CN114563569A - Application of signal amplification technology in PGP9.5 detection kit - Google Patents

Abstract

The invention provides an application of a signal amplification technology in a PGP9.5 detection kit, and relates to the field of biotechnology. The kit includes a detection reagent strip; wherein, the detection reagent strip includes a reagent A, a reagent B and a reagent C; the The raw material of reagent A includes PGP9.5 antibody 1; the raw material of reagent B includes PGP9.5 antibody 2; the reagent C includes anti-FITC antibody. The present invention improves 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 a signal amplification technology in PGP9.5 detection kit

technical field

The invention relates to the field of biotechnology, in particular to the application of a signal amplification technology in a PGP9.5 detection kit.

Background technique

Traumatic brain injury (TBI) is brain damage caused by external forces that disrupts normal brain function, resulting in impaired cognitive or physical functioning. Among all types of TBI, the most common sequelae were headache (47.9%) and memory abnormalities (42%) (British Journal of Neurosurgery, 2018), and about 30% of patients required psychological counseling or neurological treatment. TBI is the most common disease in neurosurgery and one of the major causes of death and disability worldwide, and its sequelae have a permanent impact on the health of patients.

The medical care process for suspected TBI is divided into three steps, starting with neurological assessment using the 15-point Glasgow Coma Scale (GCS) (American College of Surgeons Trauma Committee, 1997) to assess the severity of brain injury, followed by structural neurological assessment. Imaging, most commonly a CT scan of the head, to visualize fractures and intracranial lesions. Finally, according to the CT results, a treatment plan was formulated, and the patients were hospitalized for observation or discharged.

Currently, CT scans are the only objective, simple, and reliable option widely used to help clinicians assess TBI. However, the correctness of CT results is directly related to the accuracy of CT equipment and the level of doctor's interpretation. Compared with other detection methods, it is a relatively subjective judgment method. About 90% of mild TBIs (sometimes referred to as "concussions") have negative CT scans (Toth, 2015). Less than 1% of these patients require neurosurgical intervention (Papa L, 2012). Given that the percentage of positive CT scans in these patients is very low and unnecessary imaging tests may increase the risk of radiation-induced carcinogenesis, it is of great clinical and strategic significance.

Brain-specific protein product 9.5 (PGP9.5) is a deubiquitinating enzyme and a nervous system-specific protein. Human PGP9.5 consists of 223 amino acids and is mainly distributed in mature neurons, especially in substantia nigra, and in undifferentiated central and peripheral nerves. As one of the most abundant proteins in the brain, PGP9.5 can account for 2% of the brain soluble protein. PGP9.5 regulates cell proliferation, differentiation and apoptosis through ubiquitin-related pathways. In TBI, PGP9.5 crosses the blood-brain barrier and enters the blood within 1 hour, resulting in a significant increase in serum PGP9.5. It is of great significance for the early diagnosis, differential diagnosis and prognosis of TBI, and is mainly used for the auxiliary diagnosis of traumatic brain injury in clinical practice. Patent CN201780080585.4 discloses an antibody against ubiquitin C-terminal hydrolase L1 (UCH-L1) and glial fibrillary acidic protein (GFAP) and related methods, mainly for known or suspected brain damage or damage such as mild For individuals with neurological damage such as traumatic brain injury, corresponding methods, systems and kits are provided. However, the invention focuses on protecting proteins, and the related methods have long detection time, low sensitivity and narrow linearity.

In view of the problems existing in the prior art, it is very necessary to provide a rapid and simple in vitro diagnostic kit that can provide auxiliary diagnosis for traumatic brain injury.

SUMMARY OF THE INVENTION

Aiming at the problems existing in the prior art, the present invention provides an in vitro diagnostic kit that is easy to operate and can provide auxiliary diagnosis for traumatic brain injury in order to quickly and easily detect the content of brain-specific protein products in human peripheral blood. and preparation method and use thereof. The kit in the invention is a method for quantitatively analyzing the level of brain-specific protein products in human peripheral blood by using magnetic particle chemiluminescence method, and is used for auxiliary diagnosis of neurological diseases such as traumatic brain injury. The present invention improves 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.

For achieving the above object, the technical scheme adopted in the present invention is as follows:

The present invention provides a PGP9.5 detection kit, comprising a detection reagent strip; the detection reagent strip includes a reagent A, a reagent B and a reagent C;

The raw material of the reagent A includes PGP9.5 antibody 1; the raw material of the reagent B includes PGP9.5 antibody 2; the reagent C includes anti-FITC antibody;

The PGP9.5 antibody 1 includes the heavy chain variable region sequence shown in SEQ ID NO:2 and the light chain variable region sequence shown in SEQ ID NO:4; the PGP9.5 antibody 2 includes SEQ ID NO: The heavy chain variable region sequence shown in 3 and the light chain variable region sequence shown in SEQ ID NO:5.

Further, 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 further includes alkaline phosphatase, the raw material of the reagent B also includes FITC, and the raw material of the reagent C also includes an anti-FITC antibody.

Further, calibrators and quality controls are also included.

Further, the raw materials of the calibrator and the quality control material include PGP9.5 recombinant protein.

Further, it is characterized in that: the detection reagent strip further comprises a cleaning solution, a luminescent substrate, a reading hole, an elution sleeve and a pipette tip.

The present invention also provides a preparation method of the above-mentioned PGP9.5 detection kit, comprising the following steps:

(1) Production of solvent A: after coupling alkaline phosphatase with PGP9.5 antibody 1, it is mixed with buffer to obtain reagent A;

(2) Production of solvent B: the FITC is coupled with PGP9.5 antibody 2 and mixed with buffer to obtain reagent B;

(3) Production of solvent C: The anti-FITC antibody is linked to the magnetic microparticles and mixed with the buffer to obtain the reagent C.

Further, the weight ratio of the PGP9.5 antibody 1 to alkaline phosphatase is 1:1-2, and the weight ratio of the FITC to the 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 present invention relates to the formula and preparation method of buffer:

Buffer 1

Weigh 14.8-15.1g of ethanolamine and 5.8-6.0g of NaCl into a certain amount of purified water and stir until completely dissolved, adjust the pH value between 7.3 and 7.6 and set the volume to 1000ml. Filtration was performed with a 0.22 μm filter.

Table 1 Buffer 1 formula

raw material name Weigh Ethanolamine 14.8-15.1g Sodium chloride 5.8-6.0g pH 7.3-7.6 purified water Dilute to 1000mL

Buffer 2

Weigh 75 g of glycine, add it to a certain amount of purified water, stir until it is completely dissolved, and set the volume to 1000 ml. Filtration was performed with a 0.22 μm filter.

Table 2 Buffer 2 formula

raw material name Weigh Glycine 75g purified water Dilute to 1000mL

Buffer 3

Weigh 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, add them to a certain amount of purified water and stir until completely dissolved, and adjust the pH between 7.5 and 10.0. Volume to 1000ml. Filtration was performed with a 0.22 μm filter.

Table 3 Buffer 3 Recipe

raw material name Weigh Tris(hydroxymethylaminomethane) 12.0-15.0g bovine serum albumin 20.0-60g Sodium chloride 2.0-10.0g sucrose 10.0-100g pH 7.5-10.0 purified water Dilute to 1000mL

Buffer 4

Weigh 12.0-15.0g of Tris, 9.0g of NaCl, 1.0-50g of bovine serum albumin, 5.0-20.0g of glycerin, 5.0-40g of glycine, add them to a certain amount of purified water and stir until completely dissolved, adjust the pH value at 7.5 Between ~9.5 and dilute to 1000ml. Filtration was performed with a 0.22 μm filter.

Table 4 Buffer 4 Recipe

raw material name Weigh Tris(hydroxymethylaminomethane) 12.0-15.0g Sodium chloride 9.0g bovine serum albumin 1.0-50g glycerin 5.0-20.0g Glycine 5.0-40g pH 7.5-9.5 purified water Dilute to 1000mL

Buffer 5

Weigh 5.6-5.9g of Na ₂ HPO ₄ ·12H ₂ O, 0.55-0.60g of 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 yellow Raw gum, 0.1-5g sodium alginate, and 1.0-15g gelatin are added to a certain amount of purified water and stirred until completely dissolved. Adjust the pH value between 6.2 and 8.0 and set the volume to 1000ml. Filtration was performed with a 0.22 μm filter.

