
the preparation method of the magnetic particle suspension comprises the following steps: first washing, activation, second washing, antibody coating and blocking;
the first washing reagent is phosphate buffer solution, and the washing times are 5 times;
the conditions of the activation are as follows: activating for 1h at room temperature in the presence of EDC and NHS;
the second washing reagent is sodium acetate buffer solution, and the washing times are 3 times;
the concentration of the coated PlGF antibody in the antibody coating step is 15-50 mug/mL, and the coating condition is that the solution is vibrated for 2 hours at room temperature;
the blocking reagent is Tris-NaCl buffer containing BSA.
Further, in the preparation of the magnetic particle suspension,
the first washing reagent is phosphate buffer solution with the pH of 0.05mol/L and 7.4;
the conditions of the activation are as follows: activating for 1h at room temperature in the presence of 10-20 mg/mL EDC and 10-20 mg/mL NHS;
the second washed reagent is 0.05mol/L sodium acetate buffer solution with pH of 4.5;
the blocking reagent is Tris-NaCl buffer solution containing 1% BSA by mass fraction.
In the invention, the magnetic particles in the magnetic particle suspension are selected from paramagnetic particles, and further, from superparamagnetic ferroferric oxide particles; the modification group on the magnetic particles includes, but is not limited to, any one of carboxyl, amino, hydroxyl, mercapto, tosyl, ethylene oxide or streptavidin-biotin; in some embodiments of the present invention, the magnetic particle modifying group is a carboxyl group, and the magnetic particles in the magnetic particle suspension are ferroferric oxide carboxyl magnetic particles; the magnetic particles may be prepared by several methods known in the art.
In the present invention, the PlGF antibody recognition site is free of the sFlt-1 receptor and the PlGF binding site, is not affected by the sFlt-1 receptor binding, and can be used for detecting the total PlGF level in serum, preferably, the PlGF antibody is a monoclonal antibody, more preferably, the antibody is a mouse monoclonal antibody.
The prior art deduces that serum total PlGF can be calculated from sFlt-1 and free PlGF levels by the principle of drug-receptor interaction, and the sFlt-1 and free PlGF levels in the same pregnant woman sample need to be detected and the total PlGF levels calculated by the formula. Since two markers need to be detected simultaneously, this increases the medical cost; furthermore, the mechanism of receptor binding in vivo is complex, and it is doubtful whether the final calculated total PlGF is close to the true result. The invention obtains the antibody capable of recognizing the serum total PlGF through antibody screening, and the antibody is applied to the kit disclosed by the invention, so that the serum total PlGF content can be directly detected, the medical cost is reduced, and the detection result is more accurate and reliable. And meanwhile, the antibody is coated on magnetic particles for chemiluminescent detection of serum total PlGF level. In the preparation step of the magnetic particle suspension, different buffers are adopted to wash the magnetic particles, so that the magnetic particles can be well and uniformly dispersed in the magnetic particle suspension, the magnetic particle suspension has a good antibody coating effect, and meanwhile, uncoated antibodies are sufficiently removed by washing for a plurality of times, so that the detection result is more accurate.
The solvent of the marker solution is Tris-NaCl buffer solution containing 1% BSA by mass fraction; enzyme labels for enzyme-labeled PlGF antibodies include, but are not limited to, horseradish peroxidase (HRP), alkaline phosphatase (ALP), ruthenium terpyridyl (Ru (bpy)) 3 2+ ) Acridinium ester (NSP-DMAE-NHS), [ N- (4-aminobutyl) -N-ethyl]-any one of chloro-2, 3-dihydrophenothiazine-1, 4-dione (ABEI); in some embodiments of the invention, the label is horseradish peroxidase.
The kit also comprises a sample diluent and a calibrator;
the sample diluent is Tris buffer solution containing 0.5-1.5% of BSA by mass fraction, 0.1-0.3% of ProClin300 by mass fraction, 0.01-0.1% of Bronidox by mass fraction and 0.1-0.3 mg/mL of blocking agent; the blocking agent is selected from HBR, MARK33, CHEMIBLOCK or TRUBLCK. The blocking agent is mainly used for eliminating the influence of an interfering substance in a sample on a detection result, and can effectively improve the detection specificity of the kit.
Further, the sample diluent is a Tris buffer containing 1.0% BSA by mass fraction, 0.2% ProClin300 by mass fraction, 0.02% Bronidox by mass fraction and 0.2mg/mL blocker by mass fraction of 0.05 mol/L; the blocking agent is HBR.
The calibrator is a matrix solution containing 0.05mol/L Tris, 2-10% trehalose by mass fraction, 0.1-0.3% ProClin300 by mass fraction and 0 pg/mL-20000 pg/mL PlGF antigen, and the matrix solution is at least one selected from bovine serum albumin matrix solution, calf serum matrix solution and hormone-removed human serum matrix solution.
