CN113960306B

CN113960306B - Reagent for stabilizing acridinium ester marker protein and application thereof

Info

Publication number: CN113960306B
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: 2023-04-28
Anticipated expiration: 2040-07-20
Also published as: CN113960306A

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 acridinium ester marker protein in a solution system can be obviously improved; the techniques of the present invention are applicable to immunodetection reactions.

Description

Reagent for stabilizing acridinium ester marker protein and application thereof

Technical Field

The invention relates to the field of protein detection. In particular, the invention relates to reagents and uses for stabilizing acridinium ester-tagged proteins.

Background

Chemiluminescence is a common method used in clinical assays. Chemiluminescence is a method of performing an immunoassay on an analyte by directly labeling an antigen or antibody with a chemiluminescent agent. Acridinium ester is an ideal luminous 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 may be linked to an antibody or antigen of the analyte by a covalent bond and then form a complex with the analyte by an immune reaction. Acridinium esters with H in alkaline conditions ₂ O ₂ The reaction produces unstable dioxyethane which decomposes to CO ₂ And N-methylacridone in the excited state and releases photons. The acridinium ester luminescent system has the advantages of high luminous efficiency, few interference factors and the like, however, the disadvantage of using the acridinium ester marker protein is poor stability.

For this purpose, the present invention is proposed.

Disclosure of Invention

The inventor finds that the pullulan can stabilize the acridinium ester marker protein in a solution through a great deal of researches, and the stability of the acridinium ester marker protein can be obviously improved through adding the pullulan into the solution.

The invention includes the following embodiments:

in some embodiments, the invention provides the use of pullulan to stabilize acridinium ester-tagged proteins, wherein the pullulan is used to prepare dilutions of acridinium ester-tagged proteins.

In the invention, the pullulan can stabilize the acridinium ester marker protein, and the addition of the pullulan can improve the stability of the acridinium ester marker protein in a solution system compared with the addition of no pullulan. Stability is generally understood as the change in activity detected after a given storage, for example after a period of storage at a different temperature, for example 4 ℃, room temperature, 37 ℃ compared to the non-stored state. The comparability for different temperature storages may be scaled according to the Arrhenius formula known in the art.

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

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

In some embodiments, the invention provides dilutions of acridinium ester-tagged proteins, including pullulan and a buffer.

In some embodiments, the pH of the buffer is 7.0-8.5; in some embodiments, the pH of the buffer is 7.4-8.5; in some embodiments, the pH of the buffer is 8.0-8.5; in some embodiments, the pH of the buffer includes, but is not limited to, e.g., 7.0, e.g., 7.2, e.g., 7.4, e.g., 7.5, e.g., 7.6, e.g., 7.8, e.g., 8.0, e.g., 8.2, e.g., 8.5.

In some embodiments, the final concentration of buffer in the diluent is 10-100mM; in some embodiments, the final concentration of buffer in the diluent is 10-75mM; in some embodiments, the final concentration of buffer in the diluent is 25-100mM; in some embodiments, the final concentration of buffer in the diluent includes, but is not limited to, e.g., 10mM, e.g., 15mM, e.g., 20mM, e.g., 25mM, e.g., 40mM, e.g., 50mM, e.g., 60mM, e.g., 75mM, e.g., 90mM, e.g., 100mM.

In some embodiments, the buffer serves to maintain the pH of the solution system in which the acridinium ester-tagged protein is present; in some embodiments, the buffer includes, for example, tris (Tris), for example, HEPES (4-hydroxyethyl piperazine ethane sulfonic acid), for example, MOPS (3- (N-morpholinyl) propane sulfonic acid), for example, PB (phosphate), for example, citrate (Citrate), but is not limited thereto.

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

In some embodiments, the diluent further comprises a surfactant; in some embodiments, the surfactant comprises, for example, CHAPS (3- ((3-cholesteryl) dimethylamine) -1-propanesulfonic acid), for example, triton X-100 (Triton 100), for example, tween20 (Tween 20), for example, brij35 (laureth), for example, PLURONIC-F127 (pramipexole F127).