Table 5 Buffer 5 Recipe

raw material name Weigh 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 6.2-8.0 purified water Dilute to 1000mL

Buffer 6

Weigh 5.6-5.9 g of Na ₂ HPO ₄ ·12H ₂ O, 0.55-0.60 g of NaH ₂ PO ₄ , 9.0 g of NaCl, 0.1-5.0 g of KCl, add purified water and dilute to 1000 ml. Filtration was performed with a 0.22 μm filter.

Table 6 Buffer 6 Recipe

raw material name Weigh 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

Weigh 12-30 g of MES (2-(N-morpholine)ethanesulfonic acid) into purified water and make up to 1000 ml. Filtration was performed with a 0.22 μm filter.

Table 7 Buffer 7 Recipe

Buffer 8

Weigh 2.5-5.0g of Tris, 9.0g of NaCl, and 5.0-20.0g of bovine serum albumin into a certain amount of purified water and stir until completely dissolved, adjust the pH value between 7.0 and 7.6 and set the volume to 1000ml. Filtration was performed with a 0.22 μm filter.

Table 8 Buffer 8 Recipe

raw material name Weigh Tris(hydroxymethylaminomethane) 2.5-5.0g Sodium chloride 9.0g bovine serum albumin 5.0-20.0g pH value 7.0-7.6 purified water Dilute to 1000mL

Buffer 9

Weigh 2.5-5.0g of Tris and 9.0g of NaCl, add them to a certain amount of purified water and stir until completely dissolved, weigh 0.2-2mL of Tween 20 and add it to the above container, adjust the pH value between 7.0 and 7.6 and set the volume to 1000ml. Filtration was performed with a 0.22 μm filter.

Table 9 Buffer 9 Recipe

raw material name Weigh Tris(hydroxymethylaminomethane) 2.5-5.0g Sodium chloride 9.0g Tween 20 0.2-2mL pH 7.0-7.6 purified water Dilute to 1000mL

Buffer 10

Weigh 8.5-15.0g of Tris, 9.0g of NaCl, 20-150g of bovine serum albumin, and 15-100g of sucrose, add them to a certain amount of purified water, and stir until they are completely dissolved. The pH value is between 7.5 and 9.2 and the volume is adjusted to 1000ml. Filtration was performed with a 0.22 μm filter.

Table 10 Buffer 9 Recipe

Buffer 11

Weigh 12-18g of Tris, add it to a certain amount of purified water and stir until it is completely dissolved, adjust the pH value between 8.0 and 9.5 and make up to 1000ml. Filtration was performed with a 0.22 μm filter.

Table 11 Buffer 9 Recipe

raw material name Weigh Tris(hydroxymethylaminomethane) 12-18g pH 8.0-9.5 purified water Dilute to 1000mL

The technical effect achieved by the present invention is:

1. This patent uses immunological detection methods for blood detection of traumatic brain injury. Compared with imaging detection methods (mainly CT), this patent can more objectively reflect the real situation of the sample, and reduce the risk caused by subjective judgment. Misjudgments and omissions.

2. The magnetic particle chemiluminescence method used in this patent can make the detection sensitivity reach picogram level (10-12g/mL), while CT relies on pixels to achieve higher resolution. Since the present invention detects specific markers of brain injury, the detection window is much earlier than CT. In the case of negative CT, it can effectively distinguish between normal and mild TBI patients.

3. The present invention uses a fully automatic instrument for detection, and only needs to add serum samples, and accurate results can be obtained within 30 minutes. The CT test takes a long time, generally need to wait 4 hours before the test results can be obtained.

4. In the present invention, the concentration value is used to judge, and the result can be obtained to know whether the patient is ill or not, and the objectivity is strong. However, CT detection requires doctors to read the images. According to the professional level of doctors, the subjective judgment is strong, which is easy to cause missed judgments and misjudgments.

5. The present 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.

Description of drawings

Fig. 1 is the schematic diagram of PGP9.5 detection reagent strip;

Fig. 2 is the reaction flow diagram of the present invention;

Fig. 3 is the process flow diagram of the present invention;

Figure 4 is a schematic diagram of the FITC-anti-FITC antibody signal amplification system.

Detailed ways

The embodiments of the present invention are described below through specific specific examples, and those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention.

Before further describing the specific embodiments of the present invention, it should be understood that the protection scope of the present invention is not limited to the following specific specific embodiments; it should also be understood that the terms used in the examples of the present invention are for describing specific specific embodiments, It is not intended to limit the protection scope of the present invention.

When numerical ranges are given in the examples, it is to be understood that, unless otherwise indicated herein, both endpoints of each numerical range and any number between the two endpoints may be selected. Unless otherwise defined, 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 worth noting that the magnetic particles used in the present invention are Sigma RSTREP-RO; FITC is sigma F7250; the anti-FITC antibody is purchased from Abcam, item number: ab19224;

Antibody 1 and antibody 2 are both self-made monoclonal antibodies; the above-mentioned preparation method of monoclonal antibody includes the following steps:

1. Immune animals

BALB/c mice were selected as host animals for immunization, and the antigen solution of 1-5 mg/mL (the antigen was PGP9.5 protein, and the sequence of the PGP9.5 protein was shown in SEQ ID NO: 1) Intraperitoneal injection was performed after mixing with Freund's complete adjuvant at a volume ratio of 1:1. On the 14th and 35th days, the antigen solution of 1-5 mg/mL was mixed with incomplete Freund's adjuvant at a volume ratio of 1:1, and the second and third immunizations were carried out. The fourth immunization was performed with 5 mg/mL antigen in PBS. Cell fusion can be performed around day 61. During this period, two titer tests were performed on the 21st day and the 42nd day respectively to observe the immune effect.

Freund's complete adjuvant: sigma, F5881 (Cat. No.)

Incomplete Freund's Adjuvant: sigma, F5506 (Cat. No.)

2. Cell fusion and culture

The extracted mouse splenocytes and myeloma cells were mixed at a count ratio of 1:3. After mixing, RPMI 1640 medium was added to make up to 40 mL, and the supernatant was removed after centrifugation at 1500 rpm for 5 minutes. Fusion was performed in a water bath at 37°C, during which 1 mL of isothermal fusion agent was added, and after standing for 1 minute, 2 mL of isothermal RPMI 1640 medium was slowly added. After mixing, centrifuge at 1500 rpm for 5 minutes and remove the supernatant. The fused cell solution was placed in a culture dish with feeder cells and cultured at 37°C in a 5% CO2 incubator.

RPMI 1640 medium: sigma R8758 (Cat. No.)

Fusion agent: sigma, 11363735001 (article number)

3. Screening and cloning of hybridoma positive clones

After 7 days of culture, the culture medium was replaced with HAT medium, the culture was continued, and the second screening was performed 14 days later. In the second screening, the cells were diluted 3-4 times by limiting dilution method, and the cells with the highest positive value were selected for cloning to obtain suitable cell lines.

HAT medium: sigma H0262 (Cat. No.)

4. Monoclonal Antibody Preparation

BALB/c mice were selected as hosts, and each mouse was injected intraperitoneally with 0.5-1 mL of pristane. Each mouse was inoculated intraperitoneally with 2 mL of log-phase hybridoma cells with a cell concentration within 200-300 cells/mL. After 14 days, the ascites was drawn from the mice, and the ascites should be drawn at an interval of 3 days until no ascites was produced or the mice were in poor condition. The mouse ascites was purified to monoclonal antibody by salting out method and affinity method.

The sequence of the PGP9.5 antibody 1 prepared by the present invention 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. Wherein, PGP9.5 antibody 1 includes the heavy chain variable region sequence shown in SEQ ID NO:2 and the light chain variable region sequence shown in SEQ ID NO:4, and the PGP9.5 antibody 2 includes SEQ ID NO. The heavy chain variable region sequence shown in: 3 and the light chain variable region sequence shown in SEQ ID NO:5, the heavy chain variable region and the light chain variable region are shown in SEQ ID NO:6 Flexible linker peptide linkage.

The rest of the raw materials are common commercial products, so there is no specific limitation on their sources.