Further, the calibrator is a bovine serum albumin matrix solution containing 0.05mol/L Tris, 5% trehalose by mass fraction, 0.2% ProClin300 by mass fraction and 0 pg/mL-20000 pg/mL PlGF antigen; the mass fraction of the bovine serum albumin in the bovine serum albumin matrix liquid is 3%.
Experimental results show that the calibrator adopts the bovine serum albumin matrix liquid as the solvent, has the advantages of simple operation, lower cost, higher reactivity compared with other types of matrix liquid, longer preservation time and further improvement of the accuracy of the detection result.
In the screening kit for the acute fatty liver disease in gestation period, the PlGF antibody recognition site does not have sFlt-1 receptor and PlGF binding site, can be used for detecting the total PlGF level in serum, and is coated on magnetic particles, so that the magnetic particles are uniformly dispersed through a special coating means, the interference of antibodies in detection is avoided, the detection result is more accurate, the use of a blocking agent in a sample diluent further improves the detection specificity, and the screening of a calibrator matrix liquid improves the reactivity of a calibrator and prolongs the preservation time.
The invention provides a detection method of acute fatty liver diseases in gestation period, which is to detect samples by using the kit disclosed by the invention;
the detecting step includes: the sample is mixed with the magnetic particle suspension, the sample diluent and the enzyme conjugate solution in sequence, incubated, washed and then subjected to luminescence detection;
the volume ratio of the sample, the magnetic particle suspension, the sample diluent and the enzyme conjugate solution is 5:2:5:10;
the conditions of the incubation were 37 ℃ for 34 minutes;
the washed reagent is phosphate buffer; the number of washes was 5.
Further, the sample comprises serum and/or plasma.
The invention provides a screening kit for acute fatty liver diseases in gestation period, which comprises magnetic particle suspension coated with a recognition total PlGF antibody, a horseradish peroxidase-labeled recognition total PlGF antibody solution, a sample diluent and a calibrator. Compared with the traditional kit for obtaining serum total PlGF by identifying sFlt-1 and free PlGF, the kit can directly obtain the serum total PlGF level, reduces medical cost, has accurate and stable test result, high sensitivity, short reaction time, simple operation and high anti-interference performance, can more intuitively and rapidly predict the risk of pregnant women suffering from gestational acute fatty liver, provides basis for medical decision of doctors, and has good clinical application prospect.
Drawings
FIG. 1 shows calculation of the total PlGF equation from free PlGF and sFlt-1;
figure 2 shows a schematic representation of the presence of PlGF in humans;
fig. 3 shows a standard graph in an embodiment of the invention.
Detailed Description
The invention provides a screening kit for acute fatty liver diseases in gestation period, and a person skilled in the art can properly improve the technological parameters by referring to the content of the text. It is expressly noted that all such similar substitutions and modifications will be apparent to those skilled in the art, and are deemed to be included in the present invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those skilled in the relevant art that the invention can be practiced and practiced with modification and alteration and combination of the methods and applications herein without departing from the spirit and scope of the invention.
The test materials adopted by the invention are all common commercial products and can be purchased in the market.
The invention is further illustrated by the following examples:
example 1 preparation method of screening kit for gestational acute fatty liver disease
(1) Preparation of PlGF antibody coated magnetic particle suspension
The magnetic particle stock solution is fully and uniformly mixed before being taken, the mixture is washed for 5 times by using a buffer solution, the supernatant is discarded, 10-20 mg/mL EDC and 10-20 mg/mL NHS are sequentially added for activating for 1h at room temperature, and carboxyl functional groups on the surfaces of the magnetic particles are fully exposed. After the completion of the activation, the supernatant was discarded and washed 2 times with the buffer solution. PlGF coated with mouse monoclonal antibody of 15-50 mu g/mL is added, and the mixture is subjected to shaking reaction at room temperature for 2h. And then blocking four times by using Tris-NaCl buffer solution containing 1% of BSA by mass fraction, removing the supernatant after finishing, adding the Tris-NaCl buffer solution containing 1% of BSA by mass fraction to fix the volume, and storing at 2-8 ℃ for standby.
The buffer solution in the above embodiment may be any one or more of phosphate buffer, sodium citrate buffer and sodium acetate buffer, and phosphate buffer (0.05 mol/L, pH 7.4) and sodium acetate buffer (0.05 mol/L, pH 4.5) are respectively used before and after the embodiment.
(2) Preparation of marker solution coupled with PlGF antibody
Adding the PlGF mouse monoclonal antibody marked by horseradish peroxidase into Tris-NaCl buffer solution containing 1% of BSA by mass fraction in a ratio of 1 (1000-2000), fully and uniformly mixing, and storing at 2-8 ℃ for standby.
