CN113960306A

CN113960306A - Reagent for stabilizing acridinium ester marker protein and application thereof

Info

Publication number: CN113960306A
Application number: CN202010701353.XA
Authority: CN
Inventors: 彭程; 张翼; 阳馨滢
Original assignee: Fapon Biotech Inc; Guangdong Fapon Biotech Co Ltd
Current assignee: Fapon Biotech Inc; Guangdong Fapon Biotech Co Ltd
Priority date: 2020-07-20
Filing date: 2020-07-20
Publication date: 2022-01-21
Anticipated expiration: 2040-07-20
Also published as: CN113960306B

Abstract

The invention relates to a reagent for stabilizing acridinium ester marker protein and application thereof, and relates to the field of protein detection. According to the invention, the polysaccharide substance is added into the diluent, so that the stability of the acridine ester marker protein in a solution system can be obviously improved; the technology of the invention can be applied to immunodetection reaction.

Description

Reagent for stabilizing acridinium ester marker protein and application thereof

Technical Field

The present invention relates to the field of protein detection. Specifically, the invention relates to a reagent for stabilizing acridinium ester marker protein and application thereof.

Background

Chemiluminescence is a common method used in clinical testing. The chemiluminescence method is used for immunoassay of an analyte by directly labeling an antigen or an antibody with a chemiluminescent agent. Acridinium ester is an ideal luminescent substrate applied to chemiluminescence, and can detect biological macromolecules such as antigens, antibodies, enzymes and the like after being combined with an immunoassay method. The acridinium ester can be connected with an antibody or antigen of a test object through a covalent bond and then forms a complex with the test object through an immune reaction. Acridinium ester reacts with H under alkaline environment₂O₂Reaction to form instabilityOf (2) dioxyethane which decomposes to form CO₂And an excited state of N-methylacridone and release a photon. The acridinium ester luminescent system has the advantages of high luminous efficiency, few interference factors and the like, however, the acridinium ester marker protein has the defect of poor stability.

The invention is therefore proposed.

Disclosure of Invention

The inventor finds that the pullulan can stabilize the acridine ester labeled protein in the solution, and the stability of the acridine ester labeled protein can be obviously improved by adding the pullulan in the solution.

The invention includes the following embodiments:

in some embodiments, the invention provides the use of pullulan for stabilizing acridinium ester marker proteins, wherein the pullulan is used for preparing a dilution solution of the acridinium ester marker proteins.

In the invention, the pullulan can stabilize the acridine ester marker protein, and the condition that the stability of the acridine ester marker protein in a solution system can be improved by adding the pullulan compared with the condition that no pullulan is added is understood. Stability is generally understood to mean the change in activity detected after a predetermined storage, for example, after storage at a different temperature for a certain period of time, for example, storage at 4 ℃, storage at room temperature, or storage at 37 ℃, compared with the state in which the storage is not performed. The equivalence with respect to different temperature holds can be scaled according to the arrhenius formula, which is well known in the art.

In some embodiments, the final concentration of pullulan in the diluent is 5-40 g/L; in some embodiments, the final concentration of pullulan in the diluent is 10-40 g/L; in some embodiments, the final concentration of pullulan in the diluent is 20-40 g/L; in some embodiments, the final concentration of pullulan in the diluent comprises, for example, 5g/L, for example 10g/L, for example 15g/L, for example 20g/L, for example 25g/L, for example 30g/L, for example 35g/L, for example 40g/L, but is not limited thereto.

In some embodiments, the acridinium ester marker protein comprises an acridinium ester marker antigen or an acridinium ester marker antibody; in some embodiments, the antibody comprises an antibody specific for a tumor associated antigen; in some embodiments, the antibody comprises, e.g., a CA125 antibody, e.g., an AFP antibody, or e.g., a GRP antibody.

In some embodiments, the invention provides a dilution of an acridinium ester marker protein comprising pullulan and a buffer.

