WO2019024311A1

WO2019024311A1 - Solution and method for preserving fluorescent antibody

Info

Publication number: WO2019024311A1
Application number: PCT/CN2017/110455
Authority: WO
Inventors: Guoxin Wang; Tao LIAO; Minwen CHEN; Meijie TANG; Su Zhao
Original assignee: Wwhs Biotech, Inc; Nirmidas Biotech, Inc.
Priority date: 2017-08-02
Filing date: 2017-11-10
Publication date: 2019-02-07
Also published as: CN107602695A; CN107602695B

Abstract

Provided are a solution and method for preserving a fluorescent antibody, a kit including the solution, use of the solution and the kit in an immunoassay. The solution includes gelatin, cysteine, gentamicin and sodium citrate.

Description

SOLUTION AND METHOD FOR PRESERVING FLUORESCENT ANTIBODY, KIT INCLUDING THE SOLUTION, USE OF THE SOLUTION AND KIT

CROSS-REFERENCE TO RELATED APPLICATION

This application claims a priority to and benefits of Chinese Patent Application Serial No. 201710653048.6, filed with the State Intellectual Property Office of P. R. China on August 2, 2017, the entire content of which is incorporated herein by reference.

FIELD

The present disclosure relates to the field of biotechnology, and more particularly to a solution and method for preserving a fluorescent antibody, a kit including the solution, use of the solution and the kit.

BACKGROUND

Fluorescein, a synthetic organic compound, has been applied widely in various fields. For example, in the fluorescent antibody technology, the fluorescein as a tracer is combined with a monoclonal antibody or polyclonal antibody to form a fluorescein-labeled antibody to detect a corresponding antigen. Such detection has become an important technology.

However, fluorescent antibody reagents need to be preserved at low temperature and away from light, which results in high requirements to transport and storage conditions, thereby increasing costs consumed by the reagents. These shortcomings adversely affect commercialization of the fluorescent antibody reagents.

Therefore, it still needs to develop a solution for preserving the fluorescent antibody, which can effectively maintain bioactivity and fluorescence stability of the fluorescent antibody, and allows the fluorescent antibody to be easily preserved and transported.

SUMMARY

Embodiments of the present disclosure seek to solve at least one of the problems existing in the related art to at least some extent. For this, an object of the present disclosure is to provide a solution and method for preserving a fluorescent antibody, kit having the solution, use of the solution and the kit in an immunoassay. The solution of the present disclosure has at least one of the following advantages of maintaining bioactivity and fluorescence stability of the fluorescent antibody for a long term and tolerating high temperature.

In one aspect, the present disclosure provides in embodiments a solution for preserving a fluorescent antibody. In an embodiment of the present disclosure, the solution includes gelatin, cysteine, gentamicin and sodium citrate. The inventors have unexpectedly discovered through a large number of experiments that, the solution including gelatin, cysteine, gentamicin and sodium citrate can protect the fluorescent antibody well, and maintain bioactivity and fluorescence stability at the room temperature for a long term. Specifically, the gelatin provides an increased concentration of protein for the solution, thus avoiding the fluorescent antibody from hydrolysis; the cysteine not only effectively prevents oxidation of the fluorescent antibody, but also provides a buffering effect for adjusting a pH value of the solution; the gentamicin effectively inhibits growth of microorganisms, thereby prolonging the preservation period; and the sodium citrate also provides the buffering effect for adjusting the pH value of the solution. Therefore, the solution according to an embodiment of the present disclosure has at least one of the following advantages of maintaining bioactivity and fluorescence stability of the fluorescent antibody for a long term, and tolerating high temperature.

In an embodiment of the present disclosure, the gelatin is of a concentration of 5 g/L to 10 g/L, so that the present solution containing the gelatin maintains the stability of the fluorescent antibody for a long term further, thereby avoiding degradation of the fluorescent antibody.

In an embodiment of the present disclosure, the cysteine is of a concentration of 1 g/L to 5 g/L, thereby further avoiding oxidation of the fluorescent antibody, so that the present solution containing the cysteine maintains the bioactivity and fluorescence stability of the fluorescent antibody for a long term.

In an embodiment of the present disclosure, the gentamicin is of a concentration of 40 μg/L to 80 μg/L, thereby effectively inhibiting the growth of microorganisms.

