CN114563569B - Application of signal amplification technology in PGP9.5 detection kit - Google Patents

Application of signal amplification technology in PGP9.5 detection kit Download PDF

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CN114563569B
CN114563569B CN202210110138.1A CN202210110138A CN114563569B CN 114563569 B CN114563569 B CN 114563569B CN 202210110138 A CN202210110138 A CN 202210110138A CN 114563569 B CN114563569 B CN 114563569B
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buffer
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CN114563569A (en
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王法龙
李锋
杨涛
孙佳
张广俊
张明琛
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Beijing Meilian Taike Biotechnology Co ltd
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Abstract

The invention provides an application of a signal amplification technology in a PGP9.5 detection kit, relating to the technical field of biology, wherein the kit comprises a detection reagent strip; the detection reagent strip comprises a reagent A, a reagent B and a reagent C; the raw material of the reagent A comprises PGP9.5 antibody 1; the raw material of the reagent B comprises PGP9.5 antibody 2; the reagent C includes an anti-FITC antibody. The invention is improved on the basis of the original invention, adds a FITC-anti-FITC antibody signal amplification system, increases the sensitivity of the kit, reduces the usage amount of main raw materials and reduces the material cost.

Description

Application of signal amplification technology in PGP9.5 detection kit
Technical Field
The invention relates to the field of biotechnology, in particular to application of a signal amplification technology in a PGP9.5 detection kit.
Background
Traumatic Brain Injury (TBI) is brain injury caused by external forces that disrupt normal brain function, resulting in impaired cognitive or physical performance in humans. Among all types of TBI, the most common sequelae are headache (47.9%) and memory abnormalities (42%) ("british journal of neurosurgery, 2018), with about three adults in need of psychological counseling or neurological treatment. TBI is the most common disease in neurosurgery and is also one of the leading causes of death and disability worldwide, with sequelae that have a permanent impact on patient health.
The suspected TBI medical care procedure is divided into three steps, first with nerve evaluation using the 15-minute Glasgow Coma Scale (GCS) (american society of surgeons trauma committee, 1997) to assess brain injury severity, followed by structural neuroimaging examination, most commonly by visualization of bone fractures and intracranial lesions by head CT scanning. And finally, making a treatment scheme, and performing hospital stay observation or discharge according to the CT result.
Currently, CT scanning is the only objective, simple and reliable option widely used to help clinicians assess TBI. However, the correctness of the CT result is directly related to the accuracy of the CT device and the level of interpretation of the physician, and is a relatively subjective judgment method compared with other detection methods. CT scans with about 90% mild TBI (sometimes referred to as "concussion") were negative (Toth, 2015). Less than 1% of these patients require neurosurgical intervention (Papa L, 2012). In view of the very low percentage of CT scan positivity and the unnecessary imaging detection of these patients may increase the risk of radiation-induced carcinogenesis, it is of great clinical and strategic importance to find and develop other brain injury diagnostic methods to accurately determine the extent of craniocerebral injury and to assess prognosis.
Brain specific protein product 9.5(PGP9.5) is a deubiquitinase and also a nervous system specific protein. Human PGP9.5 consists of 223 amino acids, is distributed mainly in mature neurons, especially in the substantia nigra, with distribution in both undifferentiated central and peripheral nerves. PGP9.5 is one of the most abundant proteins in the brain, and its content may be 2% of brain soluble proteins. PGP9.5 exerts regulatory functions on cell proliferation, differentiation and apoptosis via ubiquitin-related pathways. In TBI, PGP9.5 will cross the blood brain barrier into the blood within 1 hour, resulting in a significant elevation of serum PGP 9.5. Has important significance for early diagnosis, differential diagnosis and prognosis judgment of TBI, and is mainly used for auxiliary diagnosis of brain trauma clinically. Patent CN201780080585.4 discloses an antibody against ubiquitin C-terminal hydrolase L1(UCH-L1) and Glial Fibrillary Acidic Protein (GFAP) and related methods, providing corresponding methods, systems and kits, mainly for individuals known or suspected to suffer from brain injury or damage, such as nerve damage as mild traumatic brain injury. However, the invention mainly protects the protein, and the related method has long detection time, low sensitivity and narrow linearity.
Aiming at the problems in the prior art, it is necessary to provide an in vitro diagnostic kit which is rapid, simple and convenient and can provide auxiliary diagnosis for traumatic brain injury.
Disclosure of Invention
Aiming at the problems in the prior art, the invention provides an in vitro diagnostic kit which is simple and convenient to operate and can provide auxiliary diagnosis for traumatic brain injury, a preparation method and application thereof, and aims to quickly and simply detect the content of a brain specific protein product in human peripheral blood. The kit of the invention is a method for quantitatively analyzing the level of the human peripheral blood brain specific protein product by adopting a magnetic particle chemiluminescence method, and is used for auxiliary diagnosis of nervous system diseases such as traumatic brain injury and the like. The invention is improved on the basis of the original invention, adds a FITC-anti-FITC antibody signal amplification system, increases the sensitivity of the kit, reduces the usage amount of main raw materials and reduces the material cost.
In order to achieve the purpose, the technical scheme adopted by the invention is as follows:
the invention provides a PGP9.5 detection kit, which comprises a detection reagent strip; the detection reagent strip comprises a reagent A, a reagent B and a reagent C;
the raw material of the reagent A comprises PGP9.5 antibody 1; the raw material of the reagent B comprises PGP9.5 antibody 2; the reagent C comprises an anti-FITC antibody;
the PGP9.5 antibody 1 comprises a heavy chain variable region sequence shown by SEQ ID NO. 2 and a light chain variable region sequence shown by SEQ ID NO. 4; the PGP9.5 antibody 2 comprises a heavy chain variable region sequence shown in SEQ ID NO. 3 and a light chain variable region sequence shown in SEQ ID NO. 5.
Furthermore, the sequence of the PGP9.5 antibody 1 is the amino acid sequence shown in SEQ ID NO. 7, and the sequence of the PGP9.5 antibody 2 is the amino acid sequence shown in SEQ ID NO. 8.
Further, the raw material of the reagent A also comprises alkaline phosphatase, the raw material of the reagent B also comprises FITC, and the raw material of the reagent C also comprises an anti-FITC antibody.
Further, the device also comprises a calibration material and a quality control material.
Further, the raw materials of the calibrator and the quality control product both comprise PGP9.5 recombinant protein.
Further, it is characterized in that: the detection reagent strip also comprises a cleaning solution, a luminescent substrate, a reading hole, an elution sleeve and a suction head.
The invention also provides a preparation method of the PGP9.5 detection kit, which comprises the following steps:
(1) production of solvent A: coupling alkaline phosphatase with PGP9.5 antibody 1, and mixing with buffer solution to obtain reagent A;
(2) production of solvent B: coupling FITC and PGP9.5 antibody 2, and then mixing with a buffer solution to obtain a reagent B;
(3) production of solvent C: and connecting the anti-FITC antibody to the magnetic particles, and mixing with a buffer solution to obtain a reagent C.
Further, the weight ratio of PGP9.5 antibody 1 to alkaline phosphatase is 1:1-2, and the weight ratio of FITC to PGP9.5 antibody 2 is 3-15: 100.
Further, the weight ratio of the anti-FITC antibody to the magnetic particles is 100: 3-20.
Further, the invention relates to a formula and a preparation method of the buffer solution, wherein the formula comprises the following steps:
buffer solution 1
14.8-15.1g of ethanolamine and 5.8-6.0g of NaCl are weighed and added into a certain amount of purified water to be stirred until the ethanolamine and the NaCl are completely dissolved, the pH value is adjusted to be 7.3-7.6, and the volume is fixed to 1000 ml. Filtration was performed with a 0.22 μm filter.
TABLE 1 buffer 1 formulation
Name of raw materials Weighing volume
Ethanolamine 14.8-15.1g
Sodium chloride 5.8-6.0g
pH value 7.3-7.6
Purified water The volume is up to 1000mL
Buffer solution 2
75g of glycine is weighed and added into a certain amount of purified water to be stirred until the glycine is completely dissolved, and the volume is up to 1000 ml. Filtration was performed with a 0.22 μm filter.
TABLE 2 buffer 2 formulation
Name of raw materials Weighing volume
Glycine 75g
Purified water The volume is up to 1000mL
Buffer solution 3
Weighing 12.0-15.0g of Tris, 20.0-60g of bovine serum albumin, 2.0-10.0g of NaCl and 10.0-100g of sucrose, adding into a certain amount of purified water, stirring until the mixture is completely dissolved, adjusting the pH value to 7.5-10.0, and fixing the volume to 1000 ml. Filtration was performed with a 0.22 μm filter.
TABLE 3 buffer 3 formulation
Name of raw materials Weighing volume
Tris (hydroxymethyl) aminomethane 12.0-15.0g
Bovine serum albumin 20.0-60g
Sodium chloride 2.0-10.0g
Sucrose 10.0-100g
pH value 7.5-10.0
Purified water The volume is up to 1000mL
Buffer 4
Weighing 12.0-15.0g of Tris, 9.0g of NaCl, 1.0-50g of bovine serum albumin, 5.0-20.0g of glycerol and 5.0-40g of glycine, adding into a certain amount of purified water, stirring until the materials are completely dissolved, adjusting the pH value to 7.5-9.5 and fixing the volume to 1000 ml. Filtration was performed with a 0.22 μm filter.