5. Antibody Affinity Detection

Use ForteBio Octet QKe biomolecular interaction analyzer to detect above-mentioned antibody and antigen (described antigen is PGP9.5 protein, and the sequence of described PGP9.5 protein is as shown in SEQ ID NO:1) by biolayer interferometry. ) affinity (see the instructions for details or refer to Tobias et al., Biomolecular Binding KineticAssays in the Octet Platform, Application Note 14, ForteBio, Div. of Pall LifeSciences, 2013). According to the detection, the KD(M) value of the antibody 1 described in this application was 0.97E-12, and the KD(M) value of the antibody 2 was 1.14E-12.

1. Detection principle

This kit uses the double antibody sandwich method to determine the content of PGP9.5. PGP9.5 in the sample binds to antibody 1 in reagent A and antibody 2 of PGP9.5 in reagent B to form a "sandwich" structure. An excess of reagent C is added, and the anti-FITC antibody in reagent C reacts with the FITC in reagent B to generate a complex of "alkaline phosphatase-antibody 1-PGP9.5-antibody 2-FITC-anti-FITC antibody-magnetic particle". After washing, the luminescent substrate is catalyzed by enzymes in the complex to form an unstable excited-state intermediate, which emits photons when the excited-state intermediate returns to the ground state. The number of photons produced is positively correlated with the concentration of PGP9.5 in the sample. The reaction flow diagram of the present invention is shown in FIG. 2 , the process flow diagram of the present invention is shown in FIG. 3 , and the schematic diagram of the FITC-anti-FITC antibody signal amplification system is shown in FIG. 4 .

2. Components

2.1 Kit Components

The PGP9.5 kit consists of test reagent strips, calibrators, quality control products, and QR codes. The detection reagent strip is composed of a series of solutions and accessories as a whole, which can independently detect a sample. The calibrator is prepared with two concentrations of PGP9.5 antigen and buffer for calibration of the standard curve; the quality control material is prepared with two concentrations of PGP9.5 antigen and buffer; entered in the QR code standard curve for the current batch.

Table 12 Main components of the kit

Main components of the kit quantity Detection reagent strip 10 quality control 200μL×1 Calibrator 1 200μL×1 Calibrator 2 200μL×1 box sign QR code 1

1.2 Reagent strip components

The detection reagent strip consists of reagent A, reagent B, reagent C, cleaning solution (1.3-2.2g tris(hydroxymethylaminomethane), 8.5-13.2g NaCl, 0.7-1mL Tween 20), and make up to 1000mL Luminescent substrate: Sigma 69086 ), luminescent substrate, reading wells, elution sleeves, and tips. Reagent A is a solution containing a certain concentration of alkaline phosphatase-labeled PGP9.5 antibody 1; Reagent B is a solution containing a certain concentration of FICT-labeled PGP9.5 antibody 2; Reagent C is a certain concentration of anti-FITC antibody-labeled magnetic particles; The liquid is used for the cleaning of the reaction process; the luminescent substrate is the luminescent substrate catalyzed by ALP; the reading hole is used for the final detection reading value. The location and components of the reagent strips are shown in Figure 1.

Table 13 Main components of reagent strips

3. Production process

3.1 Production of calibrators and quality control products

The PGP9.5 recombinant protein (sequence shown in SEQ ID NO: 1) was used as the raw material of the calibrator, dissolved in buffer 3, mixed thoroughly and prepared into 2 calibrators, with concentrations of 160pg/mL and 1280pg/mL respectively. mL.

The PGP9.5 recombinant protein was used as the raw material of the quality control product, dissolved in buffer 3, and mixed thoroughly to prepare the quality control product with a concentration of 320 pg/mL.

3.2 Buffer preparation and production of reagents A, B and C

Example 1

(1) Preparation of buffer solution

Buffer 1: Weigh 14.8g of ethanolamine and 5.8g of NaCl into a certain amount of purified water and stir until completely dissolved, adjust the pH value between 7.3 and 7.6 and set the volume to 1000ml. Filtration was performed with a 0.22 μm filter.

Buffer 2: Weigh 75g of glycine, add it to a certain amount of purified water, stir until completely dissolved, and make up to 1000ml. Filtration was performed with a 0.22 μm filter.

Buffer 3: Weigh 12.0g of Tris, 20.0g of bovine serum albumin, 2.0g of NaCl, and 10.0g of sucrose, add them to a certain amount of purified water and stir until completely dissolved, adjust the pH value between 7.5 and 10.0 and set the volume to 1000ml. Filtration was performed with a 0.22 μm filter.

Buffer 4: Weigh 12.0g of Tris, 9.0g of NaCl, 1.0g of bovine serum albumin, 5.0g of glycerol, and 5.0g of glycine, add them to a certain amount of purified water and stir until completely dissolved, adjust the pH value at 7.5 to 9.5 between and make up to 1000ml. Filtration was performed with a 0.22 μm filter.

Buffer 5: Weigh out 5.6 g of Na ₂ HPO ₄ ·12H ₂ O, 0.55 g of NaH ₂ PO ₄ , 9.0 g of NaCl, 1.0 g of bovine serum albumin, 80.0 g of sucrose, 0.1 g of xanthan gum, 0.1 g of Add g sodium alginate and 1.0 g gelatin to a certain amount of purified water and stir until completely dissolved, adjust the pH value between 6.2 and 8.0 and set the volume to 1000 ml. Filtration was performed with a 0.22 μm filter.

Buffer 6: Weigh 5.6 g of Na ₂ HPO ₄ ·12H ₂ O, 0.55 g of NaH ₂ PO ₄ , 9.0 g of NaCl, and 0.1 g of KCl, add purified water, and make up to 1000 ml. Filtration was performed with a 0.22 μm filter.

Buffer 7: Weigh 12 g of MES (2-(N-morpholino)ethanesulfonic acid) into purified water and make up to 1000 ml. Filtration was performed with a 0.22 μm filter.

Buffer 8: Weigh 2.5g of Tris, 9.0g of NaCl, and 5.0g of bovine serum albumin into a certain amount of purified water and stir until completely dissolved, adjust the pH between 7.0 and 7.6 and set the volume to 1000ml. Filtration was performed with a 0.22 μm filter.

Buffer 9: Weigh 2.5g of Tris and 9.0g of NaCl, add them to a certain amount of purified water and stir until completely dissolved, measure 0.2mL of Tween 20 and add it to the above container, and adjust the pH between 7.0 and 7.6. Volume to 1000ml. Filtration was performed with a 0.22 μm filter.

Buffer 10: Weigh 8.5g of Tris, 9.0g of NaCl, 20g of bovine serum albumin, and 15g of sucrose, add them to a certain amount of purified water and stir until they are completely dissolved, measure 1mL of ProClin300 and add it to the above container, adjust the pH value at 7.5 Between ~9.2 and dilute to 1000ml. Filtration was performed with a 0.22 μm filter.

Buffer 11: Weigh 12g of Tris, add it to a certain amount of purified water, stir until it is completely dissolved, adjust the pH value between 8.0 and 9.5, and adjust the volume to 1000ml. Filtration was performed with a 0.22 μm filter.

(2) Production of Reagent A

The enzyme-labeled PGP9.5 antibody 1 conjugate was used as the raw material of reagent A, and it was thoroughly mixed with buffer 4 to prepare reagent A.

Specifically include:

Step A1: Activation of Antibody 1: Activation of Antibody 1 needs to be carried out in a class 100,000 clean room. 4-8 mg of 2-iminosulfane hydrochloride (2IT) was weighed and dissolved in buffer 1 to 13.76 mg/mL. The 2IT solution was added to the antibody 1 solution at a ratio of 15:1 molar ratio of 2-IT to antibody 1 (ie: 1 mg of antibody 1 added to 15 μl of 2IT solution) for activation. After shaking and mixing, react at room temperature for 30 minutes. To terminate activation, add 1 mg of antibody 1 to 5 μl of buffer 2, add buffer 2 to antibody 1 solution, and react at room temperature for 10 min. The excess 2IT was removed using a PD10 desalting column, and the activated antibody 1 was collected.

