CN114487433A - Free IgE chemiluminescence immunoassay kit - Google Patents
Free IgE chemiluminescence immunoassay kit Download PDFInfo
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Abstract
The invention provides a free IgE chemiluminescence immunoassay kit. The free IgE chemiluminescence immunoassay kit comprises an R1 reagent, an R2 reagent and an R3 reagent, wherein the R1 reagent is a buffer solution containing biotin-labeled Fc epsilon RI/Fc epsilon RIA protein and a first protective agent, and the first protective agent comprises casein, animal serum and bovine serum albumin L; the R2 reagent is a buffer solution containing an anti-human IgE antibody marked by a luminescent marker and a second protective agent, wherein the second protective agent comprises casein and animal serum; the R3 reagent is a buffer containing streptavidin-labeled magnetic microparticles. The kit has the advantages of good stability, high accuracy, high sensitivity and the like.
Description
Technical Field
The invention relates to the technical field of antibody detection, and particularly relates to a free IgE chemiluminescence immunoassay kit.
Background
Allergic asthma is a chronic inflammatory respiratory disease caused by the binding of inhaled antigens to specific IgE. The complex formed by the combination of the inhalation antigen and IgE can release inflammatory mediators after being crosslinked with high-affinity receptors on the surfaces of effector cells such as mast cells, basophils and the like, thereby causing respiratory tract contraction and inflammatory reaction. Humanized anti-IgE monoclonal antibody omalizumab (omalizumab) is the first innovative medicine for treating asthma worldwide and is used for treating moderate-to-severe persistent allergic asthma. Omalizumab inhibits IgE binding to mast cell and basophil surface IgE receptors (fcsri) by binding to the Fc fragment C epsilon 3 of the IgE molecule, thereby preventing degranulation of effector cells and inhibiting the release of inflammatory mediators.
Clinical study data show that after omalizumab administration, serum free IgE levels are highly correlated with clinical manifestations, including total asthma symptom score, morning peak expiratory flow, and rescue medication usage. At the same time, clinical symptoms of asthma reappear as free IgE returns to baseline concentrations. It is currently generally accepted that the decrease in serum free IgE concentration after omalizumab administration can serve as a pharmacodynamic surrogate marker for omalizumab. In addition, although total IgE is not directly related to pharmacodynamic results, the level of total IgE can indicate that omalizumab changes the clearance rule and distribution rule of IgE in vivo.
Therefore, in preclinical evaluation of an anti-IgE antibody drug, the examination of the levels of free IgE and total IgE in serum has great significance for overall evaluation of pharmacodynamic properties, pharmacokinetics-pharmacodynamics (PK-PD), toxicology evaluation, or similarity or superiority and inferiority with the original drug, and can provide important data and theoretical support for research of biomarkers in later clinical pharmacology evaluation.
At present, the domestic and foreign literature has no detailed description on the detection method of the levels of free IgE and total IgE in serum, and the commercially available kit cannot distinguish the free IgE from the total IgE, so that the relevant detection in the preclinical evaluation of the anti-IgE antibody causes great trouble.
Disclosure of Invention
The invention aims to provide a kit capable of quantitatively detecting free IgE.
In order to solve the technical problem, the invention adopts the following technical scheme:
a free IgE chemiluminescence immunoassay kit comprises an R1 reagent, an R2 reagent and an R3 reagent,
the reagent R1 is a buffer solution containing biotin-labeled Fc epsilon RI/Fc epsilon RIA protein and a first protective agent, the first protective agent comprises casein with the content of 0.4-0.6% of the total mass of the reagent R1, animal serum with the content of 0.2-0.5% of the total mass of the reagent R1 and bovine serum albumin with the content of 0.1-0.3% of the total mass of the reagent R1, and the content of the biotin-labeled Fc epsilon RI/Fc epsilon RIA protein is 0.02-0.08 mu g/mL;
the R2 reagent is a buffer solution containing a luminescent marker labeled anti-human IgE antibody and a second protective agent, the second protective agent comprises casein with the content of 0.5-0.8% of the total mass of the R2 reagent and animal serum with the content of 0.2-0.5% of the total mass of the R2 reagent, and the content of the luminescent marker labeled anti-human IgE antibody is 0.08-0.12 mu g/mL;
the R3 reagent is a buffer solution containing streptavidin-labeled magnetic particles, and the content of the streptavidin-labeled magnetic particles is 0.5-2% of the total mass of the R3 reagent.