TABLE 4 buffer 4 formulation
Name of raw materials Weighing volume
Tris (hydroxymethyl) aminomethane 12.0-15.0g
Sodium chloride 9.0g
Bovine serum albumin 1.0-50g
Glycerol 5.0-20.0g
Glycine 5.0-40g
pH value 7.5-9.5
Purified water The volume is up to 1000mL
Buffer solution 5
Weighing 5.6-5.9g of Na 2 HPO 4 ·12H 2 O, 0.55-0.60g NaH 2 PO 4 9.0g of NaCl, 1.0-50g of bovine serum albumin, 80.0-140g of sucrose, 0.1-5.0g of xanthan gum, 0.1-5g of sodium alginate and 1.0-15g of gelatin are added into a certain amount of purified water and stirred until the materials are completely dissolved, the pH value is adjusted to be 6.2-8.0, and the volume is fixed to 1000 ml. Filtration was performed with a 0.22 μm filter.
TABLE 5 buffer 5 formulation
Name of raw materials Weighing volume
Disodium hydrogen phosphate dodecahydrate 5.6-5.9g
Sodium dihydrogen phosphate 0.55-0.60g
Sodium chloride 9.0g
Bovine serum albumin 1.0-50g
Sucrose 80.0-140g
Xanthan gum 0.1-5.0g
Sodium alginate 0.1-5g
Gelatin 1.0-15g
pH value 6.2-8.0
Purified water The volume is up to 1000mL
Buffer 6
Weighing 5.6-5.9g of Na 2 HPO 4 ·12H 2 O, 0.55-0.60g NaH 2 PO 4 9.0g of NaCl and 0.1-5.0g of KCl are added into purified water and the volume is 1000 ml. Filtration was performed with a 0.22 μm filter.
TABLE 6 buffer 6 formulation
Name of raw materials Weighing volume
Disodium hydrogen phosphate dodecahydrate 5.6-5.9g
Sodium dihydrogen phosphate 0.55-0.60g
Sodium chloride 9.0g
Potassium chloride 0.1-5.0g
Buffer 7
Weighing 12-30g MES (2- (N-morpholine) ethanesulfonic acid) and adding purified water to reach 1000 ml. Filtration was performed with a 0.22 μm filter.
TABLE 7 buffer 7 formulation
Figure BDA0003494852660000051
Figure BDA0003494852660000061
Buffer solution 8
Weighing 2.5-5.0g of Tris, 9.0g of NaCl and 5.0-20.0g of bovine serum albumin, adding the materials into a certain amount of purified water, stirring until the materials are completely dissolved, adjusting the pH value to 7.0-7.6 and fixing the volume to 1000 ml. Filtration was performed with a 0.22 μm filter.
TABLE 8 buffer 8 formulation
Name of raw materials Weighing volume
Tris (hydroxymethyl) aminomethane 2.5-5.0g
Sodium chloride 9.0g
Bovine serum albumin 5.0-20.0g
pH value 7.0-7.6
Purified water The volume is up to 1000mL
Buffer solution 9
Weighing 2.5-5.0g of Tris and 9.0g of NaCl into a certain amount of purified water, stirring until the Tris and the NaCl are completely dissolved, weighing 0.2-2mL of Tween 20, adding into the container, adjusting the pH value to 7.0-7.6, and fixing the volume to 1000 mL. Filtration was performed with a 0.22 μm filter.
TABLE 9 buffer 9 formulation
Name of raw materials Weighing volume
Tris (hydroxymethyl) aminomethane 2.5-5.0g
Sodium chloride 9.0g
Tween 20 0.2-2mL
pH value 7.0-7.6
Purified water The volume is up to 1000mL
Buffer solution 10
Weighing 8.5-15.0g of Tris, 9.0g of NaCl, 20-150g of bovine serum albumin and 15-100g of sucrose, adding into a certain amount of purified water, stirring until the mixture is completely dissolved, weighing 1-20mL of ProClin300, adding into the container, adjusting the pH value to 7.5-9.2, and fixing the volume to 1000 mL. Filtration was performed with a 0.22 μm filter.
TABLE 10 buffer 9 formulation
Figure BDA0003494852660000062
Figure BDA0003494852660000071
Buffer solution 11
Weighing 12-18g of Tris into a certain amount of purified water, stirring until the Tris is completely dissolved, adjusting the pH value to be 8.0-9.5 and metering the volume to 1000 ml. Filtration was performed with a 0.22 μm filter.
TABLE 11 buffer 9 formulation
Name of raw materials Weighing volume
Tris (hydroxymethyl) aminomethane 12-18g
pH value 8.0-9.5
Purified water The volume is up to 1000mL
The technical effects obtained by the invention are as follows:
1. compared with the imaging detection means (mainly CT), the blood detection method can objectively reflect the real situation of the sample, and reduce misjudgment and missed judgment caused by subjective judgment.
2. The magnetic particle chemiluminescence method used in the method can enable the detection sensitivity to reach a picogram level (10-12g/mL), and CT relies on pixels to achieve higher resolution. Because the invention detects the brain injury specific marker, the detection window is much earlier than CT. Under the condition of CT negative, normal people and patients with mild TBI can be effectively distinguished.
3. The invention uses a full-automatic instrument for detection, and accurate results can be obtained only by adding a serum sample for 30 minutes. The CT detection time is long, and generally needs to wait for 4 hours before the detection result can be obtained.
4. The invention uses the concentration value to judge, and the obtained result can know whether the patient is sick or not, thus having stronger objectivity. The CT detection needs doctors to read the films, and has strong subjective judgment according to the business level of the doctors, so that the missed judgment and the misjudgment are easily caused.
5. The invention improves the reaction system of the brain specific protein product 9.5(PGP9.5) detection kit, improves the sensitivity, increases the signal value and reduces the material cost.
Drawings
FIG. 1 is a schematic view of a PGP9.5 detection reagent strip;
FIG. 2 is a reaction scheme of the present invention;
FIG. 3 is a process flow diagram of the present invention;
FIG. 4 is a schematic diagram of a FITC-anti-FITC antibody signal amplification system.
Detailed Description
The embodiments of the present invention are described below with reference to specific embodiments, and other advantages and effects of the present invention will be easily understood by those skilled in the art from the disclosure of the present specification. The invention is capable of other and different embodiments and of being practiced or of being carried out in various ways, and its several details are capable of modification in various respects, all without departing from the spirit and scope of the present invention.
Before the present embodiments are further described, it is to be understood that the scope of the invention is not limited to the particular embodiments described below; it is also to be understood that the terminology used in the examples is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention.
When numerical ranges are given in the examples, it is understood that both endpoints of each of the numerical ranges and any value therebetween can be selected unless the invention otherwise indicated. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
It is to be noted that the magnetic particles used in the present invention are specifically Sigma RSTREP-RO; FITC is sigma F7250; anti-FITC antibodies were purchased from Abcam, cat No.: ab 19224;
the antibody 1 and the antibody 2 are self-made monoclonal antibodies; the preparation method of the monoclonal antibody comprises the following steps:
1. immunizing animals
BALB/c mice are selected as host animals for immunization, 1-5mg/mL antigen solution (the antigen is PGP9.5 protein, the sequence of the PGP9.5 protein is shown as SEQ ID NO: 1) and Freund's complete adjuvant are mixed according to the volume ratio of 1:1 for intraperitoneal injection. Mixing 1-5mg/mL antigen solution with Freund's incomplete adjuvant at a volume ratio of 1:1 on days 14 and 35, and performing second and third immunization, and dissolving 1-5mg/mL antigen in PBS on day 56 to perform fourth immunization. Cell fusion was performed around day 61. In the meantime, two titer tests were performed on days 21 and 42, respectively, and the immune effect was observed.
Freund's complete adjuvant: sigma, F5881 (goods number)
Freund's incomplete adjuvant: sigma, F5506 (Commodity number)
2. Cell fusion and culture
Mixing the extracted spleen cells and myeloma cells of the mice according to a counting ratio of 1: 3. After mixing, the mixture was added to RPMI 1640 medium to a volume of 40mL, centrifuged at 1500rpm for 5 minutes, and the supernatant was removed. Fusion was carried out in a 37 ℃ water bath during which 1mL of isothermal fusogenic agent was added, and after standing for 1 minute, 2mL of isothermal RPMI 1640 medium was added slowly. After mixing, the mixture was centrifuged at 1500rpm for 5 minutes, and the supernatant was removed. The fused cell liquid was placed in a culture dish with feeder cells and cultured in a 5% CO2 incubator at 37 ℃.
RPMI 1640 medium: sigma R8758 (goods number)
Fluxing agent: sigma, 11363735001 (goods number)
3. Screening and cloning of hybridoma positive clones
After 7 days of culture, the culture medium was replaced with HAT medium, and the culture was continued, and after 14 days, the second selection was performed. And the second screening adopts a limiting dilution method to dilute the cells for 3-4 times, and selects the cells with the highest positive value for cloning to obtain a proper cell strain.
HAT medium: sigma H0262 (goods number)
4. Preparation of monoclonal antibodies
BALB/c mice were selected as hosts and each mouse was intraperitoneally injected with 0.5-1mL of pristane. Each mouse was inoculated intraperitoneally with 2mL of log phase hybridoma cells at a cell concentration within 200-300 cells/mL. Ascites extraction was started after 14 days, and 3 days were allowed to elapse between each ascites extraction until no ascites was produced or mice were in a poor condition. Mouse ascites was purified as a monoclonal antibody using a salting-out method and an affinity method.