In an embodiment of the present disclosure, the sodium citrate is of a concentration of 0.02 mol/L to 0.1 mol/L, thereby providing the solution suitable acidity and alkalinity, so that the solution containing the sodium citrate maintains the bioactivity and fluorescence stability of the fluorescent antibody for a long term.

In an embodiment of the present disclosure, the solution is of a pH value of 6.0 to 7.2, thereby further maintaining the bioactivity and fluorescence stability of the fluorescent antibody for a long term.

In another aspect, the present disclosure provides in embodiments a kit including the solution described hereinbefore. As the solution of the present disclosure can maintain the bioactivity and fluorescence stability of the fluorescent antibody for a long term, and tolerate high temperature, the kit including the solution can be used in immunodetection effectively.

In still another aspect, the present disclosure provides in embodiments use of the solution and the kit described above in the immunodetection. As the solution of the present disclosure can maintain the bioactivity and fluorescence stability of the fluorescent antibody for a long term, and tolerate high temperature, the solution or the kit including the solution can be effectively used in immunodetection.

In still another aspect, the present disclosure provides in embodiments a method for preserving the fluorescent antibody. In an embodiment of the present disclosure, the method includes: providing the fluorescent antibody in the solution described above. The fluorescent antibody preserved in the solution of the present disclosure can be maintained with the bioactivity and fluorescence stability for a long term, and tolerate high temperature.

In an embodiment of the present disclosure, the fluorescent antibody is labeled with fluorescein IR800 or CF647, so that the fluorescent antibody is allowed with stronger fluorescence stability.

Additional aspects and advantages of embodiments of present disclosure will be given in part in the following descriptions, become apparent in part from the following descriptions, or be learned from the practice of the embodiments of the present disclosure.

BRIEF DESCRIPTION OF THE DRAWINGS

These and other aspects and advantages of embodiments of the present disclosure will become apparent and more readily appreciated from the following descriptions made with reference to the drawings, in which:

Fig. 1 shows a comparison among fluorescence scans of IR800-CRP Ab preserved in solution 1 and solution 2 according to example 1 of the present disclosure;

Fig. 2 is a scattergram showing fluorescence intensities of IR800-CRP Ab preserved in solution 1 and solution 2 according to example 1 of the present disclosure;

Fig. 3 shows a comparison among fluorescence scans of CF647-CRP Ab preserved in solution 1 and solution 2 according to example 2 of the present disclosure;

Fig. 4 is a scattergram showing fluorescence intensities of CF647-CRP Ab preserved in solution 1 and solution 2 according to example 2 of the present disclosure;

Fig. 5 shows a comparison among fluorescence scans of IR800-CRP Ab preserved in solution 1 according to example 1 of the present disclosure and in solution of comparative example 1; and

Fig. 6 is a scattergram showing fluorescence intensities of IR800-CRP Ab preserved in solution 1 according to example 1 of the present disclosure and in solution of comparative example 1.

DETAILED DESCRIPTION

Reference will be made in detail to embodiments of the present disclosure. The embodiments described herein with reference to drawings are explanatory, illustrative, and used to generally understand the present disclosure. The embodiments shall not be construed to limit the present disclosure.

It should be noted that, the present disclosure is achieved based on the following discoveries.

At present, the existing solution for preserving the fluorescent antibody is poor in maintenance, with bioactivity degraded and fluorescence gradually quenched, thus adversely affecting the use of the fluorescent antibody.

In view of this, the inventors have unexpectedly discovered through a large number of experiments that, a solution including gelatin, cysteine, gentamicin and sodium citrate can protect the fluorescent antibody well, and maintain bioactivity and fluorescence stability of the fluorescent antibody at the room temperature for a long term. Therefore, the solution according to an embodiment of the present disclosure has at least one of the following advantages of maintaining bioactivity and fluorescence stability of the fluorescent antibody for a long term, and tolerating high temperature.

Embodiments of the present disclosure provide a solution and method for preserving a fluorescent antibody, a kit including the solution, use of the solution and the kit in an immunoassay, which will be described in detail as follows.