In some embodiments, the final concentration of surfactant in the diluent is 0.2 to 2.5g/L; in some embodiments, the surfactant is present in the diluent at a final concentration of 0.2 to 1.2g/L; in some embodiments, the final concentration of the surfactant in the diluent 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 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 1-20g/L; in some embodiments, the inert protein is present in the diluent at a final concentration of 1-10g/L; in some embodiments, the final concentration of the inert protein in the diluent includes, but is not limited to, for example, 1g/L, for example, 3g/L, for example, 5g/L, for example, 8g/L, for example, 10g/L, for example, 15g/L, for example, 20g/L.

In some embodiments, the diluent further comprises mannitol; in some embodiments, the final concentration of mannitol in the diluent is 25-165mmol/L; in some embodiments, the final concentration of mannitol in the diluent is 25-110mmol/L; in some embodiments, the final concentration of mannitol in the diluent includes, 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 acridine ester-labeled protein comprises an acridine ester-labeled antigen or an acridine ester-labeled 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 dilutions of the present invention are used in the preparation of a detection kit or for immunodetection. The kit according to the invention is to be understood in a broad sense and mainly refers to a tool carrying reagents relevant for the immunological detection. It will be appreciated by those skilled in the art that the dilutions of the present invention may be used in an immunoassay for antibodies or antigens. In some embodiments, for example, the acridinium ester-labeled CA125 antibodies, after dilution with a diluent of the invention, bind to the CA125 antigen in the sample and effect detection of CA125 by the acridinium ester-labeled.

In some embodiments, an immunoassay kit of the present invention comprises a diluent of any one of the preceding embodiments. The kit according to the invention is to be understood in a broad sense and mainly refers to a tool carrying reagents relevant for the immunological detection. In some embodiments, the immunoassay kit may further include a number of kit reagents, such as, but not limited to, standards, such as luminescent substrates, such as proteins cross-linked to solid phase carriers (e.g., magnetic beads, such as an elisa plate).

The acridinium ester marker protein of the invention refers to acridinium ester-labeled proteins; among these, acridinium esters are to be understood in a broad sense as chemiluminescent labels comprising an acridine structure.

The invention has the following beneficial effects:

according to the invention, the stability of the acridinium ester marker protein in a solution system can be remarkably improved by adding the pullulan into the diluent. The diluent of the invention can be further added with buffer, amino acid, surfactant, inert protein and mannitol selectively, thus obtaining better effect. The diluent of the invention can be applied to an immunoassay reaction.

Detailed Description

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

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

The description of the concentration unit "mM" in the present invention is expressed as "mmol/L", that is, for example, 10mM is expressed as 10mmol/L, and the description of the concentration unit "M" is expressed as "mol/L", that is, for example, 10M is expressed as 10mol/L.

Example 1

1. Preparation of acridinium ester-labeled CA125 antibody reagent

1. 0.5mg of murine monoclonal antibody CA125 was dialyzed into 20mM PH7.2 PBS buffer, dialyzed for 4 hours at 4℃with 1 change every 1 hour; adding dissolved 10mM acridine ester (AE for short), and performing rotary reaction at room temperature for 15min; adding glycine with 10 times of excess amount of 0.1M of AE, and performing rotary reaction at room temperature for 30min; re-dialyzed into 20mM PH7.2 PBS buffer, dialyzed overnight at 4deg.C; and taking out the protein marked by AE into a centrifuge tube, and adding half-volume glycerol for light-shielding at-20 ℃.

2. Preparing a diluent of the acridine ester-labeled CA125 antibody, which is used for diluting the acridine ester-labeled CA125 antibody obtained in the step 1, and specifically comprises the following steps: adding 5 mu L of the original acridinium ester marked CA125 antibody into 2.5mL of diluent, and uniformly mixing for later use to obtain the reagent 2.