The sequence of the PGP9.5 antibody 1 prepared by the invention is an amino acid sequence shown by SEQ ID NO. 7, and the sequence of the PGP9.5 antibody 2 is an amino acid sequence shown by SEQ ID NO. 8. The PGP9.5 antibody 1 comprises a heavy chain variable region sequence shown by SEQ ID NO. 2 and a light chain variable region sequence shown by SEQ ID NO. 4, the PGP9.5 antibody 2 comprises a heavy chain variable region sequence shown by SEQ ID NO. 3 and a light chain variable region sequence shown by SEQ ID NO. 5, and the heavy chain variable region and the light chain variable region are connected through flexible connecting peptide shown by SEQ ID NO. 6.
The other raw materials are common commercial products, and thus the sources thereof are not particularly limited.
5. Antibody affinity detection
The affinity of the above antibodies to an antigen (PGP9.5 protein, the sequence of PGP9.5 protein is shown in SEQ ID NO: 1) was determined by biolayer interferometry using a ForteBio Octet QKe Biomolecular interaction analyzer (see instructions or reference Tobias et al, biological Binding Kinetic Assays in the Octet Platform, Application Note 14, ForteBio, div. of Pall Life Sciences, 2013). As can be seen by detection, the KD (M) value of the antibody 1 is 0.97E-12, and the KD (M) value of the antibody 2 is 1.14E-12.
1. Principle of detection
The kit adopts a double-antibody sandwich method to determine the content of PGP 9.5. PGP9.5 in the sample binds to antibody 1 in reagent a and PGP9.5 antibody 2 in reagent B to form a "sandwich" structure. An excess of reagent C was added, and the anti-FITC antibody in reagent C reacted with FITC in reagent B to produce a "alkaline phosphatase-antibody 1-PGP 9.5-antibody 2-FITC-anti-FITC antibody-magnetic microparticle" complex. Upon washing, the luminescent substrate is enzymatically cleaved by the enzymes in the complex to form unstable excited state intermediates which emit photons when they return to the ground state. The number of photons generated is positively correlated with the concentration of PGP9.5 in the sample. The reaction flow chart of the invention is shown in FIG. 2, the process flow of the invention is shown in FIG. 3, and the schematic diagram of FITC-anti-FITC antibody signal amplification system is shown in FIG. 4.
2. Components
2.1 kit Components
The PGP9.5 kit consists of a detection reagent strip, a calibrator, a quality control product and a two-dimensional code. The detection reagent strip is composed of a series of solutions and accessories into a whole and can independently detect a sample. The calibrator is prepared from PGP9.5 antigen with two concentrations and buffer solution and is used for calibrating a standard curve; the quality control product is prepared from PGP9.5 antigen containing two concentrations and buffer solution; the two-dimensional code records a standard curve of the current batch.
TABLE 12 major Components of the kit
The main components of the kit Loading capacity
Detection reagent strip 10 strips
Quality control product 200μL×1
Calibration article 1 200μL×1
Calibration article 2 200μL×1
Box label two-dimensional code 1 is provided with
1.2 reagent strip Components
The detection reagent strip consists of a reagent A, a reagent B, a reagent C, a cleaning solution (1.3-2.2g of tris, 8.5-13.2g of NaCl, 0.7-1mL of Tween 20, and a luminescent substrate (Sigma 69086) with constant volume of 1000 mL), a luminescent substrate, a reading hole, an elution sleeve and a suction head. The reagent A is a PGP9.5 antibody 1 solution containing alkaline phosphatase marks with certain concentration; the reagent B is PGP9.5 antibody 2 solution containing FICT marks with certain concentration; the reagent C is magnetic particles containing a certain concentration of anti-FITC antibody labels; the cleaning solution is used for cleaning the reaction process; the luminescent substrate is an ALP catalyzed luminescent substrate; assay wells were used for final assay readings. The reagent strip location and components are shown in FIG. 1.
TABLE 13 reagent strip principal Components
Figure BDA0003494852660000111
Figure BDA0003494852660000121
3. Production process
3.1 production of calibrator and quality control Material
PGP9.5 recombinant protein (shown in SEQ ID NO: 1) is used as a raw material of a calibrator, and is dissolved by buffer solution 3, and the solution is fully mixed to prepare 2 calibrators with the concentrations of 160pg/mL and 1280pg/mL respectively.
The PGP9.5 recombinant protein is used as a raw material of a quality control product, dissolved by a buffer solution 3, and fully mixed to prepare the quality control product with the concentration of 320 pg/mL.
3.2 buffer formulation and production of reagents A, B and C
Example 1
(1) Preparation of buffer solution
Buffer 1: 14.8g of ethanolamine and 5.8g of NaCl are weighed and added into a certain amount of purified water to be stirred until the ethanolamine and the NaCl are completely dissolved, the pH value is adjusted to be 7.3-7.6, and the volume is fixed to 1000 ml. Filtration was performed with a 0.22 μm filter.
Buffer 2: 75g of glycine is weighed and added into a certain amount of purified water to be stirred until the glycine is completely dissolved, and the volume is up to 1000 ml. Filtration was performed with a 0.22 μm filter.
Buffer 3: weighing 12.0g of Tris, 20.0g of bovine serum albumin, 2.0g of NaCl and 10.0g of sucrose, adding the mixture into a certain amount of purified water, stirring until the mixture is completely dissolved, adjusting the pH value to be 7.5-10.0, and fixing the volume to 1000 ml. Filtration was performed with a 0.22 μm filter.
Buffer 4: weighing 12.0g of Tris, 9.0g of NaCl, 1.0g of bovine serum albumin, 5.0g of glycerol and 5.0g of glycine, adding the mixture into a certain amount of purified water, stirring until the mixture is completely dissolved, adjusting the pH value to be 7.5-9.5, and fixing the volume to 1000 ml. Filtration was performed with a 0.22 μm filter.
And (4) buffer solution 5: 5.6g of Na were weighed 2 HPO 4 ·12H 2 O, 0.55g of NaH 2 PO 4 9.0g of NaCl, 1.0g of bovine serum albumin, 80.0g of sucrose, 0.1g of xanthan gum, 0.1g of sodium alginate and 1.0g of gelatin are added into a certain amount of purified water and stirred until the materials are completely dissolved, the pH value is adjusted to be between 6.2 and 8.0, and the volume is fixed to 1000 ml. Filtration was performed with a 0.22 μm filter.
Buffer 6: 5.6g of Na were weighed 2 HPO 4 ·12H 2 O, 0.55g of NaH 2 PO 4 9.0g NaCl, 0.1g KCl was added to purified water and made to 1000 ml. Filtration was performed with a 0.22 μm filter.
And (3) buffer solution 7: 12g of MES (2- (N-morpholino) ethanesulfonic acid) was weighed into purified water and brought to a volume of 1000 ml. Filtration was performed with a 0.22 μm filter.
Buffer 8: weighing 2.5g of Tris, 9.0g of NaCl and 5.0g of bovine serum albumin, adding the Tris, 9.0g of NaCl and 5.0g of bovine serum albumin into a certain amount of purified water, stirring until the Tris, the NaCl and the bovine serum albumin are completely dissolved, adjusting the pH value to be 7.0-7.6 and fixing the volume to 1000 ml. Filtration was performed with a 0.22 μm filter.
Buffer 9: weighing 2.5g of Tris and 9.0g of NaCl into a certain amount of purified water, stirring until the Tris and the NaCl are completely dissolved, weighing 0.2mL of Tween 20, adding into the container, adjusting the pH value to 7.0-7.6, and fixing the volume to 1000 mL. Filtration was performed with a 0.22 μm filter.
Buffer 10: weighing 8.5g of Tris, 9.0g of NaCl, 20g of bovine serum albumin and 15g of sucrose, adding the Tris into a certain amount of purified water, stirring until the Tris is completely dissolved, weighing 1mL of ProClin300, adding the ProClin300 into the container, adjusting the pH value to be 7.5-9.2 and fixing the volume to 1000 mL. Filtration was performed with a 0.22 μm filter.
Buffer 11: weighing 12g of Tris, adding the Tris into a certain amount of purified water, stirring until the Tris is completely dissolved, adjusting the pH value to be 8.0-9.5, and metering the volume to 1000 ml. Filtration was performed with a 0.22 μm filter.
(2) Production of reagent A
The enzyme-labeled PGP9.5 antibody 1 conjugate is used as a raw material of the reagent A, and the reagent A is prepared by fully and uniformly mixing the raw material with the buffer solution 4.
The method specifically comprises the following steps:
step A1: activation of antibody 1: the activation of antibody 1 needs to be carried out in a hundred thousand grade clean room. 4-8 mg of 2-iminothiolane hydrochloride (2IT) was weighed and dissolved in buffer 1 to 13.76 mg/mL. The 2IT solution was added to the antibody 1 solution for activation at a 2-IT to antibody 1 molar ratio of 15:1 (i.e., 1mg of antibody 1 was added to 15. mu.l of 2IT solution). After shaking and mixing, the mixture was reacted at room temperature for 30 minutes. After the termination of the activation, buffer 2 was added to the antibody 1 solution in a ratio of 1mg of antibody 1 to 5. mu.l of buffer 2, and the reaction was carried out at room temperature for 10 min. Excess 2IT was removed using a PD10 desalting column and activated antibody 1 was collected.