In some embodiments, the buffer has a pH of 7.0 to 8.5; in some embodiments, the buffer has a pH of 7.4 to 8.5; in some embodiments, the buffer has a pH of 8.0 to 8.5; in some embodiments, the pH of the buffer includes, for example, 7.0, for example 7.2, for example 7.4, for example 7.5, for example 7.6, for example 7.8, for example 8.0, for example 8.2, for example 8.5, but is not limited thereto.

In some embodiments, the buffer is present in the diluent at a final concentration of 10 to 100 mM; in some embodiments, the buffer is present in the diluent at a final concentration of 10 to 75 mM; in some embodiments, the buffer is present in the diluent at a final concentration of 25 to 100 mM; in some embodiments, the buffer comprises, for example, 10mM, such as 15mM, such as 20mM, such as 25mM, such as 40mM, such as 50mM, such as 60mM, such as 75mM, such as 90mM, such as 100mM, at the final concentration in the dilution, but is not limited thereto.

In some embodiments, the buffer serves to maintain the pH of the solution system in which the acridinium ester marker protein is present; in some embodiments, buffers include, but are not limited to, Tris (Tris hydroxymethyl aminomethane), such as HEPES (4-hydroxyethylpiperazine ethanesulfonic acid), such as MOPS (3- (N-morpholino) propanesulfonic acid), such as PB (phosphate), such as Citrate.

In some embodiments, the diluent further comprises an amino acid; in some embodiments, the amino acid comprises, for example, serine, for example proline, or for example lysine.

In some embodiments, the amino acid is in a final concentration of 5 to 800mmol/L in the diluent; in some embodiments, the amino acid is at a final concentration of 9.5 to 760mmol/L in the diluent; in some embodiments, the amino acid final concentration in the dilution is 190-760 mmol/L; in some embodiments, the amino acid has a final concentration in the dilution of 380-760 mmol/L; in some embodiments, the final concentration of amino acid in the dilution comprises, e.g., 5mmol/L, e.g., 7mmol/L, e.g., 9mmol/L, e.g., 20mmol/L, e.g., 50mmol/L, e.g., 100mmol/L, e.g., 180mmol/L, e.g., 200mmol/L, e.g., 300mmol/L, e.g., 350mmol/L, e.g., 400mmol/L, e.g., 500mmol/L, e.g., 600mmol/L, e.g., 700mmol/L, e.g., 750mmol/L, e.g., 770mmol/L, e.g., 790mmol/L, e.g., 800 mmol/L.

In some embodiments, the diluent further comprises a surfactant; in some embodiments, the surfactant comprises, for example, CHAPS (3- ((3-bile aminopropyl) dimethylamine) -1-propanesulfonic acid), for example Triton X-100 (Triton 100), for example Tween20 (Tween 20), for example Brij35 (laureth), for example PLURONIC-F127 (PLURONIC F127).

In some embodiments, the surfactant is present in the diluent at a final concentration of 0.2 to 2.5 g/L; in some embodiments, the surfactant is present in the diluent at a final concentration of 0.2 to 1.2 g/L; in some embodiments, the surfactant includes, but is not limited to, for example, 0.2g/L, for example 0.3g/L, for example 0.5g/L, for example 0.6g/L, for example 0.8g/L, for example 1g/L, for example 1.2g/L, for example 1.5g/L, for example 1.8g/L, for example 2.0g/L, for example 2.3g/L, for example 2.5g/L, in the diluent at a final concentration.

In some embodiments, the diluent further comprises an inert protein; in some embodiments, the inert protein comprises, for example, gelatin, e.g., hydrolyzed fish gelatin, e.g., BSA, e.g., casein.

In some embodiments, the inert protein is present in the diluent at a final concentration of 1 to 20 g/L; in some embodiments, the inert protein is present in the diluent at a final concentration of 1 to 10 g/L; in some embodiments, the inert protein in the diluent final concentration includes such as 1g/L, such as 3g/L, such as 5g/L, such as 8g/L, such as 10g/L, such as 15g/L, such as 20g/L, but not limited to.