2. Preparation of other principal reagents

1. Preparation of CA125 antibodies

Dialyzing 2mg of mouse monoclonal antibody CA125 into 20mM PH7.4PB diluent, dialyzing at 4deg.C for 2 hours, and changing liquid 1 time every 30min; adding 10mmol/L activated biotin (biotin for short), and performing rotary reaction at room temperature for 1h; re-dialyzing to 20mM PH7.4PB dilution, dialyzing at 4deg.C for 2 hr, and changing liquid 1 time every 30min; taking out the protein marked by the biotin into a centrifuge tube, adding 375 mu L of NBS and 875 mu L of glycerol, uniformly mixing, and preserving at-20 ℃ for a long time; diluting 600 times before using, and mixing uniformly to obtain the reagent 1.

2. Standard substance

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

3. Detection method

Adding standard solution into the microplate, adding reagent 1 and reagent 2, incubating at 37deg.C for 10min, adding reagent 3 (0.025% magnetic beads labeled with affinity streptomycin), incubating at 37deg.C for 10min, and washing with PBS washing solution for 6 times. To each well, 100. Mu.L of chemiluminescent substrate A was added and the luminescent signal value was measured.

4. Index evaluation

Stability: the change rate of the luminescence signal value of the reagent 2 for detection after the examination at 37 ℃ and the luminescence signal value of the reagent 2 for detection which is matched at present is averaged by the difference rate of the concentration standard substance of 6.9U/mL-5000U/mL.

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

Activity: the activity was characterized by luminescence signal values.

Example 2

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

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

Dilution 2:20mM HEPES pH7.4.

Dilution 3:20mM MOPS pH7.0.

Dilution 4:20mM PB pH6.0.

Dilution 5:20mM Citrate pH4.7.

Table 1, test results of example 2

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Example 3

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

Dilution 6:10mM HEPES pH8.0.

Dilution 7:20mM HEPES pH8.0.

Dilution 8:50mM HEPES pH8.0.

Dilution 9:75mM HEPES pH8.0.

Dilution liquid 10:25mM Tris-HCl pH8.5.

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

Dilution liquid 12:100mM Tris-HCl pH8.5.

Table 2, test results of example 3

Example 4

The following dilutions of various acridinium ester-labeled CA125 antibodies were prepared to prepare reagent 2, and the now-prepared reagent 2 and the reagent 2 subjected to accelerated thermal examination at 37 ℃ for 18 hours were used for detection, and the results are shown in table 3, and pullulan polysaccharide was used to improve the stability of acridinium ester-labeled protein.

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

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

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

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

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

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

Dilution 19:20mM HEPES pH8.0.

Table 3, test results of example 4

Example 5

The following dilutions of various acridinium ester-labeled CA125 antibodies were prepared to prepare reagent 2, and the now-prepared reagent 2 and the reagent 2 subjected to accelerated thermal examination at 37℃for 18 hours were used for detection, and the results are shown in Table 4, in which pullulan was used to improve the stability of acridinium ester-labeled protein in the concentration range of 5g/L to 40g/L.

Dilution liquid 20:20mM HEPES pH8.0+5g/L pullulan.

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

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

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

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

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

Dilution 26:20mM HEPES pH8.0.

Table 4, test results of example 5

Example 6

The following dilutions of various acridinium ester-labeled CA125 antibodies were prepared to prepare reagent 2, and the now-prepared reagent 2 and the reagent 2 subjected to accelerated thermal assessment at 37 ℃ for 18 hours were used for detection, and the results are shown in table 5, in which various surfactants can improve the stability of acridinium ester-labeled proteins.

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

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

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

Dilution liquid 30:25mM Tris-HCl pH8.5+10g/L pullulan+0.95 g/L Brij35.