Preferably, the animal serum is a composition of fetal calf serum and horse serum, and the mass ratio of the fetal calf serum to the horse serum is 10-20: 1.
Further preferably, the mass ratio of the fetal calf serum to the horse serum is 14-16: 1.
Preferably, the first protective agent further comprises polyethylene glycol 6000 in an amount of 0.1-0.3% of the total mass of the R1 reagent and trehalose in an amount of 1-2% of the total mass of the R1 reagent.
Preferably, the buffer used in the R1 reagent is TRIS buffer.
Preferably, the buffer used in the R2 reagent is HEPES buffer.
Preferably, the buffer solution adopted by the R3 reagent is PBS buffer solution.
Preferably, the pH value of the R1 reagent is 7.5-8.5.
Preferably, the pH value of the R2 reagent is 6-7.
Preferably, the pH value of the R3 reagent is 6.5-7.5.
Preferably, the mass ratio of biotin to fcsri/fcsria protein in said biotin-labeled fcsri/fcsria protein is 1: 8 to 20.
Preferably, in the anti-human IgE antibody labeled with the luminescent marker, the mass ratio of the anti-human IgE antibody to the luminescent marker is 1: 1 to 5.
Preferably, the luminescent marker is acridinium ester, luminol, isoluminol, alkaline phosphatase or ruthenium terpyridyl.
Preferably, the magnetic fine particles have a particle size of 0.8 to 1.5 μm.
Preferably, the R1 reagent further comprises salts with the content of 50-200 mmol/L, a preservative with the content of 0.01-0.1% of the total mass of the R1 reagent, an HBR-1 blocking agent with the content of 0.1-0.5 mg/mL and a surfactant with the content of 0.5-1%.
Preferably, the R2 reagent further comprises salts with the content of 50-200 mmol/L, a preservative with the content of 0.05-0.1% of the total mass of the R2 reagent, an alkaline phosphatase blocking agent with the content of 0.01-0.05 mg/mL and a surfactant with the content of 0.5-1%.
According to some embodiments, the R1 reagent consists of:
the R2 reagent comprises the following components:
the R3 reagent comprises the following components:
more preferably, the salts are NaCl, KCl and MgCl2One or more of (a).
Further preferably, the preservative is Proclin 300.
Further preferably, the surfactant is one or more of polyoxyethylene lauryl ether, triton X-100, tween 20 and tween-80.
Preferably, the free IgE chemiluminescence immunoassay kit further comprises a calibrator, and the calibrator comprises free IgE solutions with concentrations of 0.00ng/mL, 6.25ng/mL, 12.5ng/mL, 25.00ng/mL, 50.00ng/mL, 100.00ng/mL, 200.00ng/mL and 400.00ng/mL respectively.
Preferably, the free IgE chemiluminescence immunoassay kit further comprises chemiluminescence substrate liquid, wherein the chemiluminescence substrate liquid is APS-5 buffer solution.
Compared with the prior art, the invention has the following advantages:
the invention adopts a magnetic particle chemiluminescence immunoassay method, designs a kit for detecting the concentration of free IgE, and the kit has the advantages of good stability, high accuracy, high sensitivity and the like.
Drawings
FIG. 1 is a standard curve of the free IgE chemiluminescent detection kit in the examples for the detection of calibrator relative to light units, with calibrator concentration in ng/mL on the abscissa and Relative Light Units (RLU) on the ordinate.
Detailed Description
The present invention will be further described with reference to the following examples. However, the present invention is not limited to the following examples. The implementation conditions adopted in the embodiments can be further adjusted according to different requirements of specific use, and the implementation conditions not mentioned are conventional conditions in the industry. The technical features of the embodiments of the present invention may be combined with each other as long as they do not conflict with each other.