In some embodiments, the diluent further comprises mannitol; in some embodiments, the final concentration of mannitol in the dilution is 25-165 mmol/L; in some embodiments, the final concentration of mannitol in the diluent is 25-110 mmol/L; in some embodiments, the final concentration of mannitol in the diluent comprises, but is not limited to, e.g., 25mmol/L, e.g., 27mmol/L, e.g., 30mmol/L, e.g., 50mmol/L, e.g., 80mmol/L, e.g., 100mmol/L, e.g., 110mmol/L, e.g., 120mmol/L, e.g., 150mmol/L, e.g., 160mmol/L, e.g., 165 mmol/L.

In some embodiments, the acridinium ester marker protein comprises an acridinium ester marker antigen or an acridinium ester marker antibody; in some embodiments, the antibody comprises an antibody specific for a tumor associated antigen; in some embodiments, the antibody is, for example, a CA125 antibody, such as an AFP antibody, or such as a GRP antibody.

In some embodiments, the diluent of the present invention is used in the preparation of a test kit or for immunoassay. The kit of the present invention should be understood in a broad sense, and mainly refers to a tool for carrying reagents related to immunoassay. It will be appreciated by those skilled in the art that the dilutions of the present invention may be used in the immunodetection reaction of antibodies or antigens. In some embodiments, for example, an acridinium ester-labeled CA125 antibody, diluted with a diluent of the present invention, binds to CA125 antigen in the sample and, via acridinium ester labeling, effects detection of CA 125.

In some embodiments, the immunoassay kit of the present invention comprises a diluent of any of the preceding embodiments. The kit of the present invention should be understood in a broad sense, and mainly refers to a tool for carrying reagents related to immunoassay. In some embodiments, the immunoassay kit may further comprise a kit reagent, such as a standard, e.g., a luminescent substrate, e.g., a protein crosslinked to a solid support (e.g., a magnetic bead, e.g., an elisa plate), but is not limited thereto.

The acridinium ester marker protein is a protein marked by acridinium ester; among these, acridinium esters are to be understood in a broad sense and are to be understood as chemiluminescent labels comprising an acridinium structure.

The invention has the following beneficial effects:

according to the invention, the stability of the acridine ester marker protein in a solution system can be obviously improved by adding the pullulan polysaccharide into the diluent. The diluent of the present invention may further contain selectively buffering agent, amino acid, surfactant, inert protein and mannitol to obtain excellent effect. The diluent of the present invention can be applied to immunodetection reaction.

Detailed Description

The present invention will be described in further detail with reference to specific examples. The following examples are provided to illustrate embodiments of the present invention and are not intended to limit the invention. The invention may optionally include embodiments that are not illustrated by the examples.

Methods not described in detail in the detailed description may refer to methods conventional in the art or to manufacturer's instructions; reagents not described in the embodiments are commercially available.

The description herein with respect to the concentration unit "mM" is denoted by "mmol/L", i.e., 10mM is denoted by 10mmol/L, for example, and with respect to the concentration unit "M" is denoted by "mol/L", i.e., 10M is denoted by 10mol/L, for example.

Example 1

Preparation of acridinium ester labeled CA125 antibody reagent

1. Dialyzing 0.5mg of murine monoclonal antibody CA125 into 20mM PBS buffer solution with pH7.2, dialyzing at 4 ℃ for 4 hours, and changing the solution 1 time every 1 hour; adding dissolved 10mM acridine ester (AE for short), and rotating for reaction at room temperature for 15 min; adding 0.1M glycine in 10 times excess of AE, and rotating at room temperature for 30 min; dialyzed against 20mM PBS pH7.2 overnight at 4 ℃; and taking the protein marked by AE out of a centrifuge tube, adding half-volume glycerol, and storing at-20 ℃ in a dark place.

2. Preparing a diluent of the acridinium ester labeled CA125 antibody, wherein the diluent is used for diluting the acridinium ester labeled CA125 antibody obtained in the step 1, and the specific steps are as follows: and adding 5 mu L of the proacridinium ester labeled CA125 antibody into 2.5mL of diluent, and uniformly mixing for later use to obtain a reagent 2.