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

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

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

Table 5, test results of example 6

Example 7

The following dilutions of various acridinium ester-labeled CA125 antibodies were prepared to prepare reagent 2, and the now-prepared reagent 2 and the reagent 2 subjected to accelerated thermal examination at 37℃for 18 hours were used for detection, and the results are shown in Table 6, in which the surfactant was capable of improving the stability of acridinium ester-labeled protein in the concentration range of 0.2g/L to 2.5g/L.

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

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

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

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

Table 6, test results of example 7

Example 8

The following dilutions of various acridinium ester-labeled CA125 antibodies were prepared to prepare reagent 2, and the now-prepared reagent 2 and reagent 2 subjected to accelerated thermal examination at 37 ℃ for 18 hours or 3 days were used for detection, respectively, and the results are shown in table 7, and the stability of the acridinium ester-labeled protein was improved by the amino acid.

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

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

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

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

Dilution 42:20mM HEPES pH8.0+30g/L pullulan+1.1 g/L Tween 20+10g/L gelatin.

Dilution 43:20mM HEPES pH8.0+30g/L pullulan+1.1 g/L Tween 20+10g/L hydrolyzed fish gelatin.

Dilution 44:20mM HEPES pH8.0+30g/L pullulan+1.1 g/L Tween 20+10g/L BSA.

Dilution 45:20mM HEPES pH8.0+30g/L pullulan+1.1 g/L Tween 20+10g/L casein.

Table 7, test results of example 8

Example 9

The following dilutions of various acridinium ester-labeled CA125 antibodies were prepared, and reagent 2 was used for detection at 37 ℃ for 18 hours or 3 days in the presence of reagent 2 and reagent 2 for accelerated thermal testing, respectively, and the results are shown in table 8.

Dilution 46:20mM HEPES pH8.0+30g/L pullulan+1.1 g/L Tween 20+10g/L casein.

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

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

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

Dilution 50:20mM HEPES pH8.0+30g/L pullulan+1.1 g/L Tween 20+10g/L casein+10 g/L hydrolyzed fish gelatin.

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

Table 8, test results of example 9

Dilution liquid

46

47

48

49

50

51

Stability for 37-18 h

-15.81％

-1.79％

-18.57％

-6.31％

-19.98％

-8.31％

Stability for 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 reagent 2, and the now-prepared reagent 2 and the reagent 2 subjected to accelerated thermal testing at 37℃for 3 days were used for detection, and the results are shown in Table 9, in which casein was used in the range of 1g/L to 20g/L.

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

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

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

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

Table 9, test results of example 10

Dilution liquid	52	53	54	55
					Stability for 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 reagent 2, and the now-prepared reagent 2 and the reagent 2 subjected to accelerated thermal test at 37℃for 7 days were used for detection, and the results are shown in Table 10, in which amino acids were used in the range of 9.5mmol/L to 760mmol/L.

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

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

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

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

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

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

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

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

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

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

Table 10, test results of example 11

Dilution liquid	56	57	58	59	60
						Stability for 37-7 days	-33.29％	-32.69％	-32.97％	-28.30％	-29.06％
Dilution liquid	61	62	63	64	65
						Stability for 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, and the prepared reagent 2 and the reagent 2 subjected to accelerated thermal test at 37 ℃ for 7 days were used for detection, and the results are shown in table 11.

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

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

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

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

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

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

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

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

Table 11, test results of example 12

Example 13

The AFP antibody was diluted with the diluent 66, the CA125 antibody was replaced with the AFP antibody, and the other methods were used for detection by referring to the above, using the now-prepared reagent 2 and the reagent 2 subjected to accelerated thermal examination at 37 ℃ for 7 days, respectively, and the results are shown in table 12.

Table 12, test results of example 13

Example 14

The acridinium ester-labeled GRP antibody was diluted with the diluent 66, the CA125 antibody was replaced with the GRP antibody, and the other methods were used for detection by referring to the above, using the now-prepared reagent 2 and the reagent 2 subjected to accelerated thermal examination at 37 ℃ for 7 days, respectively, and the results are shown in table 13.