(3) Preparation of sample dilutions
Dissolving 1% BSA (BSA) by mass fraction, 0.2% ProClin300 by mass fraction, 0.02% Bronidox by mass fraction and 0.2mg/mL blocker into Tris buffer solution with pH of 7.5 and 0.05mol/L, mixing to obtain sample diluent, and preserving at 2-8 ℃; the blocking agent is HBR.
(4) Preparation of a calibrator
The PlGF pure antigen is diluted into 6 gradients by a matrix solution containing 5% of trehalose, 3% of BSA, 0.2% of ProClin300 and Tris with the pH of 7.5 and 0.05mol/L (the matrix solution is bovine serum albumin matrix solution, the mass fraction of bovine serum albumin in the bovine serum albumin matrix solution is 3%), the concentration is about 0pg/mL, 10pg/mL, 100pg/mL, 1000pg/mL, 10000pg/mL and 20000pg/mL, the mixture is packaged into 1 mL/bottle, and the mixture is stored at the temperature of 2-8 ℃ after freeze drying. When in use, 1mL of purified water is used for redissolution and then is uniformly shaken, and the liquid calibrator after redissolution can be placed for 28 days at the temperature of 2-8 ℃.
Example 2 detection method of the kit of the present invention
(1) Whole blood samples were collected using the correct medical technique, centrifuged (rotational speed: 4000r/min, time: 10 min) and serum was extracted for testing.
(2) Sequentially adding 50 mu L of calibrator/sample into the reaction cup;
(3) Adding 20 mu L of magnetic particle suspension into each hole;
(4) 50 mu L of sample diluent is added to each hole respectively;
(5) 100. Mu.L of enzyme conjugate solution was added to each well;
(6) After mixing well, incubation was carried out at 37℃for 34 minutes;
(7) Washing the cleaning solution for 5 times;
(8) Adding 50 mu L of each of chemiluminescent substrate A and luminescent substrate B into each hole, uniformly mixing, and detecting a luminescence value;
(9) And (3) calculating results: the kit recommends a four-parameter fitting mode, and establishes a calibration curve by taking the concentration value of the calibrator as the x axis and the log value of the luminescence value of the calibrator as the y axis (figure 3). And (5) recalculating the corresponding concentration value according to the luminous value of the sample to be detected. The automatic instrument operation system can automatically calculate a sample test result through the stored calibration curve and the luminescence value obtained by the sample test.
Example 3 confirmation of the form of the detection substance of the kit of the present invention
The receptor ligand interaction was destabilized by heat treatment (10 min,70 ℃) releasing bound PlGF from multimeric sFlt-1 receptor masking. Analyzing serum samples (P1-P3) of pregnant women before and after heat treatment by using the kit; in addition, a simulated pregnant woman sample (P0) was mixed at 1:1 using a PlGF pure antigen solution with a concentration of 3565.31pg/mL and an sFlt-1 pure antigen solution with a concentration of 5365.14pg/mL, and measured after 30min at room temperature, and then measured again after heat treatment (10 min,70 ℃ C.), the results are shown in Table 1:
TABLE 1 measurement of samples of pregnant women before and after treatment
| Sample numbering | Before treatment (pg/mL) | After treatment (pg/mL) | Rate of change |
| P1 | 513.88 | 508.24 | -1.10％ |
| P2 | 207.91 | 196.52 | -5.48％ |
| P3 | 1039.70 | 1102.43 | 6.03％ |
| P0 | 1776.87 | 1769.35 | -0.42％ |
The results show that the detection values of the four samples before and after heat treatment are not changed significantly, which indicates that whether sFlt-1 is combined with PlGF or not has no influence on the detection of the PlGF content of the kit, namely the detection material form of the kit is total PlGF.
Example 4 correlation of serum total PlGF levels with AFLP:
samples of pregnant women at late pregnancy were tested as in example 2, and the statistics are shown in table 2:
TABLE 2 correlation of serum Total PlGF levels with AFLP
As shown in Table 2, the total serum PlGF of normal pregnant women was 431.74pg/mL and the total serum PlGF of AFLP pregnant women was as high as 1986.29pg/mL, the average levels of both were 4.6 times different, and there was a significant difference.
From the above experimental results, it can be seen that the serum total PlGF level of pregnant women is significantly correlated with AFLP, and that the antibodies of the invention can detect serum total PlGF. Although the incidence of AFLP is low, once the mortality rate of the ill females is extremely high, the likelihood of AFLP can be sensitively suggested to be high when the serum total PlGF level of the pregnant female is abnormally elevated. Thus, AFLP disease can be screened by detecting total serum PlGF levels in pregnant women.
EXAMPLE 5 screening of matrix fluids in the calibration Material of the kit of the invention
The reactivity and freeze-drying reconstitution stability were evaluated separately after preparing the calibrator with different matrix solutions, and the specific results are shown in tables 3 and 4 below.