The invention provides a free IgE chemiluminescence immunoassay kit, which comprises an R1 reagent, an R2 reagent and an R3 reagent,
the reagent R1 is a buffer solution containing biotin-labeled Fc epsilon RI/Fc epsilon RIA protein and a first protective agent, the first protective agent comprises casein with the content of 0.4-0.6% of the total mass of the reagent R1, animal serum with the content of 0.2-0.5% of the total mass of the reagent R1 and bovine serum albumin with the content of 0.1-0.3% of the total mass of the reagent R1, and the content of the biotin-labeled Fc epsilon RI/Fc epsilon RIA protein is 0.02-0.08 mu g/mL;
the R2 reagent is a buffer solution containing a luminescent marker labeled anti-human IgE antibody and a second protective agent, the second protective agent comprises casein with the content of 0.5-0.8% of the total mass of the R2 reagent and animal serum with the content of 0.2-0.5% of the total mass of the R2 reagent, and the content of the luminescent marker labeled anti-human IgE antibody is 0.08-0.12 mu g/mL;
the R3 reagent is a buffer solution containing streptavidin-labeled magnetic particles, and the content of the streptavidin-labeled magnetic particles is 0.5-2% of the total mass of the R3 reagent.
According to an embodiment, the R1 reagent consists of:
according to the embodiment, the R2 reagent comprises the following components:
according to the embodiment, the R3 reagent comprises the following components:
preferably, the salts are NaCl, KCl and MgCl2One or more of (a).
Preferably, the preservative is Proclin 300.
Preferably, the surfactant is one or more of polyoxyethylene lauryl ether, triton X-100, Tween 20 and Tween-80.
Preferably, the pH value of the R1 reagent is 7.5-8.5.
Preferably, the pH value of the R2 reagent is 6-7.
Preferably, the pH value of the R3 reagent is 6.5-7.5.
Preferably, the mass ratio of biotin to fcsri/fcsria protein in said biotin-labeled fcsri/fcsria protein is 1: 8 to 20.
Preferably, in the anti-human IgE antibody labeled with the luminescent marker, the mass ratio of the anti-human IgE antibody to the luminescent marker is 1: 1 to 5.
Preferably, the magnetic fine particles have a particle size of 0.8 to 1.5 μm.
According to an embodiment, the free IgE chemiluminescence immunoassay kit further comprises a calibrator comprising free IgE solutions at concentrations of 0.00ng/mL, 6.25ng/mL, 12.5ng/mL, 25.00ng/mL, 50.00ng/mL, 100.00ng/mL, 200.00ng/mL, and 400.00ng/mL, respectively.
According to the embodiment, the free IgE chemiluminescence immunoassay kit further comprises chemiluminescence substrate liquid, wherein the chemiluminescence substrate liquid is APS-5 buffer solution.
The free IgE chemiluminescence immunoassay kit can use a full-automatic chemiluminescence immunoassay analyzer as a detection instrument to complete the detection of the free IgE. The chemiluminescence immunoassay kit is matched with an instrument, so that the time required by clinical detection is shortened, and the detection accuracy is higher.
The detection method of the free IgE chemiluminescence immunoassay kit comprises the following steps:
a full-automatic chemiluminescence immunoassay analyzer (i2900) is used as a detection instrument, and the detection principle of the kit is an indirect method, namely, 10 mu L of sample, 30 mu L R1 reagent and 30 mu L R3 reagent are sequentially added into the instrument, after reaction is carried out for 7min, magnetic separation is carried out, and unbound substances are washed. After reaction for 7min, 50. mu. L R2 reagent was added, magnetic separation was performed, unbound material was washed, luminescence substrate solution was added for reaction, and finally Relative Light Units (RLU) were recorded.
The present invention will be described in further detail with reference to specific examples.
In the following examples and comparative examples, raw materials, reagents and the like used were all conventional commercially available products, if specifically noted.
In the following examples and comparative examples, streptavidin magnetic beads were purchased from Agilent technologies, Inc. (Cat. PL 6727-1001).
In the following examples and comparative examples, "%" is given as a mass percentage, if specifically indicated.
Example 1
This example provides a free IgE chemiluminescent immunoassay kit comprising R1 reagent, R2 reagent, and R3 reagent.