Preparation of the second, other major reagents

1. Preparation of biotinylated CA125 antibody

Dialyzing 2mg of murine monoclonal antibody CA125 into 20mM pH7.4 PB diluent, dialyzing at 4 ℃ for 2 hours, and changing the solution 1 time every 30 min; adding 10mmol/L activated biotin (biotin for short), and carrying out rotary reaction at room temperature for 1 h; dialyzing again to 20mM pH7.4 PB dilution at 4 deg.C for 2 hr, and changing the solution every 30min for 1 time; taking the protein marked by the biotin out of a centrifuge tube, adding 375 mu L of NBS and 875 mu L of glycerol, mixing uniformly, and storing at-20 ℃ for a long time; diluting by 600 times before use, and mixing uniformly for later use to obtain the reagent 1.

2. Standard article

Taking a CA125 standard substance, and diluting to prepare the following concentration standard substances: 1666.7U/mL, 555.6U/mL, 185.2U/mL, 61.7U/mL, 20.6U/mL, 6.9U/mL.

Third, detection method

Adding a standard solution into a microplate, adding a reagent 1 and a reagent 2, incubating at 37 ℃ for 10min, adding a reagent 3 (0.025% magnetic beads labeled with streptomycin), incubating at 37 ℃ for 10min, and washing with PBS (phosphate buffer solution) for 6 times. To each well 100. mu.L of chemiluminescent substrate A was added and the luminescence signal was measured.

Fourth, index evaluation

Stability: the change rate of the luminescence signal value detected by the reagent 2 after the examination at 37 ℃ and the luminescence signal value detected by the prepared reagent 2 is averaged by the difference rate of the standard substance with the concentration of 6.9U/mL-5000U/mL.

High value sensitivity: the ratio of the luminescence signal value of 5000U/mL standard to the luminescence signal value of 0U/mL standard added.

Activity: the activity is characterized by the luminescence signal value.

Example 2

The following dilutions of each acridinium ester labeled CA125 antibody were prepared, and reagent 2 was prepared according to the method of example 1, and the reagent 2 prepared in situ and reagent 2 subjected to accelerated thermal examination at 37 ℃ for 18 hours were used for the detection, and as a result, the buffer system with pH7.0-8.5 was more suitable for maintaining the stability of the acridinium ester labeled protein, as shown in Table 1.

Diluent 1: 25mM Tris-HCl pH8.5.

Diluent 2: 20mM HEPES pH7.4.

Diluent 3: 20mM MOPS pH7.0.

Diluent 4: 20mM PB, pH6.0.

Diluent 5: 20mM Citrate pH 4.7.

Table 1, test results of example 2

Example 3

The following dilutions of various acridinium ester labeled CA125 antibodies were prepared to prepare reagent 2, and the reagent 2 prepared in situ and reagent 2 for accelerated thermal examination at 37 ℃ for 18h were used for detection, and the results are shown in Table 2, wherein a 10-100mM buffer system can maintain the stability of acridinium ester labeled protein.

Diluent 6: 10mM HEPES pH8.0.

Diluent 7: 20mM HEPES pH8.0.

Diluent 8: 50mM HEPES pH8.0.

Diluent 9: 75mM HEPES pH8.0.

10, diluent: 25mM Tris-HCl pH8.5.

Diluent 11: 50mM Tris-HCl pH8.5.

Diluent 12: 100mM Tris-HCl pH8.5.

Table 2 and example 3 test results

Example 4

The following dilutions of various acridinium ester labeled CA125 antibodies are prepared to prepare a reagent 2, the reagent 2 prepared in situ and the reagent 2 for accelerated thermal examination at 37 ℃ for 18h are respectively used for detection, and the result is shown in Table 3, and the pullulan polysaccharide can be used for improving the stability of the acridinium ester labeled protein.

Diluent 13: 20mM HEPES pH8.0+20g/L sucrose.