Step A2: Activation of Alkaline Phosphatase (ALP): Activation of ALP needs to be carried out in a class 100,000 clean room. 2-4 mg of (N-maleimidomethyl)cyclohexane-1-carboxylic acid succinimidyl ester (SMCC) was weighed, 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 15:1. After shaking and mixing, react at room temperature for 30 minutes. To terminate the activation, add 10 μl of buffer 2 to 1 mg of ALP, add buffer 2 to the ALP solution, and react at room temperature for 10 min. Excess SMCC was removed using a PD10 desalting column, and the activated ALP was collected.

Step A3: Connection of Antibody 1 and ALP: The connection of Antibody 1 and ALP should be carried out in a clean room of class 100,000. The ALP solution was added to the antibody 1 solution at a ratio of antibody 1 to ALP mass ratio of 1:2 (ie: 1.0 mg of antibody 1 was added to 2.0 mg of ALP). After shaking and mixing, the mixture was reacted at 2°C to 8°C for 12-18 hours.

Step A4: Termination and purification of the antibody 1 conjugate: The termination and purification of the antibody 1 conjugate should be carried out in a class 100,000 clean room. 1-10 mg of maleimide was weighed and dissolved in DMF to 9.7 mg/mL. Dilute with buffer 1 at a ratio of 1/10 to obtain a 0.97 mg/mL maleimide solution. The solution was added in a ratio of 1 mg of antibody 1 to 4 μl of 0.97 mg/mL maleimide solution, and the reaction was carried out at room temperature for 15 minutes. Accurately measure 6 μL of ethanolamine and dissolve it to 100 mM with buffer 1. That is, add 994 μL of buffer 1 to 6 μL of ethanolamine. The solution was added in a ratio of 1 mg of antibody 1 to 10 μl of 100 mM ethanolamine solution, and the solution was shaken and mixed. Concentrate the antibody 1 conjugate to be purified to 0.5 mg/mL using an ultrafiltration concentrator tube. Antibody purification was performed using a purified protein analyzer and a Superdex 200 preparative grade 2.6/60 gel column with buffer 2 as the eluent. The purified liquid is an enzyme-labeled antibody 1 conjugate.

Step A5: Fully mix the obtained conjugate of enzyme-labeled antibody 1 and buffer 4 to prepare reagent A.

(3) Production of Reagent B

The FITC-labeled PGP9.5 antibody 2 conjugate was used as the raw material of reagent B, and it was fully mixed with buffer 4 to prepare reagent B.

Specifically include the following steps:

Step B1: Desalting of Antibody 2: Use buffer 6 as equilibration and eluent for PD10 desalting column. Use 15mL of buffer 6 to wash the PD10 desalting column. When the liquid from the last wash has completely entered the column, add the antibody 2 that needs to be desalted. Intra-column volume was made up to 2 mL with buffer 2. When the liquid in the column completely entered the column material, 3 mL of buffer 6 was added to start elution. The protein eluate was collected in the corresponding volume (2 mL) of the first make-up.

The desalted Antibody 2 was added to an ultrafiltration concentration tube to concentrate to 0.5 mg/mL. The parameters of the high-speed refrigerated centrifuge are: 6000 rpm, centrifugation for 10 minutes.

Step B2: Antibody 2 is linked to FITC: Weigh FITC and mix with buffer 11 to form a FITC concentrate with a final concentration of 0.5 mg/mL. FITC was added to the antibody 2 solution for the reaction. After shaking and mixing, react at room temperature (20-30°C) for 18 hours.

(4) Production of Reagent C

The anti-FITC antibody-labeled magnetic microparticles were used as the raw material of reagent C, which was thoroughly mixed with buffer 5 to prepare reagent C.

Specifically include the following steps:

Step C1: Cleaning of Magnetic Particles

Select 0.5μm magnetic particle raw material and mix well for 0.5 hours. Magnetic beads were resuspended in buffer 7 at a concentration of 5 mg/ml. Mix the cleaned magnetic beads thoroughly for 10 minutes, and use a Sepmag magnetic separator to separate the magnetic particles. Each gram of magnetic beads is separated for 2 minutes, but not less than 5 minutes. Repeat the resuspension and separation steps three times in total.

Step C2: Resuspension of Magnetic Microparticles

The magnetic particles washed three times were resuspended in buffer 7 at a concentration of 5 mg/ml. Mix the resuspended magnetic particles thoroughly for 5 minutes.

Step C3: Selection and Desalting of Anti-FITC Antibodies

The anti-FITC antibody (affinity purification) and the FITC antibody (salting-out purification) were mixed at a ratio of 1:4 by mass ratio to form a new anti-FITC antibody. Buffer 6 was used as equilibration and eluent for the PD10 desalting column. Wash the PD10 desalting column with 15 mL of buffer 6, and add the anti-FITC antibody that needs to be desalted when the liquid from the last wash has completely entered the column. Intra-column volume was made up to 2 mL with buffer 2. When the liquid in the column completely entered the column material, 3 mL of buffer 6 was added to start elution. The protein eluate was collected in the corresponding volume (2 mL) of the first make-up.

The desalted anti-FITC antibody was added to an ultrafiltration concentration tube for concentration to 0.5 mg/mL. The parameters of the high-speed refrigerated centrifuge are: 6000 rpm, centrifugation for 20 minutes.

Step C4: Activation of Magnetic Microparticles

The carboxyl magnetic particles with a particle size of 0.5 μm were selected for activation. (that is, magnetic particles with a large number of carboxyl functional groups on the surface) Dissolve a certain amount of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) with buffer 7 to 4 mg/ mL. According to the ratio of magnetic particles to EDC mass ratio of 100:1 (ie, 1 mg of magnetic particles is added to 10 ug EDC), EDC is added to the magnetic particles for reaction, and the mixture is mixed at room temperature for 40 minutes.

Step C5: Cleaning of activated magnetic particles

The activated magnetic particles were resuspended in buffer 7 at a concentration of 5 mg/ml. Mix the cleaned magnetic beads thoroughly for 10 minutes, and use a Sepmag magnetic separator to separate the magnetic particles. Each gram of magnetic beads is separated for 2 minutes, but not less than 5 minutes.

Repeat the steps of resuspension and separation three times in total, and finally use buffer 7 to resuspend the magnetic particles at a concentration of 5 mg/ml.

Step C6: Anti-FITC Antibody Attached to Magnetic Microparticles

According to the ratio of magnetic particles: anti-FITC antibody mass ratio of 100:3 (i.e. 1mg magnetic beads plus 30ug anti-FITC antibody), add anti-FITC antibody to the activated magnetic particles for reaction, and continue to mix and react at 2 °C for 24 hours .

Step C7: Blocking of Anti-FITC Antibody Magnetic Microparticle Conjugates

After the conjugation reaction was completed, the anti-FITC antibody magnetic particle conjugate was magnetically separated. Separation of 2 minutes per gram of conjugate, but not less than 5 minutes. The linker was resuspended in buffer 8 at a concentration of 5 mg/ml. Mix the resuspended linker at room temperature for 1.5 hours.

Step C8: Washing of Anti-FITC Antibody Magnetic Microparticle Conjugates

After blocking was completed, the anti-FITC antibody magnetic particle conjugate was magnetically separated. Separation of 2 minutes per gram of conjugate, but not less than 5 minutes. The conjugate was resuspended with buffer 9 at a concentration of 5 mg/ml, and the washed conjugate was thoroughly mixed for 10 minutes.

The steps of magnetic separation, resuspension, and mixing were repeated three times in total.

Step C9: Resuspension of Anti-FITC Antibody Microparticle Conjugates

After washing, the anti-FITC antibody magnetic particle conjugates were magnetically separated. Separation of 2 minutes per gram of conjugate, but not less than 5 minutes. The conjugate was resuspended in buffer 9 at a concentration of 5 mg/ml and mixed for 5 minutes. Add 1/20 of the total volume of Buffer 10 and mix for 25 minutes. The linker should be stored at 2°C to 8°C.

Example 2

(1) Preparation of buffer solution

Buffer 1: Weigh 15.1g of ethanolamine and 6.0g of NaCl into a certain amount of purified water and stir until completely dissolved, adjust the pH value between 7.3 and 7.6 and set the volume to 1000ml. Filtration was performed with a 0.22 μm filter.

Buffer 2: Weigh 75g of glycine, add it to a certain amount of purified water, stir until completely dissolved, and make up to 1000ml. Filtration was performed with a 0.22 μm filter.