Composition of R1 reagent:
the preparation method of the R1 reagent comprises the following steps:
the labeling process of the biotin-labeled Fc epsilon RI/Fc epsilon RIA protein comprises the following steps: adding 440 mu L of DMSO into a 2mg active biotin bottle, uniformly mixing, then adding 1mg of Fc epsilon RI/Fc epsilon RIA to be marked into an ultrafiltration tube, adding a proper volume of marking buffer solution to a constant volume of 0.5mL to enable the final concentration to be 2mg/mL, uniformly mixing, centrifuging by using a high-speed refrigerated centrifuge at a rotating speed of 12000r/min and at a temperature of 2-8 ℃ for 10 minutes, adding 33.3 mu L of biotin solution and 466.7 mu L of marking buffer solution into the ultrafiltration tube after centrifuging, and lightly blowing, beating and uniformly mixing. And putting the mixture into a 37 ℃ thermostat, keeping out of the sun, incubating for 30 minutes, and centrifuging after incubation at the rotating speed of 12000r/min at the temperature of 2-8 ℃ for 10 minutes. After centrifugation, 500 mu L of marking buffer solution is added into the ultrafiltration tube, and the mixture is lightly blown and evenly mixed, and then the mixture is centrifuged for 10 minutes at the rotating speed of 12000 r/min. The steps are repeated for 3 times, after the last centrifugation, 250 mu L of marking buffer solution is added into the ultrafiltration tube and is lightly blown and uniformly mixed, the mixture is transferred into the centrifugation tube, 250 mu L of preservation solution is added, 0.5mL of biotin marking Fc epsilon RI/Fc epsilon RIA with the concentration of 2mg/mL is obtained, and the mixture is preserved at the temperature of 2-8 ℃.
According to the composition of the R1 reagent, the biotin-labeled Fc epsilon RI/Fc epsilon RIA protein and other components are mixed and encapsulated in a reagent bottle to obtain the R1 reagent.
Composition of R2 reagent:
0.1. mu.g/mL of an anti-human IgE antibody labeled with a luminescent marker (acridinium ester is used as the luminescent marker, and the mass ratio of the luminescent marker to the anti-human IgE antibody is 1: 3).
The preparation method of the R2 reagent comprises the following steps:
the labeling process of the anti-human IgE antibody labeled by the chemiluminescence label comprises the following steps: putting 1mg of anti-human IgE antibody into a centrifuge tube, adding carbonic acid buffer solution with the final concentration of 2mg/mL, fully and uniformly mixing, adding 10 mu L of 2mg/mL DMF solution of terpyridyl ruthenium, centrifuging for 30s by a high-speed refrigerated centrifuge at the rotating speed of 12000 r/min. Sealing the centrifugal tube with a sealing film, placing into a gas bath constant temperature oscillator (23 ℃), and mixing for 4 h. Then 0.5mL of 30% lysine blocking solution was added, and the mixture was put into a gas bath constant temperature oscillator (23 ℃), mixed well and then blocked for 2 hours. The blocked antibody was purified on Sephadex G250 column, eluted with PBS buffer and collected in fractions. And storing the collected antibody solution at the temperature of 2-8 ℃.
The R2 reagent was obtained by mixing the anti-human IgE antibody labeled with a chemiluminescent label with the other components according to the composition of the R2 reagent, and filling the mixture in a reagent bottle.
Composition of R3 reagent:
the preparation method of the R3 reagent comprises the following steps:
preparation of reagent R3:
the streptavidin magnetic particle process comprises the following steps: taking 1mL of streptavidin magnetic particle solution with the concentration of 100mg/mL, adding 10mL of TBST solution, fully mixing for 15 minutes, placing on a magnetic separator until the supernatant is not turbid, discarding the supernatant, taking the magnetic particles, and repeating the process for cleaning for 3 times. Adding the mixture into 10mL of buffer solution containing 50mM MES, 0.05% Tween-20, 0.05% Proclin300 and pH6.5, calibrating the concentration to be 10mg/mL, and storing at 2-8 ℃.
The R3 reagent was obtained by mixing streptavidin magnetic particles with other components according to the composition of the R3 reagent, and filling the mixture in a reagent bottle.