Solution for preserving fluorescent antibody

In one aspect, the present disclosure provides in embodiments a solution for preserving a fluorescent antibody. In an embodiment of the present disclosure, the solution includes gelatin, cysteine, gentamicin and sodium citrate. The solution of the present disclosure has at least one of the following advantages of maintaining the bioactivity and fluorescence stability of the fluorescent antibody for a long term, and tolerating high temperature.

The inventors have unexpectedly discovered through a large number of experiments that, it is important for the solution to have a certain concentration of proteins, which contributes to maintain stability of the fluorescent antibody. The fluorescent antibody is quite stable in the solution containing proteins at high concentration, but liable to hydrolyze in the solution containing proteins at low concentration. The gelatin, a protein obtained when collagen is partially hydrolyzed, provides a high concentration of proteins for the solution, thereby maintaining stability of the fluorescent antibody for a long term.

An antioxidant in the solution is key to prevent the fluorescent antibody from being invalid because of oxidization. The inventors have found that, the cysteine is the only amino acid having a reductive sulfydryl group (-SH) among more than 20 amino acids that make up a protein. The cysteine is a good natural antioxidant, and can reduce oxides to prevent the fluorescent antibody from being invalid because of oxidization. Further, the inventors have surprisingly found that, the cysteine provides the buffering effect for adjusting the pH value of the solution. Furthermore, the solution of the present disclosure also includes sodium citrate, a buffer substance. The combination of the sodium citrate and the cysteine will prevent the solution from changing in acidity and alkalinity, thereby providing the fluorescent antibody suitable acidity and alkalinity, which is important to maintain stability of the fluorescein and activity of the fluorescent antibody, and prevent the fluorescent antibody from aggregating, for a long term. In contrast, other amino acids cannot provide such positive effects, like cysteine. For example, although being capable of adjusting the acidity and alkalinity of the solution effectively, glycine is incapable of inhibiting the oxidation of fluorescent antibody owing to lack of reducibility, such that an additional antioxidant, such as vitamin C, vitamin E and dithiothreitol, is required.

For further improving the stability of the solution, an antibacterial substance is added to the solution. At present, there are a plenty of antibacterial substances inhibiting growth of microorganisms, which achieve different outcomes. Some of the antimicrobial substances, such as sodium azide, cause great harm to human body during bacteriostasis, so that good protective measures are required for users; some other of the antibacterial substances will adversely affect activity or fluorescence characteristics of the antibody, and thus adversely influence its use. In view of this, the inventors have creatively found through a large number of experiments that, the growth of a variety of microorganisms can be inhibited after the addition of the gentamicin. In addition, the inventors have surprisingly found that the gentamicin provides better thermal stability for the solution, for example, the solution of the present disclosure may tolerate the temperature of 37 ℃ within a short term, and thus is suitable for preservation at room temperature, thereby avoiding inconvenience to transport and preservation at low temperature.

The formulation of gelatin, cysteine, gentamicin and sodium citrate have been found by the inventors through a large number of experiments, the solution thus obtained can maintain the bioactivity and fluorescence stability of the fluorescent antibody for a long term and enable the fluorescent antibody to be preserved at room temperature for a long term, such as up to one year. Moreover, the solution of the present disclosure may tolerate high temperature. In addition, the gelatin, cysteine, gentamicin and sodium citrate will not adversely affect the characteristics of the fluorescein or the antibody per se.

In an embodiment of the present disclosure, the gelatin is of a concentration of 5 g/L to 10 g/L, including all values and ranges therebetween. In some embodiment of the present disclosure, the concentration of the gelatin may be 5 g/L, 6 g/L, 7 g/L, 8 g/L, 9 g/L or 10 g/L. Therefore, the gelatin may provide the fluorescent antibody with a suitable environment having a high protein concentration, so that the solution containing the gelatin maintains the stability of the fluorescent antibody for a long term, thereby avoiding the fluorescent antibody from degradation. Specifically, if the concentration of the gelatin is too low, the fluorescent antibody will be in an environment with proteins at low concentration and thus will be liable to degrade, resulting in losing its activity; and if the concentration of the gelatin is too high, the solution will be too viscous and even solidified, which is not convenient for subsequent detection.