Buffer 3: Weigh 15.0g of Tris, 60g of bovine serum albumin, 10.0g of NaCl, and 100g of sucrose, add them to a certain amount of purified water and stir until they are completely dissolved, adjust the pH between 7.5 and 10.0 and set the volume to 1000ml. Filtration was performed with a 0.22 μm filter.

Buffer 4: Weigh 15.0g of Tris, 9.0g of NaCl, 50g of bovine serum albumin, 20.0g of glycerol, and 40g of glycine into a certain amount of purified water and stir until completely dissolved, and adjust the pH between 7.5 and 9.5 And make up to 1000ml. Filtration was performed with a 0.22 μm filter.

Buffer 5: Weigh 5.9 g of Na ₂ HPO ₄ ·12H ₂ O, 0.60 g of NaH ₂ PO ₄ , 9.0 g of NaCl, 50 g of bovine serum albumin, 140 g of sucrose, 5.0 g of xanthan gum, 5 g of alginic acid Add sodium and 15g gelatin to a certain amount of purified water and stir until completely dissolved, adjust the pH value between 6.2 and 8.0 and set the volume to 1000ml. Filtration was performed with a 0.22 μm filter.

Buffer 6: Weigh 5.9 g of Na ₂ HPO ₄ ·12H ₂ O, 0.60 g of NaH ₂ PO ₄ , 9.0 g of NaCl, and 5.0 g of KCl, add purified water, and dilute to 1000 ml. Filtration was performed with a 0.22 μm filter.

Buffer 7: Weigh 30 g of MES (2-(N-morpholine)ethanesulfonic acid) into purified water and make up to 1000 ml. Filtration was performed with a 0.22 μm filter.

Buffer 8: Weigh 5.0g of Tris, 9.0g of NaCl, and 20.0g of bovine serum albumin into a certain amount of purified water and stir until completely dissolved, adjust the pH value between 7.0 and 7.6 and set the volume to 1000ml. Filtration was performed with a 0.22 μm filter.

Buffer 9: Weigh 5.0g of Tris and 9.0g of NaCl, add them to a certain amount of purified water and stir until completely dissolved, measure 2mL of Tween 20 and add it to the above container, adjust the pH value between 7.0 and 7.6 and set the volume to 1000ml. Filtration was performed with a 0.22 μm filter.

Buffer 10: Weigh 15.0g of Tris, 9.0g of NaCl, 150g of bovine serum albumin, and 100g of sucrose, add them to a certain amount of purified water, and stir until they are completely dissolved. Measure 20mL of ProClin300 and add it to the above container. Adjust the pH to 7.5. Between ~9.2 and dilute to 1000ml. Filtration was performed with a 0.22 μm filter.

Buffer 11: Weigh 18g of Tris, add it to a certain amount of purified water, stir until it is completely dissolved, adjust the pH value between 8.0 and 9.5, and adjust the volume to 1000ml. Filtration was performed with a 0.22 μm filter.

(2) Production of Reagent A

The enzyme-labeled PGP9.5 antibody 1 conjugate was used as the raw material of reagent A, and it was thoroughly mixed with buffer 4 to prepare reagent A.

Specifically include:

Step A1: Activation of Antibody 1: Activation of Antibody 1 needs to be carried out in a class 100,000 clean room. 4-8 mg of 2-iminosulfane 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 ratio of 2-IT to antibody 1 molar ratio of 30:1 (ie: 1 mg of antibody 1 added to 30 μl of 2IT solution). After shaking and mixing, react at room temperature for 30 minutes. To terminate the activation, add 1 mg of antibody 1 to 20 μl of buffer 2, add buffer 2 to the solution of antibody 1, and react at room temperature for 10 min. The excess 2IT was removed using a PD10 desalting column, and the activated antibody 1 was collected.

Step A2: Activation of Alkaline Phosphatase (ALP): Activation of ALP needs to be carried out in a class 100,000 clean room. 2-4 mg of (N-maleimidomethyl)cyclohexane-1-carboxylic acid succinimidyl ester (SMCC) was weighed, 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 60:1. After shaking and mixing, react at room temperature for 30 minutes. To terminate the activation, add 50 μl of buffer 2 according to the ratio of 1 mg ALP, add buffer 2 to the ALP solution, and react at room temperature for 10 min. Excess SMCC was removed using a PD10 desalting column, and the activated ALP was collected.

Step A3: Connection of Antibody 1 and ALP: The connection of Antibody 1 and ALP should be carried out in a clean room of class 100,000. Add the ALP solution to the antibody 1 solution according to the ratio of antibody 1 to ALP mass ratio of 1:1 (ie: 1.0 mg of antibody 1 to 1.0 mg of ALP). After shaking and mixing, the mixture was reacted at 2°C to 8°C for 12-18 hours.

Step A4: Termination and purification of the antibody 1 conjugate: The termination and purification of the antibody 1 conjugate should be carried out in a class 100,000 clean room. 1-10 mg of maleimide was weighed and dissolved in DMF to 9.7 mg/mL. Dilute with buffer 1 at a ratio of 1/10 to obtain a 0.97 mg/mL maleimide solution. The solution was added in a ratio of 1 mg of antibody 1 to 20 μl of 0.97 mg/mL maleimide solution, and the reaction was carried out at room temperature for 15 minutes. Accurately measure 6 μL of ethanolamine and dissolve it to 100 mM with buffer 1. That is, add 994 μL of buffer 1 to 6 μL of ethanolamine. The solution was added in the ratio of 1 mg of antibody 1 to 50 μl of 100 mM ethanolamine solution, and the solution was shaken and mixed. Concentrate the antibody 1 conjugate to be purified to 2 mg/mL using an ultrafiltration concentrator tube. Antibody purification was performed using a purified protein analyzer and a Superdex 200 preparative grade 2.6/60 gel column with buffer 2 as the eluent. The purified liquid is an enzyme-labeled antibody 1 conjugate.

Step A5: Fully mix the obtained conjugate of enzyme-labeled antibody 1 and buffer 4 to prepare reagent A.

(3) Production of Reagent B

The FITC-labeled PGP9.5 antibody 2 conjugate was used as the raw material of reagent B, and it was fully mixed with buffer 4 to prepare reagent B.

Specifically include the following steps:

Step B1: Desalting of Antibody 2: Use buffer 6 as equilibration and eluent for PD10 desalting column. Use 15mL of buffer 6 to wash the PD10 desalting column. When the liquid from the last wash has completely entered the column, add the antibody 2 that needs to be desalted. Intra-column volume was made up to 2.6 mL with buffer 2. When the liquid in the column completely entered the column material, 3 mL of buffer 6 was added to start elution. The protein eluate was collected in the corresponding volume (2.6 mL) of the first make-up.

The desalted antibody 2 was added to an ultrafiltration concentrating tube and concentrated to 4 mg/mL. The parameters of the high-speed refrigerated centrifuge are: 8000rpm, centrifugation for 10 minutes.

Step B2: Antibody 2 is linked to FITC: Weigh FITC with buffer 11 and mix it into a FITC concentrate with a final concentration of 0.5 mg/mL. According to the mass ratio of FITC to antibody 2 of 15:100 (that is, 1 mg of antibody is added to 150 μg of FITC), FITC was added to the antibody 2 solution for the reaction. After shaking and mixing, react at room temperature (20-30°C) for 18 hours.

(4) Production of Reagent C

The anti-FITC antibody-labeled magnetic microparticles were used as the raw material of reagent C, which was thoroughly mixed with buffer 5 to prepare reagent C.

Specifically include the following steps:

Step C1: Cleaning of Magnetic Particles

Select 3μm magnetic particle raw material and mix well for 0.5 hours. Magnetic beads were resuspended in buffer 7 at a concentration of 10 mg/ml. Mix the cleaned magnetic beads thoroughly for 10 minutes, and use a Sepmag magnetic separator to separate the magnetic particles. Each gram of magnetic beads is separated for 2 minutes, but not less than 5 minutes. Repeat the resuspension and separation steps three times in total.

Step C2: Resuspension of Magnetic Microparticles

The magnetic particles washed three times were resuspended in buffer 7 at a concentration of 10 mg/ml. Mix the resuspended magnetic particles thoroughly for 5 minutes.