Step A2: activation of alkaline phosphatase (ALP): the activation of ALP is carried out in a hundred thousand grade clean room. 2-4 mg of succinimidyl (N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC) was weighed out and dissolved in Dimethylformamide (DMF) to 6.69 mg/mL. The SMCC solution is added into the ALP solution according to the molar ratio of the SMCC to the ALP of 15: 1. After shaking and mixing, the mixture was reacted at room temperature for 30 minutes. After termination of the activation, buffer 2 was added to the ALP solution in a ratio of 1mg of ALP to 10. mu.l of buffer 2, and the reaction was carried out at room temperature for 10 min. Excess SMCC was removed using a PD10 desalting column and the ALP was collected after activation.
Step A3: linking of antibody 1 and ALP: the ligation of antibody 1 and ALP was carried out in a hundred thousand clean room. ALP solution was added to the antibody 1 solution at a mass ratio of 1:2 of antibody 1 to ALP (i.e., 1.0mg of antibody 1 to 2.0mg of ALP). After shaking and mixing evenly, the mixture reacts for 12 to 18 hours at the temperature of between 2 and 8 ℃.
Step A4: termination and purification of antibody 1 conjugates: termination and purification of the antibody 1 conjugate was carried out in a hundred thousand grade clean room. 1-10mg of maleimide was weighed out and dissolved in DMF to 9.7 mg/mL. At a ratio of 1/10, the solution was diluted with buffer 1 to give a 0.97mg/mL maleimide solution. This solution was added in a ratio of 1mg of antibody 1 to 4. mu.l of 0.97mg/mL maleimide solution, and reacted at room temperature for 15 minutes. mu.L of ethanolamine was measured accurately and dissolved in buffer 1 to 100 mM. That is, 994. mu.L of buffer 1 was added to 6. mu.L of ethanolamine. The solution was added in a ratio of 1mg of antibody 1 to 10. mu.l of 100mM ethanolamine solution, and mixed by shaking. The antibody 1 conjugate to be purified was concentrated to 0.5mg/mL using an ultrafiltration concentration tube. Antibody purification was performed using a purified protein analyzer and Superdex 200 preparative 2.6/60 gel column, buffer 2 as eluent. The purified liquid is the enzyme-labeled antibody 1 conjugate.
Step A5: and fully and uniformly mixing the obtained enzyme-labeled antibody 1 conjugate with a buffer solution 4 to prepare a reagent A.
(3) Production of reagent B
The FITC labeled PGP9.5 antibody 2 conjugate is used as a raw material of a reagent B, and the reagent B is prepared by fully and uniformly mixing the raw material with a buffer solution 4.
The method specifically comprises the following steps:
step B1: desalting of antibody 2: buffer 6 was used as the equilibration and elution solution for the PD10 desalting column. The PD10 desalting column was washed with 15mL of buffer 6, and when the last wash was completed in the column, the antibody 2 to be desalted was added. The column volume was made up to 2mL using buffer 2. When the liquid in the column completely entered the column material, 3mL of buffer 6 was added to start elution. The protein eluate was collected in the corresponding volume (2mL) to first fill up.
The antibody 2 after desalting was concentrated to 0.5mg/mL by adding to an ultrafiltration concentration tube. The parameters of the high-speed refrigerated centrifuge are as follows: centrifuge at 6000rpm for 10 minutes.
Step B2: antibody 2-conjugated FITC: FITC is weighed and prepared into FITC concentrated solution with the final concentration of 0.5mg/mL by using buffer solution 11, the FITC is added into an antibody 2 solution for reaction according to the mass ratio of FITC to antibody 2 of 3:100 (namely, 30 mu g of FITC is added into 1mg of antibody). After shaking and mixing, the mixture is reacted for 18 hours at room temperature (20-30 ℃).
(4) Production of reagent C
The anti-FITC antibody labeled magnetic particles are used as raw materials of the reagent C, and the raw materials are fully and uniformly mixed by using a buffer solution 5 to prepare the reagent C.
The method specifically comprises the following steps:
step C1: cleaning of magnetic particles
The magnetic particle material of 0.5 μm was selected and mixed well for 0.5 hour. The beads were resuspended in buffer 7 at a concentration of 5 mg/ml. And (3) fully and uniformly mixing the washed magnetic beads for 10 minutes, separating the magnetic particles by using a Sepmag magnetic separator, and separating each gram of magnetic bead raw material for 2 minutes but not less than 5 minutes. The resuspension and separation steps were repeated a total of three times.
Step C2: resuspension of magnetic microparticles
The magnetic particles after three washes were resuspended in 5mg/ml using buffer 7. The resuspended magnetic microparticles were mixed well for 5 minutes.
Step C3: selection and desalination of anti-FITC antibodies
The anti-FITC antibody (affinity purification) and the FITC antibody (salting-out purification) are mixed according to the mass ratio of 1:4 to form a new anti-FITC antibody. Buffer 6 was used as the equilibration and elution solution for the PD10 desalting column. The PD10 desalting column was washed with 15mL of buffer 6, and when the last wash was completed in the column, the anti-FITC antibody to be desalted was added. The column volume was made up to 2mL using buffer 2. When the liquid in the column completely entered the column charge, 3mL of buffer 6 was added to start elution. The protein eluate was collected in the corresponding volume (2mL) to first fill up.
And adding the desalted anti-FITC antibody into an ultrafiltration concentration tube for concentration to 0.5 mg/mL. The parameters of the high-speed refrigerated centrifuge are as follows: centrifuge at 6000rpm for 20 minutes.
Step C4: activation of magnetic particles
The carboxyl magnetic fine particles having a particle size of 0.5 μm were selected for activation. (i.e., magnetic particles having a large number of carboxyl functional groups on the surface) a certain amount of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) was dissolved to 4mg/mL with buffer 7. EDC was added to the magnetic particles to react at a ratio of 100:1 (i.e., 1mg of magnetic particles added to 10ug of EDC) by mass, and mixed for 40 minutes at room temperature.
Step C5: cleaning of activated magnetic particles
The activated magnetic particles were resuspended in 5mg/ml using buffer 7. And (3) fully and uniformly mixing the washed magnetic beads for 10 minutes, separating the magnetic particles by using a Sepmag magnetic separator, and separating each gram of magnetic bead raw material for 2 minutes but not less than 5 minutes.
The resuspension and separation steps were repeated a total of three times, and finally the magnetic particles were resuspended with buffer 7 at a concentration of 5 mg/ml.
Step C6: anti-FITC antibody-linked magnetic microparticles
According to the magnetic particles: and (3) adding the anti-FITC antibody into the activated magnetic particles for reaction according to the mass ratio of the anti-FITC antibody to 100:3 (namely adding 30ug of anti-FITC antibody into 1mg of magnetic beads), and continuously mixing uniformly at the temperature of 2 ℃ for reaction for 24 hours.
Step C7: blocking of anti-FITC antibody magnetic microparticle conjugates
After the coupling reaction is completed, the anti-FITC antibody magnetic particle conjugate is subjected to magnetic separation. The ligation was separated for 2 minutes per gram but not less than 5 minutes. The ligation was resuspended in 5mg/ml using buffer 8. The resuspended ligation was mixed well at room temperature for 1.5 hours.
Step C8: cleaning of anti-FITC antibody magnetic particle conjugate
After blocking was complete, the anti-FITC antibody magnetic microparticle conjugate was subjected to magnetic separation. The ligation was separated for 2 minutes per gram but not less than 5 minutes. The ligation mixture was resuspended in buffer 9 at a concentration of 5mg/ml, and the washed ligation mixture was thoroughly mixed for 10 minutes.
The steps of magnetic separation, heavy suspension and uniform mixing are repeated for three times.
Step C9: resuspension of anti-FITC antibody microparticle conjugates
After washing was completed, the anti-FITC antibody magnetic microparticle conjugate was subjected to magnetic separation. The ligation was separated for 2 minutes per gram but not less than 5 minutes. The ligation was resuspended in 5mg/ml buffer 9 and mixed for 5 minutes. Add total volume 1/20 of buffer 10 and mix for 25 minutes. The connection object is preserved in the environment of 2-8 ℃.
Example 2
(1) Preparation of buffer solution
Buffer 1: weighing 15.1g of ethanolamine and 6.0g of NaCl, adding the ethanolamine and the NaCl into a certain amount of purified water, stirring until the ethanolamine and the NaCl are completely dissolved, adjusting the pH value to 7.3-7.6, and fixing the volume to 1000 ml. Filtration was performed with a 0.22 μm filter.
Buffer 2: 75g of glycine is weighed and added into a certain amount of purified water to be stirred until the glycine is completely dissolved, and the volume is up to 1000 ml. Filtration was performed with a 0.22 μm filter.
Buffer 3: weighing 15.0g of Tris, 60g of bovine serum albumin, 10.0g of NaCl and 100g of sucrose, adding into a certain amount of purified water, stirring until the mixture is completely dissolved, adjusting the pH value to 7.5-10.0, and fixing the volume to 1000 ml. Filtration was performed with a 0.22 μm filter.