In an embodiment of the present disclosure, the cysteine is of a concentration of 1 g/L to 5 g/L, including all values and ranges therebetween. In some embodiment of the present disclosure, the concentration of the cysteine may be 1 g/L, 2 g/L, 3 g/L, 4 g/L or 5 g/L, thereby inhibiting the oxidation of the fluorescent antibody, and avoiding the fluorescent antibody from being invalid because of oxidation. Moreover, the cysteine is capable of adjusting the pH value of the solution of the present disclosure, such that the solution containing the cysteine is provided with the pH value in a range of 6.0 to 7.2. Specifically, the cysteine cannot inhibit the oxidation of the fluorescent antibody effectively and provide the good buffering effect for the solution if it is in an over low concentration, thus resulting in declined bioactivity of the fluorescent antibody and gradually quenched fluorescence; and if the cysteine in in an over high concentration, a disulfide bond between antibodies will be reduced, resulting in inactivate antibody.

In an embodiment of the present disclosure, the gentamicin is of a concentration of 40 μg/L to 80 μg/L, including all values and ranges therebetween. In some embodiment of the present disclosure, the concentration of the gentamicin may be 40 μg/L, 50 μg/L, 60 μg/L, 70 μg/L or 80 μg/L, thereby effectively inhibiting the growth of microorganisms in the solution of the present disclosure, and enabling the fluorescent antibody to tolerate the temperature of 37 ℃ within a short term. Specifically, the gentamicin cannot inhibit the growth of microorganisms effectively if in an over low concentration; otherwise adversely affects the bioactivity of the fluorescent antibody if in an over high concentration.

In an embodiment of the present disclosure, the sodium citrate is of a concentration of 0.02 mol/L to 0.1 mol/L, including all values and ranges therebetween. In some embodiment of the present disclosure, the concentration of the sodium citrate may be 0.02 mol/L, 0.03 mol/L, 0.04 mol/L, 0.05 mol/L, 0.06 mol/L, 0.07 mol/L, 0.08 mol/L, 0.09 mol/L or 0.1 mol/L. Therefore, the solution of the present disclosure is provided with suitable acidity and alkalinity, with the pH value in the range of 6.0 to 7.2. As the cysteine also provides the buffering effect, it is possible to decrease addition amount of the sodium citrate. Specifically, the sodium citrate cannot provide sufficient buffering action if in an over low concentration, thereby resulting in declined bioactivity of the antibody, gradually quenched fluorescence, and aggregation and precipitation of the fluorescent antibody; and if the sodium citrate is in an over high concentration, the fluorescent antibody will aggregate and precipitate owing to a salting-out effect.

In an embodiment of the present disclosure, the solution is of a pH of 6.0 to 7.2. The optimal pH is about 6 for the antibody and about 7 for the fluorescein. The inventors have found though a large number of experiments that the solution of the present disclosure with a pH value between 6.0 and 7.2 can be provided by adding the cysteine and the sodium citrate, thereby providing the fluorescent antibody stable acidity and alkalinity, and preventing the fluorescent antibody from aggregation and precipitation.

Kit

In the second aspect, the present disclosure provides in embodiments a kit including the solution as described above. As the solution of present disclosure can maintain the bioactivity and the fluorescence stability of the fluorescent antibody and tolerate high temperature, the kit including the solution can be used in immunodetection.

It should be appreciated to those skilled in the art that, the characteristics and advantages described above with respect to the solution of the present disclosure are also suitable to the kit, and will not be elaborated herein.

Use

In the third aspect, the present disclosure provides in embodiments use of the solution and the kit described above in the immunoassay. As the solution of the present disclosure can maintain the bioactivity and fluorescence stability of the fluorescent antibody for a long term, and tolerate high temperature, the solution or the kit including the solution can be used in immunodetection.

It should be noted that, term “immunodetection” used herein should be broadly understood, and mainly refers to such a technology by which an antigen (to be tested) can be detected by means of specific combination between the antigen and the corresponding antibody.

It should be appreciated to those skilled in the art that, the characteristics and advantages described above with respect to the solution and the kit of the present disclosure are also suitable to the use, and will not be elaborated herein.

Method for preserving fluorescent antibody

In the fourth aspect, the present disclosure provides in embodiments a method for preserving the fluorescent antibody. The method includes: providing the fluorescent antibody in the solution described above. The fluorescent antibody preserved in the solution of the present disclosure can be maintained with bioactivity and fluorescence stability for a long term, and tolerate high temperature.