Step C3: Selection and Desalting of Anti-FITC Antibodies

The anti-FITC antibody (affinity purification) and FITC antibody (salting-out purification) were mixed in a ratio of 4:1 by mass ratio to form a new anti-FITC antibody. Buffer 6 was used as equilibration and eluent for the PD10 desalting column. Wash the PD10 desalting column with 15 mL of buffer 6, and add the anti-FITC antibody that needs to be desalted when the liquid from the last wash has completely entered the column. Intra-column volume was made up to 2.6 mL with buffer 2. When the liquid in the column completely entered the column material, 3 mL of buffer 6 was added to start elution. The protein eluate was collected in the corresponding volume (2.6 mL) of the first make-up.

The desalted anti-FITC antibody was added to an ultrafiltration concentration tube for concentration to 4 mg/mL. The parameters of the high-speed refrigerated centrifuge are: 8000rpm, centrifugation for 10 minutes.

Step C4: Activation of Magnetic Microparticles

The carboxyl magnetic particles with a particle size of 3 μm were selected for activation. (that is, magnetic particles with a large number of carboxyl functional groups on the surface) Dissolve a certain amount of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) with buffer 7 to 15mg/ mL. According to the ratio of magnetic particles to EDC mass ratio of 100:30 (ie, 1 mg of magnetic particles is added to 300 ug EDC), EDC is added to the magnetic particles for reaction, and the mixture is mixed at room temperature for 40 minutes.

Step C5: Cleaning of activated magnetic particles

The activated magnetic particles were resuspended in buffer 7 at a concentration of 10 mg/ml. Mix the cleaned magnetic beads thoroughly for 10 minutes, and use a Sepmag magnetic separator to separate the magnetic particles. Each gram of magnetic beads is separated for 2 minutes, but not less than 5 minutes.

Repeat the steps of resuspension and separation three times in total, and finally use buffer 7 to resuspend the magnetic particles at a concentration of 10 mg/ml.

Step C6: Anti-FITC Antibody Attached to Magnetic Microparticles

According to the ratio of magnetic particles: anti-FITC antibody mass ratio of 100:20 (i.e. 1 mg of magnetic beads plus 200ug of anti-FITC antibody), add anti-FITC antibody to the activated magnetic particles for reaction, and continue to mix and react at 8°C for 16 hours .

Step C7: Blocking of Anti-FITC Antibody Magnetic Microparticle Conjugates

After the conjugation reaction was completed, the anti-FITC antibody magnetic particle conjugate was magnetically separated. Separation of 2 minutes per gram of conjugate, but not less than 5 minutes. The linker was resuspended in buffer 8 at a concentration of 10 mg/ml. Mix the resuspended linker at room temperature for 1.5 hours.

Step C8: Washing of Anti-FITC Antibody Magnetic Microparticle Conjugates

After blocking was completed, the anti-FITC antibody magnetic particle conjugate was magnetically separated. Separation of 2 minutes per gram of conjugate, but not less than 5 minutes. Use buffer 9 to resuspend the conjugate at a concentration of 10 mg/ml, and mix the washed conjugate thoroughly for 10 minutes.

The steps of magnetic separation, resuspension, and mixing were repeated three times in total.

Step C9: Resuspension of Anti-FITC Antibody Microparticle Conjugates

After washing, the anti-FITC antibody magnetic particle conjugates were magnetically separated. Separation of 2 minutes per gram of conjugate, but not less than 5 minutes. The conjugate was resuspended in buffer 9 at a concentration of 10 mg/ml and mixed for 5 minutes. Add 1/50 of the total volume of Buffer 10 and mix for 25 minutes. The linker should be stored at 2°C to 8°C.

Example 3

(1) Preparation of buffer solution

Buffer 1: Weigh 15.0g of ethanolamine and 6.0g of NaCl into a certain amount of purified water and stir until completely dissolved, adjust the pH value between 7.3 and 7.6 and set the volume to 1000ml. Filtration was performed with a 0.22 μm filter.

Buffer 2: Weigh 75g of glycine, add it to a certain amount of purified water, stir until completely dissolved, and make up to 1000ml. Filtration was performed with a 0.22 μm filter.

Buffer 3: Weigh 13.0g of Tris, 40g of bovine serum albumin, 8.0g of NaCl, and 50g of sucrose into a certain amount of purified water and stir until completely dissolved, adjust the pH between 7.5 and 10.0 and set the volume to 1000ml. Filtration was performed with a 0.22 μm filter. Buffer 4: Weigh 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 into a certain amount of purified water and stir until completely dissolved. Between 7.5 and 9.5 and dilute to 1000ml. Filtration was performed with a 0.22 μm filter.

Buffer 5: Weigh out 5.8 g of Na ₂ HPO ₄ ·12H ₂ O, 0.58 g of NaH ₂ PO ₄ , 9.0 g of NaCl, 10.0 g of bovine serum albumin, 100 g of sucrose, 2.0 g of xanthan gum, 2.0 g of Sodium alginate and 8.0g gelatin were added to a certain amount of purified water and stirred until completely dissolved. Adjust the pH value between 6.2 and 8.0 and set the volume to 1000ml. Filtration was performed with a 0.22 μm filter.

Buffer 6: Weigh 5.8 g of Na ₂ HPO ₄ ·12H ₂ O, 0.58 g of NaH ₂ PO ₄ , 9.0 g of NaCl, and 2.0 g of KCl, add purified water, and dilute to 1000 ml. Filtration was performed with a 0.22 μm filter.

Buffer 7: Weigh 20.0 g of MES (2-(N-morpholine)ethanesulfonic acid) into purified water and make up to 1000 ml. Filtration was performed with a 0.22 μm filter.

Buffer 8: Weigh 3.0g of Tris, 9.0g of NaCl, and 10.0g of bovine serum albumin into a certain amount of purified water and stir until completely dissolved, adjust the pH between 7.0 and 7.6 and set the volume to 1000ml. Filtration was performed with a 0.22 μm filter.

Buffer 9: Weigh 3.0g of Tris and 9.0g of NaCl and add them to a certain amount of purified water and stir until completely dissolved, measure 1mL of Tween 20 and add it to the above container, adjust the pH value between 7.0 and 7.6 and set the volume to 1000ml. Filtration was performed with a 0.22 μm filter.

Buffer 10: Weigh 10.0 g of Tris, 9.0 g of NaCl, 100 g of bovine serum albumin, and 80 g of sucrose, add them to a certain amount of purified water, and stir until they are completely dissolved. Measure 10 mL of ProClin300 into the above container, and adjust the pH value to 7.5. Between ~9.2 and dilute to 1000ml. Filtration was performed with a 0.22 μm filter.

Buffer 11: Weigh 15g of Tris, add it to a certain amount of purified water and stir until it is completely dissolved, adjust the pH value between 8.0 and 9.5 and adjust the volume to 1000ml. Filtration was performed with a 0.22 μm filter.

(2) Production of Reagent A

The enzyme-labeled PGP9.5 antibody 1 conjugate was used as the raw material of reagent A, and it was thoroughly mixed with buffer 4 to prepare reagent A.

Specifically include:

Step A1: Activation of Antibody 1: Activation of Antibody 1 needs to be carried out in a class 100,000 clean room. 4-8 mg of 2-iminosulfane hydrochloride (2IT) was weighed and dissolved in buffer 1 to 13.76 mg/mL. The 2IT solution was added to the antibody 1 solution at the ratio of 2-IT to antibody 1 molar ratio of 20:1 for activation. After shaking and mixing, react at room temperature for 30 minutes. To terminate the activation, add 1 mg of antibody 1 to 10 μl of buffer 2, add buffer 2 to the solution of antibody 1, and react at room temperature for 10 min. The excess 2IT was removed using a PD10 desalting column, and the activated antibody 1 was collected.

Step A2: Activation of Alkaline Phosphatase (ALP): Activation of ALP needs to be carried out in a class 100,000 clean room. 2-4 mg of (N-maleimidomethyl)cyclohexane-1-carboxylic acid succinimidyl ester (SMCC) was weighed, 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, react at room temperature for 30 minutes. To terminate the activation, add 20 μl of buffer 2 according to the ratio of 1 mg ALP, add buffer 2 to the ALP solution, and react at room temperature for 10 min. Excess SMCC was removed using a PD10 desalting column, and the activated ALP was collected.