Table 13, test results of example 14

The foregoing description of the embodiments has been provided for the purpose of illustrating the general principles of the invention, and is not meant to limit the invention thereto, but to limit the invention thereto, and any modifications, equivalents, improvements and equivalents thereof may be made without departing from the spirit and principles of the invention.

Claims

1. A dilution of an acridinium ester-labeled protein comprising an acridinium ester-labeled antigen or an acridinium ester-labeled antibody, comprising pullulan and a buffer.

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

3. The diluent of claim 2, wherein the final concentration of pullulan in the diluent is 10-40g/L.

4. A diluent according to claim 3, wherein the final concentration of pullulan in the diluent is 20-40g/L.

5. The diluent of claim 1 or 2, wherein the pH of the buffer is 7.0-8.5.

6. The diluent of claim 5 wherein the buffer has a pH of 7.4 to 8.5.

7. The diluent of claim 6 wherein the buffer has a pH of 8.0 to 8.5.

8. The diluent of claim 1 or 2, wherein the buffer is present in the diluent at a final concentration of 10-100mM.

9. The diluent of claim 8, wherein the buffer is present in the diluent at a final concentration of 10-75mM.

10. The diluent of claim 8, wherein the buffer is present in the diluent at a final concentration of 25-100mM.

11. The diluent of claim 1, wherein the buffer comprises Tris, HEPES, MOPS, PB or Citrate.

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

13. The diluent of claim 12, wherein the amino acid is present in the diluent at a final concentration of 5-800mmol/L.

14. The diluent of claim 13, wherein the amino acid is present in the diluent at a final concentration of 9.5 to 760mmol/L.

15. The diluent of claim 14, wherein the final concentration of the amino acid in the diluent is 190-760mmol/L.

16. The diluent of claim 15, wherein the amino acid is present in the diluent at a final concentration of 380-760mmol/L.

17. The diluent of claim 1, further comprising a surfactant; the surfactant comprises CHAPS, triton X-100, tween20, brij35 or PLURONIC-F127.

18. The diluent of claim 17, wherein the surfactant is present in the diluent at a final concentration of 0.2-2.5 g/L.

19. The diluent of claim 18, wherein the surfactant is present in the diluent at a final concentration of 0.2-1.2g/L.

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

21. The diluent of claim 20, wherein the inert protein is present in the diluent at a final concentration of 1-20g/L.

22. The diluent of claim 21, wherein the inert protein is present in the diluent at a final concentration of 1-10g/L.

23. The diluent of claim 1, further comprising mannitol.

24. The diluent of claim 23, wherein said mannitol is present at a final concentration of 25-165mmol/L in said diluent.

25. The diluent of claim 24, wherein said mannitol is present at a final concentration of 25-110mmol/L in said diluent.

26. The diluent of claim 1, wherein the antibody comprises an antibody specific for a tumor associated antigen.

27. The diluent of claim 26 wherein the antibody comprises a CA125 antibody, an AFP antibody, or a GRP antibody.

28. Use of pullulan for stabilizing an acridine ester-tagged protein, wherein the pullulan is used to prepare a dilution of an acridine ester-tagged protein comprising an acridine ester-tagged antigen or an acridine ester-tagged antibody.

29. The use according to claim 28, wherein the final concentration of pullulan in the diluent is 5-40g/L.

30. The use according to claim 29, wherein the final concentration of pullulan in the diluent is 10-40g/L.

31. The use according to claim 30, wherein the final concentration of pullulan in the diluent is 20-40g/L.

32. The use according to any one of claims 28 to 31, wherein the antibody comprises an antibody specific for a tumour associated antigen.

33. The use of claim 32, wherein the antibody comprises a CA125 antibody, an AFP antibody, or a GRP antibody.

34. Use of the diluent according to any one of claims 1-27 for the preparation of an immunoassay kit or for an immunoassay.

35. An immunoassay kit comprising the diluent of any one of claims 1-27.