TABLE 3 reactivity of calibration materials formulated with different matrix fluids
TABLE 4 lyophilized reconstitution stability of calibrants prepared from different matrix fluids
As can be seen from the results in tables 3 and 4, the reactivity of the calibrator prepared with the three matrix solutions was not greatly different, wherein the background signal values of the matrix solutions of 3% bovine serum albumin and 100% calf serum were low; the freeze-drying and re-dissolving stability of the calibrator prepared from 3% bovine serum albumin and 100% antihormone human serum matrix liquid is kept within 10% after 30 days. Therefore, the matrix solution of the calibrator is preferably 3% bovine serum albumin.
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.
Claims (10)
1. A kit for screening for gestational acute fatty liver disease comprising a marker solution and a magnetic particle suspension:
the label solution comprises a solvent and an enzyme-labeled PlGF antibody;
the PlGF antibody is a monoclonal antibody, and the recognition site of the monoclonal antibody does not exist between the sFlt-1 receptor and the PlGF binding site;
the magnetic particle suspension comprises a buffer solution and magnetic beads coated with PlGF antibodies;
the preparation method of the magnetic particle suspension comprises the following steps: first washing, activation, second washing, antibody coating and blocking;
the first washing reagent is phosphate buffer solution, and the washing times are 5 times;
the conditions of the activation are as follows: activating for 1h at room temperature in the presence of EDC and NHS;
the second washing reagent is sodium acetate buffer solution, and the washing times are 3 times;
the concentration of the coated PlGF antibody in the antibody coating step is 15-50 mug/mL, and the coating condition is that the solution is vibrated for 2 hours at room temperature;
the blocking reagent is Tris-NaCl buffer containing BSA.
2. The kit according to claim 1, wherein,
the first washing reagent is phosphate buffer solution with the pH of 0.05mol/L and 7.4;
the conditions of the activation are as follows: activating for 1h at room temperature in the presence of 10-20 mg/mL EDC and 10-20 mg/mL NHS;
the second washed reagent is 0.05mol/L sodium acetate buffer solution with pH of 4.5;
the blocking reagent is Tris-NaCl buffer solution containing 1% BSA by mass fraction.
3. The kit according to claim 1 or 2, wherein the magnetic particles in the magnetic particle suspension are ferroferric oxide carboxyl magnetic particles.
4. The kit according to any one of claim 1 to 3, wherein,
the solvent of the marker solution is Tris-NaCl buffer solution containing 1% BSA by mass fraction; the enzyme label of the enzyme-labeled PlGF antibody is horseradish peroxidase.
5. The kit of any one of claims 1-4, further comprising a sample diluent and a calibrator;
the sample diluent is Tris buffer solution containing 0.5-1.5% BSA by mass fraction, 0.1-0.3% ProClin300 by mass fraction, 0.01-0.1% Bronidox by mass fraction and 0.1-0.3 mg/mL blocker by mass fraction;
the calibrator is a matrix solution containing 0.05mol/L Tris, 2-10% trehalose by mass fraction, 0.1-0.3% ProClin300 by mass fraction and 0 pg/mL-20000 pg/mL PlGF antigen, and the matrix solution is at least one selected from bovine serum albumin matrix solution, calf serum matrix solution and hormone-removed human serum matrix solution.
6. The kit of any one of claims 1 to 5, wherein the sample diluent is 0.05mol/L Tris buffer containing 1.0% bsa by mass, 0.2% proclin300 by mass, 0.02% bronidox by mass and 0.2mg/mL blocker;
the blocking agent is HBR.
7. The kit according to any one of claims 1 to 6, wherein the calibrator is a bovine serum albumin matrix solution comprising 0.05mol/L Tris, 5% trehalose by mass, 0.2% proclin300 by mass and 0pg/mL to 20000pg/mL of pigf antigen; the mass fraction of the bovine serum albumin in the bovine serum albumin matrix liquid is 3%.
8. A method for detecting acute fatty liver disease in gestation period, characterized in that the kit according to any one of claims 1 to 7 is used for detecting a sample.
9. The method according to claim 8, wherein,
the detecting step includes: the sample is mixed with the magnetic particle suspension, the sample diluent and the enzyme conjugate solution in sequence, incubated, washed and then subjected to luminescence detection;
the volume ratio of the sample, the magnetic particle suspension, the sample diluent and the enzyme conjugate solution is 5:2:5:10;
the conditions of the incubation were 37 ℃ for 34 minutes;
the washed reagent is phosphate buffer; the number of washes was 5.
10. The method of claim 8 or 9, wherein the sample comprises serum and/or plasma.
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| CN117741165A (en) * | 2023-11-29 | 2024-03-22 | 浙江鼎创医疗科技有限公司 | Colloidal gold immunochromatographic test strip for high-specificity and high-sensitivity HNL detection and preparation method and use method thereof |
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