Buffer 4: weighing 15.0g of Tris, 9.0g of NaCl, 50g of bovine serum albumin, 20.0g of glycerol and 40g of glycine, adding the materials into a certain amount of purified water, stirring until the materials are completely dissolved, adjusting the pH value to be 7.5-9.5 and fixing the volume to 1000 ml. Filtration was performed with a 0.22 μm filter.
Buffer 5: 5.9g of Na were weighed 2 HPO 4 ·12H 2 O, 0.60g of NaH 2 PO 4 9.0g of NaCl, 50g of bovine serum albumin, 140g of sucrose, 5.0g of xanthan gum, 5g of sodium alginate and 15g of gelatin are added into a certain amount of purified water and stirred until the materials are completely dissolved, the pH value is adjusted to be 6.2-8.0, and the volume is fixed to 1000 ml. Filtration was performed with a 0.22 μm filter.
Buffer 6: 5.9g of Na were weighed 2 HPO 4 ·12H 2 O, 0.60g of NaH 2 PO 4 9.0g NaCl, 5.0g KCl was added to purified water and made to 1000 ml. Filtration was performed with a 0.22 μm filter.
And (3) buffer solution 7: 30g of MES (2- (N-morpholino) ethanesulfonic acid) was weighed out, added to purified water and brought to a volume of 1000 ml. Filtration was performed with a 0.22 μm filter.
Buffer 8: weighing 5.0g of Tris, 9.0g of NaCl and 20.0g of bovine serum albumin, adding the Tris into a certain amount of purified water, stirring until the Tris is completely dissolved, adjusting the pH value to 7.0-7.6, and fixing the volume to 1000 ml. Filtration was performed with a 0.22 μm filter.
Buffer 9: weighing 5.0g of Tris and 9.0g of NaCl into a certain amount of purified water, stirring until the Tris and the NaCl are completely dissolved, weighing 2mL of Tween 20, adding into the container, adjusting the pH value to 7.0-7.6, and fixing the volume to 1000 mL. Filtration was performed with a 0.22 μm filter.
Buffer 10: weighing 15.0g of Tris, 9.0g of NaCl, 150g of bovine serum albumin and 100g of sucrose, adding into a certain amount of purified water, stirring until the mixture is completely dissolved, weighing 20mL of ProClin300, adding into the container, adjusting the pH value to be 7.5-9.2, and fixing the volume to 1000 mL. Filtration was performed with a 0.22 μm filter.
Buffer 11: weighing 18g of Tris, adding the Tris into a certain amount of purified water, stirring until the Tris is completely dissolved, adjusting the pH value to be 8.0-9.5, and metering to 1000 ml. Filtration was performed with a 0.22 μm filter.
(2) Production of reagent A
The enzyme-labeled PGP9.5 antibody 1 conjugate is used as a raw material of the reagent A, and the reagent A is prepared by fully and uniformly mixing the raw material with the buffer solution 4.
The method specifically comprises the following steps:
step A1: activation of antibody 1: the activation of antibody 1 needs to be carried out in a hundred thousand grade clean room. 4-8 mg of 2-iminothiolane hydrochloride (2IT) was weighed and dissolved in buffer 1 to 13.76 mg/mL. The 2IT solution was added to the antibody 1 solution for activation at a 30:1 molar ratio of 2-IT to antibody 1 (i.e., 1mg of antibody 1 was added to 30. mu.l of 2IT solution). After shaking and mixing, the mixture was reacted at room temperature for 30 minutes. After the termination of the activation, buffer 2 was added to the antibody 1 solution in a ratio of 1mg of antibody 1 to 20. mu.l of buffer 2, and the reaction was carried out at room temperature for 10 min. Excess 2IT was removed using a PD10 desalting column and the activated antibody 1 was collected.
Step A2: activation of alkaline phosphatase (ALP): the activation of ALP is carried out in a hundred thousand grade clean room. 2-4 mg of succinimidyl (N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC) was weighed out and dissolved in Dimethylformamide (DMF) to 6.69 mg/mL. The SMCC solution is added into the ALP solution according to the molar ratio of 60:1 of the SMCC to the ALP. After shaking and mixing, the mixture was reacted at room temperature for 30 minutes. After termination of the activation, buffer 2 was added to the ALP solution in a ratio of 1mg of ALP to 50. mu.l of buffer 2, and the reaction was carried out at room temperature for 10 min. Excess SMCC was removed using a PD10 desalting column and the ALP was collected after activation.
Step A3: linking of antibody 1 and ALP: the ligation of antibody 1 and ALP was carried out in a hundred thousand clean room. ALP solution was added to the antibody 1 solution at a mass ratio of antibody 1 to ALP of 1:1 (i.e., 1.0mg of antibody 1 to 1.0mg of ALP). After shaking and mixing evenly, the mixture reacts for 12 to 18 hours at the temperature of between 2 and 8 ℃.
Step A4: termination and purification of antibody 1 conjugates: termination and purification of the antibody 1 conjugate was carried out in a hundred thousand grade clean room. 1-10mg of maleimide was weighed out and dissolved in DMF to 9.7 mg/mL. At a ratio of 1/10, the solution was diluted with buffer 1 to give a 0.97mg/mL maleimide solution. This solution was added in a ratio of 20. mu.l of 0.97mg/mL maleimide solution to 1mg of antibody 1, and reacted at room temperature for 15 minutes. mu.L of ethanolamine was measured accurately and dissolved in buffer 1 to 100 mM. That is, 994. mu.L of buffer 1 was added to 6. mu.L of ethanolamine. The solution was added in a ratio of 1mg of antibody 1 to 50. mu.l of 100mM ethanolamine solution, and mixed by shaking. The antibody 1 conjugate to be purified was concentrated to 2mg/mL using an ultrafiltration concentration tube. Antibody purification was performed using a purified protein analyzer and Superdex 200 preparative 2.6/60 gel column, buffer 2 as eluent. The purified liquid is the enzyme-labeled antibody 1 conjugate.
Step A5: and fully and uniformly mixing the obtained enzyme-labeled antibody 1 conjugate with a buffer solution 4 to prepare a reagent A.
(3) Production of reagent B
The FITC labeled PGP9.5 antibody 2 conjugate is used as a raw material of a reagent B, and the reagent B is prepared by fully and uniformly mixing the raw material with a buffer solution 4.
The method specifically comprises the following steps:
step B1: desalting of antibody 2: buffer 6 was used as the equilibration and elution solution for the PD10 desalting column. The PD10 desalting column was washed with 15mL of buffer 6, and when the last wash was completed in the column, the antibody 2 to be desalted was added. The column volume was made up to 2.6mL using buffer 2. When the liquid in the column completely entered the column charge, 3mL of buffer 6 was added to start elution. The protein eluate was collected in the corresponding volume (2.6mL) to first fill up.
The antibody 2 after desalting was concentrated to 4mg/mL by adding to an ultrafiltration concentration tube. The parameters of the high-speed refrigerated centrifuge are as follows: 8000rpm, and centrifugation for 10 minutes.
Step B2: antibody 2-conjugated FITC: FITC is weighed and prepared into FITC concentrated solution with the final concentration of 0.5mg/mL by using buffer solution 11, the FITC is added into an antibody 2 solution for reaction according to the mass ratio of the FITC to the antibody 2 of 15:100 (namely 150 mu g of FITC is added into 1mg of antibody). After shaking and mixing, the mixture is reacted for 18 hours at room temperature (20-30 ℃).
(4) Production of reagent C
The anti-FITC antibody labeled magnetic particles are used as raw materials of the reagent C, and the raw materials are fully and uniformly mixed by using a buffer solution 5 to prepare the reagent C.
The method specifically comprises the following steps:
step C1: cleaning of magnetic particles
The magnetic particle material of 3 μm was selected and mixed well for 0.5 hour. The beads were resuspended in buffer 7 at a concentration of 10 mg/ml. And (3) fully and uniformly mixing the washed magnetic beads for 10 minutes, separating the magnetic particles by using a Sepmag magnetic separator, and separating each gram of magnetic bead raw material for 2 minutes but not less than 5 minutes. The resuspension and separation steps were repeated a total of three times.
Step C2: resuspension of magnetic microparticles
The magnetic particles after three washes were resuspended in 10mg/ml buffer 7. The resuspended magnetic microparticles were mixed well for 5 minutes.
Step C3: selection and desalination of anti-FITC antibodies
The anti-FITC antibody (affinity purification) and the FITC antibody (salting-out purification) are mixed according to the mass ratio of 4:1 to form a new anti-FITC antibody. Buffer 6 was used as the equilibration and elution solution for the PD10 desalting column. The PD10 desalting column was washed with 15mL of buffer 6, and when the last wash was completed in the column, the anti-FITC antibody to be desalted was added. The column volume was made up to 2.6mL using buffer 2. When the liquid in the column completely entered the column charge, 3mL of buffer 6 was added to start elution. The protein eluate was collected in the corresponding volume (2.6mL) to first fill up.
And adding the desalted anti-FITC antibody into an ultrafiltration concentration tube for concentration to 4 mg/mL. The parameters of the high-speed refrigerated centrifuge are as follows: 8000rpm, and centrifugation for 10 minutes.
Step C4: activation of magnetic particles
The carboxyl magnetic fine particles having a particle size of 3 μm were selected for activation. (i.e., magnetic particles having a large number of carboxyl functional groups on the surface) a certain amount of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) was dissolved to 15mg/mL with buffer 7. EDC was added to the magnetic particles to react at a ratio of 100:30 mass ratio of magnetic particles to EDC (i.e., 1mg of magnetic particles added to 300ug of EDC), and mixed for 40 minutes at room temperature.