Preparation of a calibrator:
serum base was used to formulate free IgE calibrators at concentrations of 0.00ng/mL, 6.25ng/mL, 12.5ng/mL, 25.00ng/mL, 50.00ng/mL, 100.00ng/mL, 200.00ng/mL, and 400.00 ng/mL. The calibrator was assayed using the kit prepared in example 1 and a standard curve was plotted, as shown in FIG. 1, with calibrator concentration in ng/mL on the abscissa and Relative Light Units (RLU) on the ordinate.
Comparative example 1
This comparative example provides a free IgE chemiluminescent immunoassay kit comprising a R1 reagent, a R2 reagent, and a R3 reagent.
Composition of R1 reagent:
the preparation method of the R1 reagent comprises the following steps:
the labeling process of the biotin-labeled Fc epsilon RI/Fc epsilon RIA protein comprises the following steps: adding 440 mu L of DMSO into a 2mg active biotin bottle, uniformly mixing, then adding 1mg of Fc epsilon RI/Fc epsilon RIA to be marked into an ultrafiltration tube, adding a proper volume of marking buffer solution to a constant volume of 0.5mL to enable the final concentration to be 2mg/mL, uniformly mixing, centrifuging by using a high-speed refrigerated centrifuge at a rotating speed of 12000r/min and at a temperature of 2-8 ℃ for 10 minutes, adding 33.3 mu L of biotin solution and 466.7 mu L of marking buffer solution into the ultrafiltration tube after centrifuging, and lightly blowing, beating and uniformly mixing. And putting the mixture into a 37 ℃ thermostat, keeping out of the sun, incubating for 30 minutes, and centrifuging after incubation at the rotating speed of 12000r/min at the temperature of 2-8 ℃ for 10 minutes. After centrifugation, 500 mu L of marking buffer solution is added into the ultrafiltration tube, and the mixture is lightly blown and evenly mixed, and then the mixture is centrifuged for 10 minutes at the rotating speed of 12000 r/min. The steps are repeated for 3 times, after the last centrifugation, 250 mu L of marking buffer solution is added into the ultrafiltration tube and is lightly blown and uniformly mixed, the mixture is transferred into the centrifugation tube, 250 mu L of preservation solution is added, 0.5mL of biotin marking Fc epsilon RI/Fc epsilon RIA with the concentration of 2mg/mL is obtained, and the mixture is preserved at the temperature of 2-8 ℃.
According to the composition of the R1 reagent, the biotin-labeled Fc epsilon RI/Fc epsilon RIA protein and other components are mixed and encapsulated in a reagent bottle to obtain the R1 reagent.
The R2 reagent was the same as in example 1.
The R3 reagent was the same as in example 1.
Comparative example 2
This comparative example provides a free IgE chemiluminescent immunoassay kit comprising a R1 reagent, a R2 reagent, and a R3 reagent.
The R1 reagent was the same as in example 1.
R2 reagent composition:
the preparation method of the R2 reagent comprises the following steps:
the process for labeling the anti-human IgE antibody by using the chemiluminescent label comprises the following steps: putting 1mg of anti-human IgE antibody into a centrifuge tube, adding carbonic acid buffer solution with the final concentration of 2mg/mL, fully and uniformly mixing, adding 10 mu L of 2mg/mL DMF solution of terpyridyl ruthenium, centrifuging for 30s by a high-speed refrigerated centrifuge at the rotating speed of 12000 r/min. Sealing the tube with sealing film, placing into gas bath constant temperature oscillator (23 deg.C), and mixing for 4 hr. Then 0.5mL of 30% lysine blocking solution was added, and the mixture was put into a gas bath constant temperature oscillator (23 ℃), mixed well and then blocked for 2 hours. The blocked antibody was purified on Sephadex G250 column, eluted with PBS buffer and collected in fractions. And storing the collected antibody solution at the temperature of 2-8 ℃.
The R2 reagent was obtained by mixing the anti-human IgE antibody labeled with a chemiluminescent label with the other components according to the composition of the R2 reagent, and filling the mixture in a reagent bottle.
The R3 reagent was the same as in example 1.
Comparative example 3
This comparative example provides a free IgE chemiluminescent immunoassay kit comprising a R1 reagent, a R2 reagent, and a R3 reagent.
The R1 reagent was the same as in example 1.
The R2 reagent was the same as in example 1.
