Method for labeling antibody CD16 by fluorescein isothiocyanate FITC

Abstract

The invention provides a method for labeling an antibody CD16 with fluorescein isothiocyanate FITC, the coupling rate of the anti-CD16 antibody-FITC conjugate prepared according to the method disclosed by the invention is remarkably improved, and the fluorescence intensity is high; products are high in accuracy through multi-sample detection, cross reaction analysis is qualified, and the quality of products among batches is stable.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology. Specifically, the present invention relates to a method for labeling antibody CD16 with fluorescein isothiocyanate (FITC). Background Art

[0002] Fluorescent labeling technology is a technology that binds some fluorescent substances to a certain group of the target molecule through covalent bonds, and provides information for the object under study according to its fluorescence characteristics. Through fluorescent labeling technology, the molecular mechanism can be dissected in detail. In solution, single molecules can be detected in real time, with high resolution and sensitivity.

[0003] Fluorescein isothiocyanate (FITC)-labeled antibody is one of the most commonly used fluorescent-labeled antibodies in the field of biological health research. Fluorescein isothiocyanate has high absorption rate, high fluorescence quantum yield, and is easily soluble in organic solvents. Under alkaline conditions, the carbamide bond on FITC can covalently bind to the free amino group on the antibody to form a fluorescent antibody. The labeled antibody still has the ability to bind to the antigen, and through fluorescence microscopy or flow cytometry analysis, the antigen can be localized, qualitatively or quantitatively analyzed.

[0004] Currently, the method for labeling antibody with FITC has problems such as low coupling efficiency, uneven antibody coupling efficiency, resulting in great differences in the subsequent detection effects of the coupling products, and poor stability between batches.

[0005] Therefore, those skilled in the art are committed to improving the method for labeling antibody with fluorescein isothiocyanate (FITC) in order to perform more efficient and higher-quality fluorescent labeling of antibodies. Summary of the Invention

[0006] The purpose of the present invention is to provide a method for labeling antibody CD16 with fluorescein isothiocyanate (FITC).

[0007] In the first aspect of the present invention, a method for labeling antibody CD16 with fluorescein isothiocyanate (FITC) is provided, and the method includes the steps of:

[0008] S1. Provide an anti-CD16 antibody solution, and the antibody concentration in the anti-CD16 antibody solution is 0.5 - 1.5 mg / mL;

[0009] S2. Provide a FITC solution dissolved in DMSO, and the FITC concentration in the FITC solution is 2 - 4 mg / mL;

[0010] S3. Mix the FITC solution and the anti-CD16 antibody solution according to a set feeding ratio and carry out a coupling reaction to obtain a coupling product.

[0011] In another preferred example, the anti-CD16 antibody solution is subjected to dialysis treatment.

[0012] In another preferred example, the anti-CD16 antibody solution is dialyzed with a carbonate solution at pH 9.0 - 9.5 and 0.1 - 0.3 M.

[0013] In another preferred example, the concentration of FITC in the FITC solution is 3 - 4 mg / mL.

[0014] In another preferred example, in step S3, the molar feeding ratio of FITC to the anti-CD16 antibody is: 10 - 200:1; preferably 20 - 100:1.

[0015] In another preferred example, in step S3, multiple sets of feeding ratios are set for coupling respectively.

[0016] In another preferred example, in step S3, three sets of feeding ratios are set for coupling respectively.

[0017] In another preferred example, in step S3, the molar feeding ratio of FITC to the anti-CD16 antibody is: 20:1, 50:1, and / or 100:1.

[0018] In another preferred example, in step S3, the coupling reaction conditions are: reaction temperature 15 - 35 °C, incubate with light-shielding and shaking for 1 - 3 h; then transfer to 0 - 10 °C and incubate for 6 - 24 h to complete the coupling reaction.

[0019] In another preferred example, in step S3, the coupling reaction conditions are: reaction temperature 20 - 25 °C, incubate with light-shielding and shaking for 1.5 - 3 h; then transfer to 2 - 8 °C and incubate for 6 - 24 h to complete the coupling reaction.

[0020] In another preferred example, the method further includes the step:

[0021] S4. The coupling product is dialyzed with PBS to remove free FITC.

[0022] In another preferred example, the method further includes the step:

[0023] S5. The coupling product after the dialysis treatment in step S4 is subjected to full wavelength scanning, and the ratio of 495 nm to 280 nm is calculated to obtain the coupling rate of FITC, and the coupling rate is controlled to be 0.5 - 2.0.

[0024] In another preferred example, in step S5, it further includes the step: adjusting the concentration of the coupling product to 10 μg / mL, using PBS as a blank control, measuring the emission spectrum at the emission wavelength of OD510, and controlling the fluorescence intensity peak value of the coupling product to be more than 100000 higher than that of the blank.

[0025] In another preferred example, the method further includes the step:

[0026] Add 0.4% BSA, 20% glycerol, and 0.1% Proclin 300 to the conjugate product and store it at low temperature.

[0027] In another preferred example, the method includes the steps of:

[0028] (1) Dialyze the anti-CD16 antibody overnight with a 0.2 M carbonate solution at pH 9.0 - 9.5, and adjust the antibody concentration to 1 mg / mL.