Diluent 14: 20mM HEPES pH8.0+20g/L trehalose.

Diluent 15: 20mM HEPES pH8.0+20g/L mannitol.

Diluent 16: 20mM HEPES pH8.0+20g/L pullulan.

Diluent 17: 20mM HEPES pH8.0+20g/L dextran.

Diluent 18: 20mM HEPES pH8.0+20g/L glycerol.

Diluent 19: 20mM HEPES pH8.0.

Table 3, example 4 test results

Example 5

The following dilutions of various acridinium ester labeled CA125 antibodies are respectively prepared to prepare a reagent 2, the reagent 2 prepared in situ and the reagent 2 for accelerated thermal examination at 37 ℃ for 18h are respectively used for detection, and the result is shown in Table 4, and the pullulan polysaccharide can be used for improving the stability of the acridinium ester labeled protein within the concentration range of 5g/L-40 g/L.

20, diluent 20: 20mM HEPES pH8.0+5g/L pullulan.

Diluent 21: 20mM HEPES pH8.0+10g/L pullulan.

Diluent 22: 20mM HEPES pH8.0+15g/L pullulan.

Diluent 23: 20mM HEPES pH8.0+20g/L pullulan.

Diluent 24: 20mM HEPES pH8.0+30g/L pullulan.

Diluent 25: 20mM HEPES pH8.0+40g/L pullulan.

Diluent 26: 20mM HEPES pH8.0.

Table 4 and example 5 show the results of detection

Example 6

The following dilutions of various acridinium ester labeled CA125 antibodies are prepared to prepare a reagent 2, the reagent 2 prepared in situ and the reagent 2 for accelerated thermal examination at 37 ℃ for 18h are respectively used for detection, and the results are shown in Table 5, and a plurality of surfactants can improve the stability of the acridinium ester labeled protein.

Diluent 27: 25mM Tris-HCl pH8.5+10g/L pullulan +1g/L CHAPS.

Diluent 28: 25mM Tris-Hcl pH8.5+10g/L pullulan +1.06g/L Triton X-100.

Diluent 29: 25mM Tris-HCl pH8.5+10g/L pullulan polysaccharide +1.1g/L Tween 20.

30, dilution liquid: 25mM Tris-HCl pH8.5+10g/L pullulan +0.95g/L Brij 35.

Diluent 31: 25mM Tris-HCl pH8.5+10g/L pullulan polysaccharide +1g/L PLURONIC-F127.

Diluent 32: 25mM Tris-HCl pH8.5+10g/L pullulan polysaccharide +1.32g/L CTAB.

Diluent 33: 25mM Tris-HCl pH8.5+10g/L pullulan.

Table 5 and example 6 detection results

Example 7

The following dilutions of various acridinium ester labeled CA125 antibodies were prepared to prepare reagents 2, and the prepared reagents 2 and the reagents 2 for accelerated thermal examination at 37 ℃ for 18h were used for detection, and the results are shown in Table 6, wherein the surfactant can improve the stability of the acridinium ester labeled protein in the concentration range of 0.2g/L-2.5 g/L.

Diluent 34: 20mM HEPES pH8.0+30g/L pullulan polysaccharide +2.2g/L Tween 20.

Diluent 35: 20mM HEPES pH8.0+30g/L pullulan polysaccharide +1.1g/L Tween 20.

Diluent 36: 20mM HEPES pH8.0+30g/L pullulan polysaccharide +0.55g/L Tween 20.

Diluent 37: 20mM HEPES pH8.0+30g/L pullulan polysaccharide +0.22g/L Tween 20.

Table 6 and example 7 detection results

Example 8

The following dilutions of various acridinium ester labeled CA125 antibodies are prepared to prepare a reagent 2, the reagent 2 prepared in situ and the reagent 2 for accelerated thermal examination at 37 ℃ for 18h or 3 days are respectively used for detection, and the results are shown in Table 7, wherein the stability of the acridinium ester labeled protein improved by amino acid is better.