Step C5: cleaning of activated magnetic particles
The activated magnetic particles were resuspended in 10mg/ml using buffer 7. And (3) fully and uniformly mixing the washed magnetic beads for 10 minutes, separating the magnetic particles by using a Sepmag magnetic separator, and separating each gram of magnetic bead raw material for 2 minutes but not less than 5 minutes.
The resuspension and separation steps were repeated a total of three times, and finally the magnetic particles were resuspended with buffer 7 at a concentration of 10 mg/ml.
Step C6: anti-FITC antibody-linked magnetic microparticles
According to the magnetic particles: and adding the anti-FITC antibody into the activated magnetic particles for reaction according to the mass ratio of the anti-FITC antibody to 100:20 (namely adding 200ug of the anti-FITC antibody into 1mg of magnetic beads), and continuously mixing uniformly at the temperature of 8 ℃ for reaction for 16 hours.
Step C7: blocking of anti-FITC antibody magnetic microparticle conjugates
After the coupling reaction is completed, the anti-FITC antibody magnetic particle conjugate is subjected to magnetic separation. The ligation was separated for 2 minutes per gram but not less than 5 minutes. The ligation was resuspended in 10mg/ml using buffer 8. The resuspended ligation was mixed well at room temperature for 1.5 hours.
Step C8: cleaning of anti-FITC antibody magnetic particle conjugate
After blocking was complete, the anti-FITC antibody magnetic microparticle conjugate was subjected to magnetic separation. The ligation was separated for 2 minutes per gram but not less than 5 minutes. The ligation mixture was resuspended in buffer 9 at a concentration of 10mg/ml, and the washed ligation mixture was thoroughly mixed for 10 minutes.
The steps of magnetic separation, heavy suspension and uniform mixing are repeated for three times.
Step C9: resuspension of anti-FITC antibody microparticle conjugates
After washing was completed, the anti-FITC antibody magnetic microparticle conjugate was subjected to magnetic separation. The ligation was separated for 2 minutes per gram but not less than 5 minutes. The ligation was resuspended in 10mg/ml buffer 9 and mixed for 5 minutes. Add total volume 1/50 of buffer 10 and mix well for 25 minutes. The connection object is preserved in the environment of 2-8 ℃.
Example 3
(1) Preparation of buffer solution
Buffer 1: weighing 15.0g of ethanolamine and 6.0g of NaCl, adding the ethanolamine and the NaCl into a certain amount of purified water, stirring until the ethanolamine and the NaCl are completely dissolved, adjusting the pH value to 7.3-7.6, and fixing the volume to 1000 ml. Filtration was performed with a 0.22 μm filter.
Buffer 2: 75g of glycine is weighed and added into a certain amount of purified water to be stirred until the glycine is completely dissolved, and the volume is up to 1000 ml. Filtration was performed with a 0.22 μm filter.
Buffer 3: weighing 13.0g of Tris, 40g of bovine serum albumin, 8.0g of NaCl and 50g of sucrose, adding into a certain amount of purified water, stirring until the mixture is completely dissolved, adjusting the pH value to 7.5-10.0, and fixing the volume to 1000 ml. Filtration was performed with a 0.22 μm filter. Buffer 4: weighing 5.8g of 13.0g of Tris, 9.0g of NaCl, 10.0g of bovine serum albumin, 10.0g of glycerol and 10.0g of glycine, adding the mixture into a certain amount of purified water, stirring until the mixture is completely dissolved, adjusting the pH value to be 7.5-9.5 and fixing the volume to 1000 ml. Filtration was performed with a 0.22 μm filter.
Buffer 5: 5.8g of Na were weighed 2 HPO 4 ·12H 2 O, 0.58g of NaH 2 PO 4 9.0g of NaCl, 10.0g of bovine serum albumin, 100g of sucrose, 2.0g of xanthan gum, 2.0g of sodium alginate and 8.0g of gelatin are added into a certain amount of purified water and stirred until the materials are completely dissolved, the pH value is adjusted to be between 6.2 and 8.0, and the volume is fixed to 1000 ml. Filtration was performed with a 0.22 μm filter.
Buffer 6: 5.8g of Na were weighed 2 HPO 4 ·12H 2 O、058g of NaH 2 PO 4 9.0g NaCl, 2.0g KCl was added to purified water and made to 1000 ml. Filtration was performed with a 0.22 μm filter.
And (3) buffer solution 7: 20.0g of MES (2- (N-morpholino) ethanesulfonic acid) was weighed into purified water and brought to 1000 ml. Filtration was performed with a 0.22 μm filter.
Buffer 8: weighing 3.0g of Tris, 9.0g of NaCl and 10.0g of bovine serum albumin, adding the Tris into a certain amount of purified water, stirring until the Tris is completely dissolved, adjusting the pH value to 7.0-7.6, and fixing the volume to 1000 ml. Filtration was performed with a 0.22 μm filter.
Buffer 9: weighing 3.0g of Tris and 9.0g of NaCl into a certain amount of purified water, stirring until the Tris and the NaCl are completely dissolved, weighing 1mL of Tween 20, adding into the container, adjusting the pH value to 7.0-7.6, and fixing the volume to 1000 mL. Filtration was performed with a 0.22 μm filter.
Buffer 10: weighing 10.0g of Tris, 9.0g of NaCl, 100g of bovine serum albumin and 80g of sucrose, adding into a certain amount of purified water, stirring until the mixture is completely dissolved, weighing 10mL of ProClin300, adding into the container, adjusting the pH value to be 7.5-9.2, and fixing the volume to 1000 mL. Filtration was performed with a 0.22 μm filter.
Buffer 11: weighing 15g of Tris into a certain amount of purified water, stirring until the Tris is completely dissolved, adjusting the pH value to be 8.0-9.5 and metering the volume to 1000 ml. Filtration was performed with a 0.22 μm filter.
(2) Production of reagent A
The enzyme-labeled PGP9.5 antibody 1 conjugate is used as a raw material of the reagent A, and the reagent A is prepared by fully and uniformly mixing the raw material with the buffer solution 4.
The method specifically comprises the following steps:
step A1: activation of antibody 1: the activation of antibody 1 needs to be carried out in a hundred thousand grade clean room. 4-8 mg of 2-iminothiolane hydrochloride (2IT) was weighed and dissolved in buffer 1 to 13.76 mg/mL. The 2IT solution is added to the antibody 1 solution for activation in a ratio of 2-IT to antibody 1 molar ratio of 20: 1. After shaking and mixing, the mixture was reacted at room temperature for 30 minutes. After the termination of the activation, buffer 2 was added to the antibody 1 solution in a ratio of 1mg of antibody 1 to 10. mu.l of buffer 2, and the reaction was carried out at room temperature for 10 min. Excess 2IT was removed using a PD10 desalting column and activated antibody 1 was collected.
Step A2: activation of alkaline phosphatase (ALP): the activation of ALP is carried out in a hundred thousand grade clean room. 2-4 mg of succinimidyl (N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC) was weighed out and dissolved in Dimethylformamide (DMF) to 6.69 mg/mL. The SMCC solution was added to the ALP solution at a molar ratio of SMCC to ALP of 40: 1. After shaking and mixing, the mixture was reacted at room temperature for 30 minutes. After termination of the activation, buffer 2 was added to the ALP solution in a ratio of 1mg of ALP to 20. mu.l of buffer 2, and the reaction was carried out at room temperature for 10 min. Excess SMCC was removed using a PD10 desalting column and the ALP was collected after activation.
Step A3: linking of antibody 1 and ALP: the ligation of antibody 1 and ALP was carried out in a hundred thousand clean room. ALP solution was added to the antibody 1 solution at a mass ratio of 1:1.5 of antibody 1 to ALP (i.e., 1.0mg of antibody 1 to 1.5mg of ALP). After shaking and mixing, the mixture was reacted at 4 ℃ for 15 hours.
Step A4: termination and purification of antibody 1 conjugates: termination and purification of the antibody 1 conjugate was carried out in a hundred thousand grade clean room. 1-10mg of maleimide was weighed out and dissolved in DMF to 9.7 mg/mL. At a ratio of 1/10, the solution was diluted with buffer 1 to give a 0.97mg/mL maleimide solution. This solution was added in a ratio of 1mg of antibody 1 to 10. mu.l of 0.97mg/mL maleimide solution, and reacted at room temperature for 15 minutes. mu.L of ethanolamine was measured accurately and dissolved in buffer 1 to 100 mM. That is, 994. mu.L of buffer 1 was added to 6. mu.L of ethanolamine. The solution was added in a ratio of 1mg of antibody 1 to 30. mu.l of 100mM ethanolamine solution, and mixed by shaking. The antibody 1 conjugate to be purified was concentrated to 1mg/mL using an ultrafiltration concentration tube. Antibody purification was performed using a purified protein analyzer and Superdex 200 preparative 2.6/60 gel column, buffer 2 as eluent. The purified liquid is the enzyme-labeled antibody 1 conjugate.
Step A5: and fully and uniformly mixing the obtained enzyme-labeled antibody 1 conjugate with a buffer solution 4 to prepare a reagent A.
(3) Production of reagent B
The FITC labeled PGP9.5 antibody 2 conjugate is used as a raw material of a reagent B, and the reagent B is prepared by fully and uniformly mixing the FITC labeled PGP9.5 antibody 2 conjugate with a buffer solution 4.