R3 reagent composition:
the preparation method of the R3 reagent comprises the following steps:
the streptavidin magnetic particle process comprises the following steps: taking 1mL of streptavidin magnetic particle solution with the concentration of 100mg/mL, adding 10mL of TBST solution, fully mixing for 15 minutes, placing on a magnetic separator until the supernatant is not turbid, discarding the supernatant, taking the magnetic particles, and repeating the process for cleaning for 3 times. Adding the mixture into 10mL of buffer solution containing 50mM MES, 0.05% Tween-20, 0.05% Proclin300 and pH6.5, calibrating the concentration to be 10mg/mL, and storing at 2-8 ℃.
The R3 reagent was obtained by mixing streptavidin magnetic particles with other components according to the composition of the R3 reagent, and filling the mixture in a reagent bottle.
1. And (3) linear evaluation:
diluting the high value sample close to the upper limit of the linear range to at least 5 concentrations according to a certain proportion, wherein the low value concentration sample is close to the lower limit of the linear range. The test was repeated 3 times for each concentration of the sample using the kits prepared in example 1, comparative example 2 and comparative example 3, the average value was calculated, the average value of the measured concentration and the theoretical concentration or the dilution ratio were fitted with a straight line by the least square method, and the linear correlation coefficient r was calculated, and the results are shown in table 1.
TABLE 1
2. Blank limit evaluation: relative Light (RLU) units are measured 20 times on a zero value calibrator by using the kits prepared in comparative example 1, comparative example 2 and comparative example 3, the average value is subtracted by twice standard deviation, and the obtained value is taken as a blank limit by being substituted into a standard curve; the results are shown in Table 2.
TABLE 2
3. And (3) repeatability evaluation: the kits prepared in example 1, comparative example 2 and comparative example 3 were used to perform repeated tests 10 times on two samples with low repeatability (6-10 ng/mL) and high repeatability (30-50 ng/mL), and the average M, standard deviation SD and CV values of 10 concentration measurements were calculated, and the results are shown in Table 3.
TABLE 3
4. Evaluation of stability: the free IgE chemiluminescence immunoassay kits prepared in example 1, comparative example 2 and comparative example 3 were stored at 2 ℃ to 8 ℃, and blank limit, linearity and repeatability indexes at 3 months, 6 months, 9 months, 12 months and 15 months were evaluated, respectively, with the results shown in table 4.
TABLE 4
5. Evaluation of clinical samples: the free IgE chemiluminescence immunoassay kit prepared in example 1, comparative example 2 and comparative example 3 was used for detecting clinical serum free IgE samples, and the results are shown in table 5.
TABLE 5
The present invention has been described in detail for the purpose of illustration and description, and it will be apparent to those skilled in the art that the invention can be practiced without limitation to such detail, and all changes and modifications that come within the spirit of the invention are desired to be protected.
Claims (10)
1. A free IgE chemiluminescence immunoassay kit is characterized in that: which comprises an R1 reagent, an R2 reagent and an R3 reagent,
the reagent R1 is a buffer solution containing biotin-labeled Fc epsilon RI/Fc epsilon RIA protein and a first protective agent, the first protective agent comprises casein with the content of 0.4-0.6% of the total mass of the reagent R1, animal serum with the content of 0.2-0.5% of the total mass of the reagent R1 and bovine serum albumin with the content of 0.1-0.3% of the total mass of the reagent R1, and the content of the biotin-labeled Fc epsilon RI/Fc epsilon RIA protein is 0.02-0.08 mu g/mL;
the R2 reagent is a buffer solution containing a luminescent marker labeled anti-human IgE antibody and a second protective agent, the second protective agent comprises casein with the content of 0.5-0.8% of the total mass of the R2 reagent and animal serum with the content of 0.2-0.5% of the total mass of the R2 reagent, and the content of the luminescent marker labeled anti-human IgE antibody is 0.08-0.12 mu g/mL;
the R3 reagent is a buffer solution containing streptavidin-labeled magnetic particles, and the content of the streptavidin-labeled magnetic particles is 0.5-2% of the total mass of the R3 reagent.
2. The free IgE chemiluminescent immunoassay kit of claim 1, wherein: the animal serum is a composition of fetal calf serum and horse serum, and the mass ratio of the fetal calf serum to the horse serum is 10-20: 1.