Diluent 38: 20mM HEPES pH8.0+30g/L pullulan polysaccharide +1.1g/L Tween 20.

Dilution liquid 39: 20mM HEPES pH8.0+30g/L pullulan +1.1g/L Tween20+95mmol/L (about 10g/L) serine.

Diluent 40: 20mM HEPES pH8.0+30g/L pullulan +1.1g/L Tween20+55mmol/L (about 10g/L) lysine.

Diluent 41: 20mM HEPES pH8.0+30g/L pullulan +1.1g/L Tween20+87mmol/L (about 10g/L) proline.

Diluent 42: 20mM HEPES pH8.0+30g/L pullulan polysaccharide +1.1g/L Tween20 +10g/L gelatin.

Diluent 43: 20mM HEPES pH8.0+30g/L pullulan polysaccharide +1.1g/L Tween20 +10g/L hydrolyzed fish gelatin.

Diluent 44: 20mM HEPES pH8.0+30g/L pullulan polysaccharide +1.1g/L Tween20 +10g/L BSA.

Diluent 45: 20mM HEPES pH8.0+30g/L pullulan polysaccharide +1.1g/L Tween20 +10g/L casein.

Table 7 and example 8 show the results of detection

Example 9

The following dilutions of various acridinium ester-labeled CA125 antibodies were prepared to prepare reagents 2, and the prepared reagent 2 and the reagent 2 for accelerated thermal examination at 37 ℃ for 18h or 3 days were used for detection, respectively, with the results shown in Table 8.

Diluent 46: 20mM HEPES pH8.0+30g/L pullulan polysaccharide +1.1g/L Tween20 +10g/L casein.

Diluent 47: 20mM HEPES pH8.0+30g/L pullulan +1.1g/L Tween20 +10g/L casein +55mmol/L (about 10g/L) lysine.

Diluent 48: 20mM HEPES pH8.0+30g/L pullulan +1.1g/L Tween20 +10g/L casein +10g/L BSA.

Diluent 49: 20mM HEPES pH8.0+30g/L pullulan +1.1g/L Tween20 +10g/L casein +95mmol/L (about 10g/L) serine.

50 parts of diluent: 20mM HEPES pH8.0+30g/L pullulan +1.1g/L Tween20 +10g/L casein +10g/L hydrolyzed fish gelatin.

Diluent 51: 20mM HEPES pH8.0+30g/L pullulan +1.1g/L Tween20 +10g/L casein +87mmol/L (about 10g/L) proline.

Table 8 and example 9 show the results of detection

Diluent liquid

46

47

48

49

50

51

Stability of 37-18 h

-15.81％

-1.79％

-18.57％

-6.31％

-19.98％

-8.31％

Stability of 37-3 days

-35.87％

-20.54％

-36.87％

-15.66％

-33.74％

-21.65％

Example 10

The following dilutions of various acridinium ester-labeled CA125 antibodies were prepared to prepare reagents 2, and the freshly prepared reagent 2 and the reagent 2 for accelerated thermal examination at 37 ℃ for 3 days were used for detection, respectively, and the results are shown in Table 9, where casein was used at a concentration of 1g/L-20 g/L.

Diluent 52: 20mM HEPES pH8.0+30g/L pullulan +1.1g/L Tween20+95mmol/L (about 10g/L) serine.

Diluent 53: 20mM HEPES pH8.0+30g/L pullulan +1.1g/L Tween20+95mmol/L (about 10g/L) serine +20g/L casein.

Diluent 54: 20mM HEPES pH8.0+30g/L pullulan +1.1g/L Tween20+95mmol/L (about 10g/L) serine +10g/L casein.

Diluent 55: 20mM HEPES pH8.0+30g/L pullulan +1.1g/L Tween20+95mmol/L (about 10g/L) serine +1g/L casein.