The method specifically comprises the following steps:
step B1: desalting of antibody 2: buffer 6 was used as the equilibration and elution solution for the PD10 desalting column. The PD10 desalting column was washed with 15mL of buffer 6, and when the last wash was completed in the column, the antibody 2 to be desalted was added. The column volume was made up to 2mL using buffer 2. When the liquid in the column completely entered the column charge, 3mL of buffer 6 was added to start elution. The protein eluate was collected in the corresponding volume (2mL) to first fill up.
The antibody 2 after desalting was concentrated to 3mg/mL by adding to an ultrafiltration concentration tube. The parameters of the high-speed refrigerated centrifuge are as follows: centrifuge at 6000rpm for 20 minutes.
Step B2: antibody 2-conjugated FITC: FITC is weighed and prepared into FITC concentrated solution with the final concentration of 0.5mg/mL by using buffer solution 11, the FITC is added into the antibody 2 solution for reaction according to the mass ratio of the FITC to the antibody 2 of 9:100 (namely, 90 mu g of FITC is added into 1mg of antibody). After shaking and mixing, the mixture is reacted for 18 hours at room temperature (20-30 ℃).
(4) Production of reagent C
The anti-FITC antibody labeled magnetic particles are used as raw materials of the reagent C, and the raw materials are fully and uniformly mixed by using a buffer solution 5 to prepare the reagent C.
The method specifically comprises the following steps:
step C1: cleaning of magnetic particles
The magnetic particle material of 1.5 μm was selected and mixed well for 0.5 hour. The beads were resuspended in buffer 7 at a concentration of 8 mg/ml. And (3) fully and uniformly mixing the washed magnetic beads for 10 minutes, separating the magnetic particles by using a Sepmag magnetic separator, and separating each gram of magnetic bead raw material for 2 minutes but not less than 5 minutes. The resuspension and separation steps were repeated a total of three times.
Step C2: resuspension of magnetic microparticles
The magnetic particles after three washes were resuspended in a concentration of 8mg/ml using buffer 7. The resuspended magnetic microparticles were mixed well for 5 minutes.
Step C3: selection and desalination of anti-FITC antibodies
The anti-FITC antibody (affinity purification) and the FITC antibody (salting-out purification) are mixed according to the mass ratio of 1:1 to form a new anti-FITC antibody. Buffer 6 was used as the equilibration and elution solution for the PD10 desalting column. The PD10 desalting column was washed with 15mL of buffer 6, and when the last wash was completed in the column, the anti-FITC antibody to be desalted was added. The column volume was made up to 2mL using buffer 2. When the liquid in the column completely entered the column charge, 3mL of buffer 6 was added to start elution. The protein eluate was collected in the corresponding volume (2mL) to first fill up.
And adding the desalted anti-FITC antibody into an ultrafiltration concentration tube for concentration to 2 mg/mL. The parameters of the high-speed refrigerated centrifuge are as follows: centrifuge at 6000rpm for 20 minutes.
Step C4: activation of magnetic particles
The carboxyl magnetic fine particles having a particle size of 1.5 μm were selected for activation. (i.e., magnetic particles with a large number of carboxyl functional groups on the surface) a defined amount of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) was dissolved to 10mg/mL with buffer 7. EDC was added to the magnetic particles to react at a ratio of 100:9 (i.e., 1mg of magnetic particles added to 90ug of EDC) by mass, and mixed for 40 minutes at room temperature.
Step C5: cleaning of activated magnetic particles
The activated magnetic particles were resuspended in a concentration of 8mg/ml using buffer 7. And (3) fully and uniformly mixing the washed magnetic beads for 10 minutes, separating the magnetic particles by using a Sepmag magnetic separator, and separating each gram of magnetic bead raw material for 2 minutes but not less than 5 minutes.
The resuspension and separation steps were repeated a total of three times, and finally the magnetic particles were resuspended with buffer 7 at a concentration of 8 mg/ml.
Step C6: anti-FITC antibody-linked magnetic microparticles
According to the magnetic particles: and adding the anti-FITC antibody into the activated magnetic particles for reaction according to the mass ratio of the anti-FITC antibody of 100:15 (namely adding 150ug of anti-FITC antibody into 1mg of magnetic beads), and continuously mixing uniformly at 4 ℃ for reaction for 20 hours.
Step C7: blocking of anti-FITC antibody magnetic microparticle conjugates
After the coupling reaction is completed, the anti-FITC antibody magnetic particle conjugate is subjected to magnetic separation. The ligation was separated for 2 minutes per gram but not less than 5 minutes. The ligation was resuspended in buffer 8 at a concentration of 8 mg/ml. The resuspended ligation was mixed well at room temperature for 1.5 hours.
Step C8: cleaning of anti-FITC antibody magnetic particle conjugate
After blocking was complete, the anti-FITC antibody magnetic microparticle conjugate was subjected to magnetic separation. The ligation was separated for 2 minutes per gram but not less than 5 minutes. The ligation mixture was resuspended in buffer 9 at a concentration of 8mg/ml and the washed ligation mixture was mixed well for 10 minutes.
The steps of magnetic separation, heavy suspension and uniform mixing are repeated for three times.
Step C9: resuspension of anti-FITC antibody microparticle conjugates
After washing was completed, the anti-FITC antibody magnetic microparticle conjugate was subjected to magnetic separation. The ligation was separated for 2 minutes per gram but not less than 5 minutes. The ligation was resuspended in 8mg/ml buffer 9 and mixed for 5 minutes. Add total volume 1/30 of buffer 10 and mix well for 25 minutes. The connection object is preserved in the environment of 2-8 ℃.
4. Detection method
The detection is carried out by adopting a full-automatic chemiluminescence immunoassay analyzer self-developed by Beijing Meiliantaceae biotechnology limited company. The amount of sample required for the reaction was 30. mu.L, and the automatic assay procedure was:
immune reaction: and adding the 30uL sample, the 50uL reagent B, the 50uL reagent A and the 50uL reagent C into the 11 th pore site in sequence, and reacting for 20min at 37 ℃.
Magnetic separation and cleaning: adding 300 mu L of cleaning solution into the No. 12 hole, sucking the mixture containing the magnetic particles out of the No. 11 hole by using magnetic force, and demagnetizing the No. 12 hole. After 2min of cleaning. Magnetic separation and washing are performed 1 time at the No. 13 and No. 14 holes respectively.
Reading the value: adding 150uL of luminescent substrate into the hole site No. 15, sucking the mixture containing magnetic particles out of the hole site No. 14 by magnetic force, and demagnetizing the hole site No. 15. The relative luminescence intensity (RLU) was measured using a self-developed instrument after the luminescence of the alkaline phosphatase-catalyzed luminescent substrate.
And obtaining a PGP9.5 concentration-luminous value standard curve according to the detected value of the calibrator. The curve was fitted using a four parameter Logistic equation.
The detection value of the sample can correspond to the unique concentration value obtained on the curve, so that the concentration detection of the unknown sample is realized.
5. Detecting the index
The correlation indexes measured in examples 1 to 3 are shown below, wherein the lot-to-lot differences, specificity and vial-to-vial differences only exemplify the correlation results of example 1.
5.1 accuracy
A brain specific protein product (PGP9.5) solution (A) having a concentration of about 3000pg/mL (tolerance. + -. 10%) was added to a sample B having a concentration ranging from 0pg/mL to 40pg/mL at a volume ratio of 1:9 between the added PGP9.5 antigen and the sample B, and the recovery ratio R was calculated according to the formula (1) and should be in the range of 90% to 110%.
Figure BDA0003494852660000251
In the formula: r is the recovery rate; v is the volume of the sample A liquid; v 0 Is the volume of serum sample B; c is the average value of 3 measurements after the serum sample B liquid is added into the A liquid; c 0 The average value of 3 measurements of the serum sample B liquid is shown; c S The concentration of the sample A solution.
5.2 blank limit
Repeating the test for 20 times to obtain concentration values of 20 test results, and calculating average value
Figure BDA0003494852660000252
And Standard Deviation (SD). Mean value of
Figure BDA0003494852660000253
The blank limit is obtained, and the result is less than or equal to 40 pg/mL.
5.3 Linear region
Mixing high value sample near the upper limit of linear zone with low value sample near the lower limit of linear zone or zero concentration sample to obtain at least 5 diluted concentrationsWherein the low value concentration sample is close to the lower limit of the linear interval. The test was repeated 3 times for each concentration of the sample to obtain the luminescence value, the measurement result of each sample was recorded, and the average value (y) of the 3 measurements of each sample was calculated i ). In diluted concentration (x) i ) As independent variable, the mean value (y) of the results is determined i ) Linear regression equations were solved for the dependent variables. And (3) calculating a correlation coefficient (r) of the linear regression according to the formula (2), wherein the correlation coefficient r is more than or equal to 0.990 within a linear interval of 80-2560 pg/mL.
Figure BDA0003494852660000261
In the formula: r is a correlation coefficient; x is the number of i Is a dilution ratio; y is i Determining a mean value for each sample;
Figure BDA0003494852660000262
is the average of the dilution ratios;
Figure BDA0003494852660000263
is the overall mean value of the sample measurement results.