Step A3: Connection of Antibody 1 and ALP: The connection of Antibody 1 and ALP should be carried out in a clean room of class 100,000. The ALP solution was added to the antibody 1 solution at a ratio of antibody 1 to ALP mass ratio of 1:1.5 (ie: 1.0 mg of antibody 1 was added to 1.5 mg of ALP). After shaking and mixing, the mixture was reacted at 4°C for 15 hours.

Step A4: Termination and purification of the antibody 1 conjugate: The termination and purification of the antibody 1 conjugate should be carried out in a class 100,000 clean room. 1-10 mg of maleimide was weighed and dissolved in DMF to 9.7 mg/mL. Dilute with buffer 1 at a ratio of 1/10 to obtain a 0.97 mg/mL maleimide solution. The solution was added in a ratio of 1 mg of antibody 1 to 10 μl of 0.97 mg/mL maleimide solution, and reacted at room temperature for 15 minutes. Accurately measure 6 μL of ethanolamine and dissolve it to 100 mM with buffer 1. That is, add 994 μL of buffer 1 to 6 μL of ethanolamine. The solution was added in a ratio of 1 mg of antibody 1 to 30 μl of 100 mM ethanolamine solution, and the solution was shaken and mixed. Concentrate the antibody 1 conjugate to be purified to 1 mg/mL using an ultrafiltration concentrator tube. Antibody purification was performed using a purified protein analyzer and a Superdex 200 preparative grade 2.6/60 gel column with buffer 2 as the eluent. The purified liquid is an enzyme-labeled antibody 1 conjugate.

Step A5: Fully mix the obtained conjugate of enzyme-labeled antibody 1 and buffer 4 to prepare reagent A.

(3) Production of Reagent B

The FITC-labeled PGP9.5 antibody 2 conjugate was used as the raw material of reagent B, and it was fully mixed with buffer 4 to prepare reagent B.

Specifically include the following steps:

Step B1: Desalting of Antibody 2: Use buffer 6 as equilibration and eluent for PD10 desalting column. Use 15mL of buffer 6 to wash the PD10 desalting column. When the liquid from the last wash has completely entered the column, add the antibody 2 that needs to be desalted. Intra-column volume was made up to 2 mL with buffer 2. When the liquid in the column completely entered the column material, 3 mL of buffer 6 was added to start elution. The protein eluate was collected in the corresponding volume (2 mL) of the first make-up.

The desalted Antibody 2 was added to an ultrafiltration concentrating tube and concentrated to 3 mg/mL. The parameters of the high-speed refrigerated centrifuge are: 6000 rpm, centrifugation for 20 minutes.

Step B2: Antibody 2 is linked to FITC: Weigh FITC and mix with buffer 11 to form a FITC concentrate with a final concentration of 0.5 mg/mL. FITC was added to the antibody 2 solution for the reaction. After shaking and mixing, react at room temperature (20-30°C) for 18 hours.

(4) Production of Reagent C

The anti-FITC antibody-labeled magnetic microparticles were used as the raw material of reagent C, which was thoroughly mixed with buffer 5 to prepare reagent C.

Specifically include the following steps:

Step C1: Cleaning of Magnetic Particles

Select 1.5μm magnetic particle raw material and mix well for 0.5 hours. Magnetic beads were resuspended in buffer 7 at a concentration of 8 mg/ml. Mix the cleaned magnetic beads thoroughly for 10 minutes, and use a Sepmag magnetic separator to separate the magnetic particles. Each gram of magnetic beads is separated for 2 minutes, but not less than 5 minutes. Repeat the resuspension and separation steps three times in total.

Step C2: Resuspension of Magnetic Microparticles

The magnetic microparticles washed three times were resuspended in buffer 7 at a concentration of 8 mg/ml. Mix the resuspended magnetic particles thoroughly for 5 minutes.

Step C3: Selection and Desalting of Anti-FITC Antibodies

The anti-FITC antibody (affinity purification) and the FITC antibody (salting-out purification) were mixed at a ratio of 1:1 by mass ratio to form a new anti-FITC antibody. Buffer 6 was used as equilibration and eluent for the PD10 desalting column. Wash the PD10 desalting column with 15 mL of buffer 6, and add the anti-FITC antibody that needs to be desalted when the liquid from the last wash has completely entered the column. Intra-column volume was made up to 2 mL with buffer 2. When the liquid in the column completely entered the column material, 3 mL of buffer 6 was added to start elution. The protein eluate was collected in the corresponding volume (2 mL) of the first make-up.

The desalted anti-FITC antibody was added to an ultrafiltration concentration tube for concentration to 2 mg/mL. The parameters of the high-speed refrigerated centrifuge are: 6000 rpm, centrifugation for 20 minutes.

Step C4: Activation of Magnetic Microparticles

The carboxyl magnetic particles with a particle size of 1.5 μm were selected for activation. (that is, magnetic particles with a large number of carboxyl functional groups on the surface) Dissolve a certain amount of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) with buffer 7 to 10 mg/ mL. According to the ratio of the mass ratio of magnetic particles to EDC of 100:9 (ie, 1 mg of magnetic particles is added to 90 ug EDC), EDC is added to the magnetic particles for reaction, and the mixture is mixed at room temperature for 40 minutes.

Step C5: Cleaning of activated magnetic particles

The activated magnetic particles were resuspended in buffer 7 at a concentration of 8 mg/ml. Mix the cleaned magnetic beads thoroughly for 10 minutes, and use a Sepmag magnetic separator to separate the magnetic particles. Each gram of magnetic beads is separated for 2 minutes, but not less than 5 minutes.

The steps of resuspension and separation were repeated three times in total, and finally the magnetic particles were resuspended with buffer 7 at a concentration of 8 mg/ml.

Step C6: Anti-FITC Antibody Attached to Magnetic Microparticles

According to the ratio of magnetic particles: anti-FITC antibody mass ratio of 100:15 (i.e. 1mg magnetic beads plus 150ug anti-FITC antibody), add anti-FITC antibody to the activated magnetic particles for reaction, and continue to mix and react at 4°C for 20 hours .

Step C7: Blocking of Anti-FITC Antibody Magnetic Microparticle Conjugates

After the conjugation reaction was completed, the anti-FITC antibody magnetic particle conjugate was magnetically separated. Separation of 2 minutes per gram of conjugate, but not less than 5 minutes. The linker was resuspended in buffer 8 at a concentration of 8 mg/ml. Mix the resuspended linker at room temperature for 1.5 hours.

Step C8: Washing of Anti-FITC Antibody Magnetic Microparticle Conjugates

After blocking was completed, the anti-FITC antibody magnetic particle conjugate was magnetically separated. Separation of 2 minutes per gram of conjugate, but not less than 5 minutes. The conjugate was resuspended with buffer 9 at a concentration of 8 mg/ml, and the washed conjugate was thoroughly mixed for 10 minutes.

The steps of magnetic separation, resuspension, and mixing were repeated three times in total.

Step C9: Resuspension of Anti-FITC Antibody Microparticle Conjugates

After washing, the anti-FITC antibody magnetic particle conjugates were magnetically separated. Separation of 2 minutes per gram of conjugate, but not less than 5 minutes. The conjugate was resuspended in buffer 9 at a concentration of 8 mg/ml and mixed for 5 minutes. Add 1/30 of the total volume of Buffer 10 and mix for 25 minutes. The linker should be stored at 2°C to 8°C.

4. Detection method

The detection was carried out using a fully automatic chemiluminescence immunoassay developed by Beijing Meilian Tech Biotechnology Co., Ltd. The sample volume required for the reaction is 30 μL, and the automatic inspection process is as follows:

Immune reaction: Add 30uL of sample, 50uL of reagent B, 50uL of reagent A, and 50uL of reagent C to well 11 in sequence, and react at 37°C for 20min.

Magnetic separation and cleaning: 300 μL of cleaning solution was added to the No. 12 hole, the mixture containing the magnetic particles was magnetically sucked out of the No. 11 hole, and demagnetized at the No. 12 hole. After cleaning for 2 minutes. Magnetic separation and cleaning were performed once in holes 13 and 14 respectively.