Table 9 and example 10 show the results of detection

Diluent liquid	52	53	54	55
					Stability of 37-3 days	-25.06％	-19.57％	-15.85％	-17.79％

Example 11

The following dilutions of various acridinium ester-labeled CA125 antibodies were prepared to prepare reagents 2, and the freshly prepared reagent 2 and the reagent 2 for accelerated thermal examination at 37 ℃ for 7 days were used for detection, respectively, and the amino acids were used in the range of 9.5mmol/L to 760mmol/L, as shown in Table 10.

Diluent 56: 20mM HEPES pH8.0+30g/L pullulan +1.1g/L Tween20 +10g/L casein +9.5mmol/L serine.

Diluent 57: 20mM HEPES pH8.0+30g/L pullulan +1.1g/L Tween20 +10g/L casein +47.5mmol/L serine.

Diluent 58: 20mM HEPES pH8.0+30g/L pullulan +1.1g/L Tween20 +10g/L casein +95mmol/L serine.

Diluent 59: 20mM HEPES pH8.0+30g/L pullulan +1.1g/L Tween20 +10g/L casein +190mmol/L serine.

Dilution liquid 60: 20mM HEPES pH8.0+30g/L pullulan +1.1g/L Tween20 +10g/L casein +285mmol/L serine.

Diluent 61: 20mM HEPES pH8.0+30g/L pullulan +1.1g/L Tween20 +10g/L casein +380mmol/L serine.

Diluent 62: 20mM HEPES pH8.0+30g/L pullulan +1.1g/L Tween20 +10g/L casein +475mmol/L serine.

Diluent 63: 20mM HEPES pH8.0+30g/L pullulan +1.1g/L Tween20 +10g/L casein +570mmol/L serine.

Diluent 64: 20mM HEPES pH8.0+30g/L pullulan +1.1g/L Tween20 +10g/L casein +665mmol/L serine.

Diluent 65: 20mM HEPES pH8.0+30g/L pullulan +1.1g/L Tween20 +10g/L casein +760mmol/L serine.

Table 10 and example 11 show the results of detection

Diluent liquid	56	57	58	59	60
						Stability of 37-7 days	-33.29％	-32.69％	-32.97％	-28.30％	-29.06％
Diluent liquid	61	62	63	64	65
						Stability of 37-7 days	-23.09％	-15.27％	-17.08％	-17.29％	-19.92％

Example 12

The following dilutions of various acridinium ester-labeled CA125 antibodies were prepared to prepare reagents 2, and the test results are shown in Table 11, in which the reagent 2 prepared in situ and the reagent 2 subjected to accelerated thermal test at 37 ℃ for 7 days were used for the tests.

Diluent 66: 20mM HEPES pH8.0+30g/L pullulan +1.1g/L Tween20 +475mmol/L serine +109mmol/L mannitol +10g/L casein.

Diluent 67: 20mM HEPES pH8.0+1.1g/L Tween20 +475mmol/L serine +109mmol/L mannitol +10g/L casein.

Diluent 68: 20mM HEPES pH8.0+30g/L pullulan +1.1g/L Tween20 +109mmol/L mannitol +10g/L casein.

Diluent 69: 20mM HEPES pH8.0+30g/L pullulan +475mmol/L serine +109mmol/L mannitol +10g/L casein.

Diluent 70: 20mM HEPES pH8.0+30g/L pullulan +1.1g/L Tween20 +475mmol/L serine +109mmol/L mannitol.

Dilution liquid 71: 20mM HEPES pH8.0+30g/L pullulan +1.1g/L Tween20 +475mmol/L serine +10g/L casein.

Diluent 72: 20mM HEPES pH8.0+30g/L pullulan +1.1g/L Tween20 +475mmol/L serine +27.4mmol/L mannitol +10g/L casein.

Dilution solution 73: 20mM HEPES pH8.0+30g/L pullulan +1.1g/L Tween20 +475mmol/L serine +165mmol/L mannitol +10g/L casein.

Table 11 and example 12 show the results of measurement

Example 13

The acridinium ester-labeled AFP antibody was diluted with the diluent 66, and the AFP antibody was used in place of the CA125 antibody, and other methods were described above, in which the ready-prepared reagent 2 and the reagent 2 for accelerated thermal examination at 37 ℃ for 7 days were used for the detection, respectively, and the results are shown in Table 12.