5.4 repeatability
The quality control product is tested repeatedly for 10 times by the same batch number kit, and the average value of 10 test results is calculated
Figure BDA0003494852660000264
And standard deviation SD. The Coefficient of Variation (CV) was calculated according to equation (3) and the result CV was less than or equal to 8%.
Figure BDA0003494852660000265
In the formula: s is the standard deviation of the sample test values;
Figure BDA0003494852660000266
is the average of the sample test values.
5.5 run-to-run differences
Respectively and repeatedly testing quality control products by using 3 batch number kits10 times, calculate the average of 30 test results
Figure BDA0003494852660000267
And standard deviation SD, and obtaining Coefficient of Variation (CV) according to formula (3), wherein the result CV is less than or equal to 12%.
5.6 specificity
Adding 5000pg/mL of Glial Fibrillary Acidic Protein (GFAP) to the sample without any analyte, taking a mean value 3 times, and calculating the result according to formula (4), the cross-reactivity should be less than 5%
R CR ＝M/C×100％(4)
In the formula: RCR is the cross-reactivity; m is the mean value of the measurement results of the cross reactants; c is the cross-reactant index.
5.7 difference between calibrator and quality control bottle
Detecting 10 bottles of calibrator (or quality control material) of the same batch for 1 time respectively, calculating according to formula (5), and determining the mean value of the results
Figure BDA0003494852660000271
And standard deviation (S1). Continuously measuring 1 bottle of the above 10 bottles of calibrator (or quality control) for 5 times, and calculating the mean value of the results
Figure BDA0003494852660000272
And standard deviation (S2), calculating the repeatability CV% between bottles according to the formulas (6) and (7), and the measurement result should meet the requirement of 2.10.
Figure BDA0003494852660000273
Figure BDA0003494852660000274
Figure BDA0003494852660000275
(Note: when S1< S2, let CV bottle be 0)
In the formula: s is the standard deviation.
6. The result of the detection
6.1 accuracy
TABLE 14
Examples of the invention Example 1 Example 2 Example 3
High value sample concentration 3000 3000 3000
Low value sample concentration 5 5 5
Low value sample parallel 1 4.123 4.521 5.026
Low value sample parallel 2 4.258 4.669 5.341
Low value sample parallel 3 4.079 4.352 5.297
Low value sample mean 4.1533 4.514 5.221
Dilution of sample parallel 1 317.502 287.218 311.594
Dilution sample parallel 2 310.629 276.552 306.286
Dilution sample parallel 3 310.996 281.934 309.765
Mean of diluted samples 313.04 281.901 309.215
Recovery rate 103.10％ 92.61％ 101.51％
6.2 blank Limit
Watch 15
Figure BDA0003494852660000281
6.3 Linear Range
TABLE 16
Figure BDA0003494852660000282
6.4 repeatability
TABLE 17
Figure BDA0003494852660000291
6.5 run-to-run differences
Watch 18
Figure BDA0003494852660000292
Figure BDA0003494852660000301
6.6 specificity
Watch 19
Figure BDA0003494852660000302
6.7 difference between bottles
Watch 20
Figure BDA0003494852660000303
Finally, it should be noted that the above-mentioned contents are only used for illustrating the technical solutions of the present invention, and not for limiting the protection scope of the present invention, and that the simple modifications or equivalent substitutions of the technical solutions of the present invention by those of ordinary skill in the art can be made without departing from the spirit and scope of the technical solutions of the present invention.
SEQUENCE LISTING
<110> Beijing Meiliantaceae Biotechnology Co., Ltd
<120> application of signal amplification technology in PGP9.5 detection kit
<130> 20211222
<160> 8
<170> PatentIn version 3.5
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Leu Leu Leu Leu Phe Pro Leu Thr Ala Gln His Glu Asn Phe Arg Lys
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Val Leu Lys Gln Phe Leu Ser Glu Thr Glu Lys Met Ser Pro Glu Asp
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Claims (9)

1. A PGP9.5 detection kit, characterized by: comprises a detection reagent strip; the detection reagent strip comprises a reagent A, a reagent B and a reagent C;
the raw materials of the reagent A comprise PGP9.5 antibody 1 and alkaline phosphatase; raw materials of the reagent B comprise PGP9.5 antibody 2 and FITC; the reagent C comprises an anti-FITC antibody;
the PGP9.5 antibody 1 comprises a heavy chain variable region sequence shown by SEQ ID NO. 2 and a light chain variable region sequence shown by SEQ ID NO. 4; the PGP9.5 antibody 2 comprises a heavy chain variable region sequence shown in SEQ ID NO. 3 and a light chain variable region sequence shown in SEQ ID NO. 5.
2. The PGP9.5 detection kit according to claim 1, characterized in that: the sequence of the PGP9.5 antibody 1 is an amino acid sequence shown by SEQ ID NO. 7, and the sequence of the PGP9.5 antibody 2 is an amino acid sequence shown by SEQ ID NO. 8.
3. The PGP9.5 detection kit according to claim 1, characterized in that: also comprises a calibrator and a quality control product.
4. The PGP9.5 detection kit according to claim 3, characterized in that: the raw materials of the calibrator and the quality control product both comprise PGP9.5 recombinant protein.
5. The PGP9.5 detection kit according to claim 1, characterized in that: the detection reagent strip also comprises a cleaning solution, a luminescent substrate, a reading hole, an elution sleeve and a suction head.
6. The method for preparing a PGP9.5 assay kit according to any one of claims 1 to 5, wherein: the method comprises the following steps:
(1) production of solvent A: coupling alkaline phosphatase with PGP9.5 antibody 1, and mixing with buffer solution to obtain reagent A;
(2) production of solvent B: coupling FITC and PGP9.5 antibody 2, and then mixing with a buffer solution to obtain a reagent B;
(3) production of solvent C: and connecting the anti-FITC antibody to the magnetic particles, and mixing with a buffer solution to obtain a reagent C.
7. The method of claim 6, wherein: the weight ratio of PGP9.5 antibody 1 to alkaline phosphatase is 1:1-2, and the weight ratio of FITC to PGP9.5 antibody 2 is 3-15: 100.
8. The method of claim 6, wherein: the weight ratio of the anti-FITC antibody to the magnetic particles is 100: 3-20.
9. The method of claim 6, wherein: the buffer solution in the steps (1) and (2) comprises tris (hydroxymethyl) aminomethane, sodium chloride, bovine serum albumin, glycerol and glycine.
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Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110366558A (en) * 2016-10-28 2019-10-22 班扬生物标记公司 For the antibody and correlation technique of ubiquitin c-terminal hydrolase-l 1 (UCH-L1) and glial fibrillary acid protein (GFAP)
CN110684740A (en) * 2019-08-09 2020-01-14 无锡傲锐东源生物科技有限公司 Monoclonal antibody of anti-human ubiquitin carboxyl terminal hydrolase-1 (UCH-L1) and application thereof
CN110892266A (en) * 2017-12-09 2020-03-17 雅培实验室 Methods of using a combination of GFAP and UCH-L1 to aid in the diagnosis and assessment of traumatic brain injury in a human subject
CN111337682A (en) * 2020-05-18 2020-06-26 博奥赛斯（天津）生物科技有限公司 Novel coronavirus IgM/IgG magnetic particle chemiluminescence immunoassay kit
CN112630430A (en) * 2020-11-16 2021-04-09 北京美联泰科生物技术有限公司 Kit for quantitatively detecting UCHL-1 and application thereof
CN113933521A (en) * 2021-09-26 2022-01-14 北京贝尔医疗设备有限公司 Magnetic particle chemiluminescence detection kit and preparation method and application thereof

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2017197028A1 (en) * 2016-05-10 2017-11-16 University Of Pittsburgh-Of The Commonwealth System Of Higher Education Compositions and methods for identifying subjects at risk for traumatic brain injury

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110366558A (en) * 2016-10-28 2019-10-22 班扬生物标记公司 For the antibody and correlation technique of ubiquitin c-terminal hydrolase-l 1 (UCH-L1) and glial fibrillary acid protein (GFAP)
CN110892266A (en) * 2017-12-09 2020-03-17 雅培实验室 Methods of using a combination of GFAP and UCH-L1 to aid in the diagnosis and assessment of traumatic brain injury in a human subject
CN110684740A (en) * 2019-08-09 2020-01-14 无锡傲锐东源生物科技有限公司 Monoclonal antibody of anti-human ubiquitin carboxyl terminal hydrolase-1 (UCH-L1) and application thereof
CN111337682A (en) * 2020-05-18 2020-06-26 博奥赛斯（天津）生物科技有限公司 Novel coronavirus IgM/IgG magnetic particle chemiluminescence immunoassay kit
CN112630430A (en) * 2020-11-16 2021-04-09 北京美联泰科生物技术有限公司 Kit for quantitatively detecting UCHL-1 and application thereof
CN113933521A (en) * 2021-09-26 2022-01-14 北京贝尔医疗设备有限公司 Magnetic particle chemiluminescence detection kit and preparation method and application thereof

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
Establishment and characterization of immortalized bovine male germline stem cell line;Qi-jing LEI 等;《Journal of Integrative Agriculture》;20171130;第16卷(第11期);第2547-2557页 *
Generation and Characterization of Monoclonal Antibodies against a Cyclic Variant of Hepatitis C Virus E2 Epitope 412-422;Annamaria Sandomenico 等;《J Virol》;20160430;第90卷(第7期);第3745-3759页 *

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