Reading value: Add 150uL of luminescent substrate to hole 15, suck the mixture containing magnetic particles out of hole 14 by magnetic force, and demagnetize it in hole 15. After the luminescent substrate catalyzed by alkaline phosphatase emits light, the relative luminescence intensity (RLU) was detected by a self-developed instrument.

A PGP9.5 concentration-luminescence value standard curve can be obtained according to the detected calibrator value. 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 this curve, so as to realize the concentration detection of the unknown sample.

5. Detection indicators

The relevant indicators of the respective detection of Examples 1-3 are specifically shown below, wherein, the inter-batch difference, specificity and inter-bottle difference are only the relevant results of Example 1.

5.1 Accuracy

The brain-specific protein product (PGP9.5) solution (A) with a concentration of about 3000pg/mL (allowable deviation ±10%) was added to the sample B with a concentration range of 0pg/mL-40pg/mL. The added PGP9.5 The volume ratio between antigen and sample B is 1:9, and the recovery rate R is calculated according to formula (1), and the recovery rate should be in the range of 90% to 110%.

In the formula: R is the recovery rate; V is the volume of sample A solution; V ₀ is the volume of serum sample B solution; C is the average value of three measurements after serum sample B solution is added to A solution; C ₀ is serum sample B solution The average value of 3 measurements; C _S is the concentration of sample A solution.

5.2 Blank limit

和标准差(SD)。平均值

即为空白限，结果应≤40pg/mL。Repeat the test 20 times for the sample without any analyte to obtain the concentration value of the 20 test results and calculate the average value

and standard deviation (SD). average value

That is the blank limit, and the result should be ≤40pg/mL.

5.3 Linear Intervals

Mix high-value samples close to the upper limit of the linear interval with low-value samples or zero-concentration samples close to the lower limit of the linear interval into no less than 5 dilution concentrations, of which the samples with low-value concentrations must be close to the lower limit of the linear interval. The samples of each concentration were tested three times to obtain the luminescence value, the measurement results of each sample were recorded, and the average value (y _i ) of the three measurements of each sample was calculated. Taking the dilution concentration (x _i ) as the independent variable and the mean value of the measurement results (y _i ) as the dependent variable, a linear regression equation was obtained. Calculate the correlation coefficient (r) of linear regression according to formula (2), in the linear interval of 80 ~ 2560pg/mL, the correlation coefficient r should be ≥ 0.990.

为稀释比例的均值；

为样本测定结果总均值。In the formula: r is the correlation coefficient; x _i is the dilution ratio; y _i is the mean value of the measurement results of each sample;

is the mean value of the dilution ratio;

The overall mean of the results for the sample determination.

5.4 Repeatability

和标准差SD。按公式(3)计算变异系数(CV)，结果CV≤8％。Repeat the test of the quality control product 10 times with the same batch of kits, and calculate the average of the 10 test results.

and standard deviation SD. The coefficient of variation (CV) was calculated according to formula (3), and the result was CV≤8%.

为样本测试值的平均值。In the formula: s is the standard deviation of the sample test value;

is the mean of the sample test values.

5.5 Batch-to-batch difference

和标准差SD，根据公式(3)得出变异系数(CV)，结果CV≤12％。Repeat the testing of the quality control products 10 times with 3 batches of kits, and calculate the average of the 30 test results.

and standard deviation SD, the coefficient of variation (CV) was obtained according to formula (3), the result CV≤12%.

5.6 Specificity

Add 5000pg/mL glial fibrillary acidic protein (GFAP) to the sample without any analyte, measure 3 times and take the average value, calculate the result according to formula (4), the cross-reaction rate should be less than 5%

R _CR =M/C×100%(4)

In the formula: RCR is the cross-reaction rate; M is the mean value of the measurement results of the cross-reactants; C is the marked value of the cross-reactants.

5.7 Difference between calibrator and quality control vials

与标准差(S1)。另取上述10瓶校准品(或质控品)中的1瓶连续测定5次，计算结果的均值

和标准差(S2)，按照公式(6)、(7)计算瓶间重复性CV％，测量结果应满足2.10要求。Test 10 bottles of calibrators (or quality control products) with the same batch number once each, calculate according to formula (5), and measure the mean value of the results

with the standard deviation (S1). In addition, 1 bottle of the above 10 bottles of calibrator (or quality control product) was continuously measured for 5 times, and the mean value of the results was calculated.

and standard deviation (S2), calculate the repeatability CV% between bottles according to formulas (6) and (7), and the measurement results should meet the requirements of 2.10.

(Note: when S1<S2, set CV between bottles = 0)

Where: S is the standard deviation.

6. Test results

6.1 Accuracy

Table 14

example Example 1 Example 2 Example 3 High sample concentration 3000 3000 3000 Low 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 sample mean 4.1533 4.514 5.221 Dilute samples in parallel 1 317.502 287.218 311.594 Dilute samples in parallel 2 310.629 276.552 306.286 Dilute samples in parallel 3 310.996 281.934 309.765 Diluted sample mean 313.04 281.901 309.215 Recovery rate 103.10% 92.61% 101.51%

6.2 Blank limit

Table 15

6.3 Linear range

Table 16

6.4 Repeatability

Table 17

6.5 Batch-to-batch difference

Table 18

6.6 Specificity

Table 19

6.7 Difference between bottles

Table 20

Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, rather than limiting the protection scope of the present invention. The essence and scope of the technical solution of the invention.

SEQUENCE LISTING

<110> Beijing Meilian Tech Biotechnology Co., Ltd.

<120> Application of a signal amplification technology in PGP9.5 detection kit

<130> 20211222

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Claims (10)

1. a PGP9.5 detection kit, is characterized in that: comprise detection reagent strip; Described detection reagent strip comprises reagent A, reagent B and reagent C; The raw material of the reagent A includes PGP9.5 antibody 1; the raw material of the reagent B includes PGP9.5 antibody 2; the reagent C includes anti-FITC antibody; The PGP9.5 antibody 1 includes the heavy chain variable region sequence shown in SEQ ID NO:2 and the light chain variable region sequence shown in SEQ ID NO:4; the PGP9.5 antibody 2 includes SEQ ID NO: The heavy chain variable region sequence shown in 3 and the light chain variable region sequence shown in SEQ ID NO:5. 2. PGP9.5 detection kit according to claim 1, is characterized in that: the sequence of described PGP9.5 antibody 1 is the amino acid sequence shown in SEQ ID NO:7, the sequence of described PGP9.5 antibody 2 is the amino acid sequence shown in SEQ ID NO:8. 3. PGP9.5 detection kit according to claim 1, is characterized in that: the raw material of described reagent A also comprises alkaline phosphatase, the raw material of described reagent B also comprises FITC, the raw material of described reagent C also comprises Including anti-FITC antibodies. 4. PGP9.5 detection kit according to claim 1, is characterized in that: also comprises calibrator and quality control substance. 5 . The PGP9.5 detection kit according to claim 4 , wherein the raw materials of the calibrator and the quality control product both comprise PGP9.5 recombinant protein. 6 . 6 . The PGP9.5 detection kit according to claim 1 , wherein the detection reagent strip further comprises a cleaning solution, a luminescent substrate, a reading hole, an elution sleeve and a suction tip. 7 . 7. the preparation method of the PGP9.5 detection kit as described in any one of claim 1-6, is characterized in that: comprises the following steps: (1) Production of solvent A: after coupling alkaline phosphatase with PGP9.5 antibody 1, it is mixed with buffer to obtain reagent A; (2) Production of solvent B: the FITC is coupled with PGP9.5 antibody 2 and mixed with buffer to obtain reagent B; (3) Production of solvent C: The anti-FITC antibody is linked to the magnetic microparticles and mixed with the buffer to obtain the reagent C. 8. preparation method according to claim 7 is characterized in that: the weight ratio of described PGP9.5 antibody 1 and alkaline phosphatase is 1:1-2, the weight ratio of described FITC and PGP9.5 antibody 2 3-15:100. 9 . The preparation method according to claim 7 , wherein the weight ratio of the anti-FITC antibody to the magnetic particles is 100:3-20. 10 . 10 . The preparation method according to claim 7 , wherein the buffers in steps (1) and (2) comprise tris, sodium chloride, bovine serum albumin, glycerin and glycine. 11 .

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