Table 12 and example 13 detection results

Example 14

The acridinium ester-labeled GRP antibody was diluted with the diluent 66, and the GRP antibody was used in place of the CA125 antibody, and other methods were performed as described above, and the test results are shown in Table 13, using the reagent 2 prepared on site and the reagent 2 subjected to accelerated thermal testing at 37 ℃ for 7 days.

Table 13 and example 14 show the results of detection

The above-mentioned embodiments are intended to illustrate the objects, technical solutions and advantages of the present invention in further detail, and it should be understood that the above-mentioned embodiments are only exemplary embodiments of the present invention and are not intended to limit the present invention, and any modifications, equivalents, improvements and the like made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. The dilution of acridinium ester marker protein is characterized by comprising pullulan and a buffer.

2. The diluent of claim 1, wherein the pullulan is present in the diluent at a final concentration of 5-40 g/L;

optionally, the final concentration of the pullulan in the diluent is 10-40 g/L;

optionally, the final concentration of the pullulan in the diluent is 20-40 g/L;

optionally, the buffer has a pH of 7.0-8.5;

optionally, the buffer has a pH of 7.4 to 8.5;

optionally, the buffer has a pH of 8.0-8.5;

optionally, the buffer is present in the dilution at a final concentration of 10-100 mM;

optionally, the buffer is present in the dilution at a final concentration of 10-75 mM;

optionally, the buffer is present in the dilution at a final concentration of 25-100 mM;

optionally, the buffer comprises Tris, HEPES, MOPS, PB or Citrate.

3. The diluent of claim 1, further comprising an amino acid comprising serine, proline, or lysine;

optionally, the final concentration of the amino acid in the diluent is 5-800 mmol/L;

optionally, the final concentration of the amino acid in the diluent is 9.5-760 mmol/L;

optionally, the final concentration of the amino acid in the diluent is 190-760 mmol/L;

optionally, the final concentration of the amino acid in the dilution is 380-760 mmol/L.

4. The diluent of claim 1, further comprising a surfactant; the surfactant comprises CHAPS, Triton X-100, Tween20, Brij35 or PLURONIC-F127;

optionally, the final concentration of the surfactant in the diluent is 0.2-2.5 g/L;

optionally, the surfactant has a final concentration of 0.2-1.2g/L in the diluent.

5. The diluent of claim 1, further comprising an inert protein; the inert protein comprises gelatin, hydrolyzed fish gelatin, BSA or casein;

optionally; the final concentration of the inert protein in the diluent is 1-20 g/L;

optionally; the final concentration of the inert protein in the diluent is 1-10 g/L.

6. The diluent of claim 1, further comprising mannitol;

optionally, the final concentration of the mannitol in the diluent is 25-165 mmol/L;

optionally, the final concentration of the mannitol in the diluent is 25-110 mmol/L.

7. The dilution according to claim 1, wherein the acridinium ester marker protein comprises an acridinium ester marker antigen or an acridinium ester marker antibody;

optionally, the antibody comprises an antibody specific for a tumor associated antigen;

optionally, the antibody comprises a CA125 antibody, an AFP antibody, or a GRP antibody.

8. The application of the pullulan polysaccharide in stabilizing the acridinium ester marker protein is characterized in that the pullulan polysaccharide is used for preparing a diluent of the acridinium ester marker protein.

9. The use according to claim 1, wherein the final concentration of pullulan in the dilution is 5-40 g/L;

optionally, the final concentration of pullulan in the diluent is 10-40 g/L;

optionally, the final concentration of pullulan in the diluent is 20-40 g/L;

optionally, the acridinium ester marker protein comprises an acridinium ester marker antigen or an acridinium ester marker antibody;

10. Use of a diluent according to any one of claims 1 to 9 in the preparation of an immunoassay kit or for immunoassay.

11. An immunoassay kit comprising a diluent according to any one of claims 1 to 9.