3. The free IgE chemiluminescent immunoassay kit of claim 1, wherein: the first protective agent also comprises polyethylene glycol 6000 with the content of 0.1-0.3% of the total mass of the R1 reagent and trehalose with the content of 1-2% of the total mass of the R1 reagent.
4. The free IgE chemiluminescent immunoassay kit of claim 1, wherein: the buffer solution used by the R1 reagent is TRIS buffer solution; and/or the buffer used by the R2 reagent is HEPES buffer; and/or the buffer solution adopted by the R3 reagent is PBS buffer solution.
5. The free IgE chemiluminescent immunoassay kit of claim 1, wherein: the pH value of the R1 reagent is 7.5-8.5; and/or the pH value of the R2 reagent is 6-7; and/or the pH value of the R3 reagent is 6.5-7.5.
6. The free IgE chemiluminescent immunoassay kit of claim 1, wherein: the mass ratio of biotin to the Fc epsilon RI/Fc epsilon RIA protein in the biotin-labeled Fc epsilon RI/Fc epsilon RIA protein is 1: 8-20; and/or the mass ratio of the anti-human IgE antibody to the luminescent marker in the luminescent marker-labeled anti-human IgE antibody is 1: 1-5, and/or the luminescent marker is acridinium ester, luminol, isoluminol, alkaline phosphatase or ruthenium terpyridyl, and/or the particle size of the magnetic particle is 0.8-1.5 μm.
7. The free IgE chemiluminescent immunoassay kit of claim 1, wherein: the R1 reagent further comprises salts with the content of 50-200 mmol/L, a preservative with the content of 0.01-0.1% of the total mass of the R1 reagent, an HBR-1 blocking agent with the content of 0.1-0.5 mg/mL and a surfactant with the content of 0.5-1%;
and/or the R2 reagent further comprises salts with the content of 50-200 mmol/L, preservatives with the content of 0.05-0.1% of the total mass of the R2 reagent, alkaline phosphatase blocking agents with the content of 0.01-0.05 mg/mL and surfactants with the content of 0.5-1%.
8. The free IgE chemiluminescent immunoassay kit of claim 7 or 9, wherein: the R1 reagent comprises the following components:
the R2 reagent comprises the following components:
the R3 reagent comprises the following components:
9. the free IgE chemiluminescent immunoassay kit of claim 7 or 9, wherein the salt is NaCl, KCl, MgCl2One or more of; and/or the preservative is Proclin300, and/or the surfactant is one or more of polyoxyethylene lauryl ether, triton X-100, Tween 20 and Tween-80.
10. The free IgE chemiluminescent immunoassay kit of claim 1, wherein: the free IgE chemiluminescence immunoassay kit further comprises a calibrator, wherein the calibrator comprises free IgE solutions with the concentrations of 0.00ng/mL, 6.25ng/mL, 12.5ng/mL, 25.00ng/mL, 50.00ng/mL, 100.00ng/mL, 200.00ng/mL and 400.00ng/mL respectively; and/or, the free IgE chemiluminescence immunoassay kit further comprises chemiluminescence substrate liquid, wherein the chemiluminescence substrate liquid is APS-5 buffer solution.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115718197A (en) * | 2022-09-03 | 2023-02-28 | 长沙海柯生物科技有限公司 | Kit for detecting content of free lgE and application thereof |
| CN116068206A (en) * | 2023-03-15 | 2023-05-05 | 安徽惠邦生物工程有限公司 | Myelin basic protein detection kit and detection method thereof |
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2021
- 2021-12-27 CN CN202111612827.4A patent/CN114487433A/en active Pending
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115718197A (en) * | 2022-09-03 | 2023-02-28 | 长沙海柯生物科技有限公司 | Kit for detecting content of free lgE and application thereof |
| CN115718197B (en) * | 2022-09-03 | 2023-07-04 | 长沙海柯生物科技有限公司 | Kit for detecting content of free lgE and application thereof |
| CN116068206A (en) * | 2023-03-15 | 2023-05-05 | 安徽惠邦生物工程有限公司 | Myelin basic protein detection kit and detection method thereof |
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