In an embodiment of the present disclosure, the fluorescent antibody is labeled with fluorescein IR800 or CF647. The inventors have found that fluorescein IR800 or CF647, having good stability, can maintain the fluorescence characteristic at room temperature for a long term, and is not easy to be quenched.

It should be appreciated to those skilled in the art that, the characteristics and advantages described above with respect to the solution of the present disclosure are also suitable to the method, and will not be elaborated herein.

In the following, the technical solution of the present disclosure will be described in detail in combination with examples. It should be appreciated to those skilled in the art that, the examples described below are explanatory, illustrative, and used to generally understand the present disclosure, and shall not be construed to limit the present disclosure. The same or similar elements and the elements having same or similar functions are denoted by like reference numerals throughout the descriptions. Examples which do not indicate specific techniques or conditions are carried out either in accordance with the techniques or conditions described in the literatures in the related art or in accordance with the product specifications. The reagents or instruments whose manufacturers are not indicated are all conventional products, which are commercially available.

Example 1

(1) Preparation of fluorescent antibody

Fluorescein IR800 activated by N-hydroxysuccinimide (NHS) was dissolved in DMSO to prepare an IR800-NHS solution with a concentration of 2 mmol/L. A reaction system was prepared with anti-human c-reactive protein (CRP) monoclonal antibody having a final concentration of 10 μmol/L, 100 μmol/L IR800-NHS and 10 mmol/L phosphate buffer (PBS) , which is add with sterile deionized water up to 100 μL. The reaction system was incubated at room temperature for 1.5 h, after which the resulting product was purified by illustra NAP-25 Columns, and finally eluted with 500 μL PBS to collect the purified fluorescent antibody (IR800-CRP Ab) .

(2) Preparation of the solution for preserving the fluorescent antibody

Solutions were prepared according to the formulations shown in table 1 by dissolving the components at room temperature under stirring, and filtering with 22 μm filter membrane for use.

Table 1 Formulations of solutions

(3) Preservation of fluorescent antibody

IR800-CRP Ab was diluted in each of the two solutions prepared in step (2) at a ratio of 1: 1. The diluted antibodies were aliquoted into small volumes and preserved away from light at 37 ℃.

(4) Detection of fluorescence intensity

Fluorescence intensity was measured on day 0, day 2, day 4, day 6, day 8 and day 10, respectively.

Table 2 Scan fluorescence intensities of IR800-CRP Ab at different preservation time periods

(5) Preparation of CRP antibody chip

The CRP antibody was diluted to 0.2 mg/mL with 10 mmol/L PBS buffer, then printed onto a plasma gold chip by GeSim Nano-Plotter (TM) 2.1 with 3 nL and 4 replicates for each point to obtain a round spot having a diameter of about 400 microns, followed by incubated at room temperature for 2 h to obtain the CRP antibody chip.

(6) Detection of CRP antibody chip

The CRP antibody chip was blocked with 1%BSA in PBS under shaken for 1 hour to reduce nonspecific binding, then washed by PBST with 150 μl/well (0.05%Tween 20) , subsequently added with 100 μl CRP antigen (1 mg/L) to each well and shaken for 0.5 h. After washed with PBST for three times, the chip was added with IR800-CRP Ab (4 nmol/L) to stain for 0.5 h in the dark under stirring, followed by successively washed with PBST three times and with pure water once, and then centrifugal dried.

The chip was scanned with MidaScan scanner at 785 nm channel under a laser intensity of 7.0 and a resolution of 20 μm. After scanning, 16-bit gray-scale images were obtained and analyzed with MidaScan Software V1.0.0 or a higher version. The intensity of each point was measured by gate array analysis mode and the lattice morphology was automatically recognized by the program. The intensity of each point was obtained by dividing the total signal strength of a selected region by area of the selected region. The average fluorescence intensity of the four parallel points on the image was defined as the measured intensity. There was a positive correlation between the activity of CRP fluorescent antibody and the fluorescence intensity on the obtained image.

The detection results of IR800-CRP Ab are shown in Figs. 1-2. It can be seen that, the fluorescent antibody preserved in solution 1 or solution 2 for 10 days still has a strong fluorescence intensity, a high bioactivity, and a high stability.

Example 2

The fluorescent antibody was prepared with the method described in Example 1, except that the fluorescein IR800 was replaced with fluorescein CF647 and the obtained chip was scanned at 670 nm channel.

The detection results of the fluorescence intensity are shown in Table 3, and the detection results of CRP antibody chip are shown in Figs. 3-4. It can be seen that, the fluorescent antibody preserved in solution 1 or solution 2 for 10 days still has a strong fluorescence intensity, a high bioactivity, and a high stability.

Table 3 Scan fluorescence intensities of CF647-CRP Ab at different preservation time periods

Comparative example 1

The fluorescent antibody was preserved and detected with the method described in Example 1, except that components of a solution used in comparative example 1 was different from that in example 1 of the present disclosure, as shown in Table 4.

The detection results of CRP antibody chip are shown in Figs. 5-6. It can be seen that, compared to Example 1, the fluorescent antibody preserved in the solution of comparative example 1 for 10 days has a significantly decreased fluorescence intensity, a significantly decreased bioactivity, and a poor stability.

Table 4 components of solutions for preserving the fluorescent antibody

Table 5 Scan fluorescence intensities of IR800-CRP Ab at different preservation time periods

Comparative example 2

The fluorescent antibody was preserved and detected with the method described in Example 1, except that the concentration of gelatin was 12 g/L.

In such a case, the fluorescent antibody cannot be dissolved after preserved in the solution for two days at 37 ℃ as the solution was coagulated because the concentration of the gelatin was over high.

Comparative example 3

The fluorescent antibody was preserved and detected with the method described in Example 1, except that the concentration of cysteine was 8 g/L.

As the concentration of cysteine is too high, the antibodies are inactivated because of the reduction of the disulfide bond between the antibodies, which results in a significant decrease in fluorescence intensity.

Comparative example 4

The fluorescent antibody was preserved and detected with the method described in Example 1, except that the concentration of gentamicin was 100 μg/mL.

In such a case, the concentration of gentamicin is too high, though the bioactivity of the fluorescent antibody can be maintained to a great extent, the initial value of fluorescence intensity is much lower than that of Example 1, indicating that gentamicin concentration is too high to adversely affect the initial activity of fluorescent antibody.

Reference throughout this specification to “an embodiment, ” “some embodiments, ” “one embodiment” , “another example, ” “an example, ” “a specific example, ” or “some examples, ” means that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the present disclosure. Thus, the appearances of the phrases such as “in some embodiments, ” “in one embodiment” , “in an embodiment” , “in another example, ” “in an example, ” “in a specific example, ” or “in some examples, ” in various places throughout this specification are not necessarily referring to the same embodiment or example of the present disclosure. Furthermore, the particular features, structures, materials, or characteristics may be combined in any suitable manner in one or more embodiments or examples.

Although explanatory embodiments have been shown and described, it would be appreciated by those skilled in the art that the above embodiments cannot be construed to limit the present disclosure, and changes, alternatives, and modifications can be made in the embodiments without departing from spirit, principles and scope of the present disclosure.

Claims

A solution for preserving a fluorescent antibody, comprising: gelatin; cysteine; gentamicin; and sodium citrate.
The solution according to claim 1, wherein the gelatin is of a concentration of 5 g/L to 10 g/L.
The solution according to claim 1 or 2, wherein the cysteine is of a concentration of 1 g/L to 5 g/L.
The solution according to any one of claims 1 to 3, wherein the gentamicin is of a concentration of 40 μg/L to 80 μg/L.
The solution according to any one of claims 1 to 4, wherein the sodium citrate is of a concentration of 0.02 mol/L to 0.1 mol/L.
The solution according to any one of claims 1 to 5, wherein the solution is of a pH value of 6.0 to 7.2.
A kit, comprising a solution according to any one of claims 1 to 6.
Use of a solution according to any one of claims 1 to 6 or a kit according to claim 7 in an immunoassay.
A method for preserving a fluorescent antibody, comprising: providing the fluorescent antibody in a solution according to any one of claims 1 to 6.
The method according to claim 9, wherein the fluorescent antibody is labeled with fluorescein IR800 or CF647.