[0029] (2) Dissolve FITC in DMSO to prepare a FITC solution with a concentration of 3 mg / mL.

[0030] (3) Set the feeding ratio of FITC to anti-CD16 antibody, that is, the FITC:CD16 molar ratio is 20:1, 50:1, and 100:1 respectively. According to the three feeding ratios, add the FITC solution to the anti-CD16 antibody solution.

[0031] (4) After mixing, incubate at 22 °C and 600 rpm / min in the dark for 2 h, then transfer to 4 °C and incubate overnight for the conjugation reaction to obtain the conjugate product CD16-FITC.

[0032] (5) Dialyze the conjugate product CD16-FITC with PBS for 24 h to remove free FITC.

[0033] (6) After concentrating the conjugate product CD16-FITC, perform a full-wavelength scan, calculate the ratio of 495 nm to 280 nm to obtain the conjugation rate of FITC, and control the conjugation rate to be 0.5 - 2.0; dilute the conjugate product CD16-FITC to 10 μg / mL, use PBS as a blank control, and measure the emission spectrum at the emission wavelength of OD510, and control the fluorescence intensity peak value of the conjugate product CD16-FITC to be more than 100,000 higher than that of the blank.

[0034] (7) Add 0.4% (w / v) BSA, 20% (v / v) glycerol, and 0.1% (w / v) Proclin 300 to the conjugate product CD16-FITC and store it at 4 °C.

[0035] It should be understood that within the scope of the present invention, the above technical features of the present invention and the technical features specifically described below (such as in the examples) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be elaborated one by one here. Description of the Drawings

[0036] Figure 1 Shows the full-wavelength scan results from 250 nm to 800 nm.

[0037] Figure 2Shows the emission spectrum of the emitted light OD510.

[0038] Figure 3 Shows the flow cytometry detection results of the CD16 antibody-FITC conjugate prepared in Comparative Example 1.

[0039] Figure 4 Shows the flow cytometry detection results of the CD16 antibody-FITC (1:20) conjugate prepared in Example 1.

[0040] Figure 5 Shows the cross-reaction analysis verification results. Detailed implementation mode

[0041] Before describing the present invention, it should be understood that the present invention is not limited to the specific methods and experimental conditions described, as such methods and conditions can vary. It should also be understood that the terms used herein are for the purpose of describing specific embodiments only and are not intended to be limiting, and the scope of the present invention will be limited only by the appended claims.

[0042] 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. As used herein, when referring to a specifically recited numerical value, the term "about" means that the value can vary by no more than 1% from the recited value. For example, as used herein, the expression "about 100" includes all values between 99 and 101 (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0043] Although any methods and materials similar or equivalent to those described in the present invention can be used in the practice or testing of the present invention, preferred methods and materials are exemplified herein.

[0044] The main advantages of the present invention are:

[0045] (1) The coupling rate of the anti-CD16 antibody-FITC conjugate prepared by the method of the present invention is significantly improved, and the fluorescence intensity is high;

[0046] (2) The anti-CD16 antibody-FITC conjugate prepared by the method of the present invention has high detection accuracy for multiple samples, qualified cross-reaction analysis, and stable product quality among batches.

[0047] The present invention will be further described in detail below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. For the experimental methods without specified detailed conditions in the following embodiments, they are generally carried out according to the conventional conditions such as those described in "Molecular Cloning: A Laboratory Manual" (translated by Huang Peitang et al., Beijing: Science Press, 2002) by Sambrook.J et al. in the United States, or according to the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are calculated by weight. The experimental materials and reagents used in the following embodiments can be obtained from commercial channels without special instructions.

[0048] Example 1

[0049] The operating steps for preparing FITC-labeled anti-CD16 antibody in this example are as follows:

[0050] (1) Dialyze the anti-CD16 antibody (purchased from Invitrogen, clone number MEM-154) overnight with 0.2M carbonate solution at pH 9.0 - 9.5, and adjust the antibody concentration to 1 mg / mL;

[0051] (2) Dissolve FITC (purchased from Invitrogen) with DMSO at a concentration of 3 mg / mL;

[0052] (3) Set three feeding ratios of FITC to antibody CD16, that is, the molar ratios of FITC:CD16 are 20:1, 50:1, and 100:1 respectively. According to the three feeding ratios, add FITC to CD16;

[0053] (4) Mix well, incubate at 22 °C, 600 rpm / min, in the dark with shaking for 2 h, and then transfer to a 4 °C vertical mixer for overnight incubation;

[0054] (5) Dialyze the conjugate CD16-FITC with PBS for 24 h, changing the solution once in the middle to fully remove free FITC;

[0055] (6) Concentrate CD16-FITC, measure the concentration, scan the full wavelength, calculate the ratio of 495 nm to 280 nm to obtain the conjugation rate of FITC. The conjugation rate can be between 0.5 - 2.0. Dilute the conjugate to 10 μg / mL, use PBS as a blank control, measure the emission spectrum at the emission wavelength of OD510. If the fluorescence intensity peak value of CD16-FITC is more than 100,000 higher than that of the blank, it meets the standard;

[0056] (7) Add 0.4% BSA, 20% glycerol, and 0.1% Proclin 300 to CD16-FITC, aliquot at 1.0 mL / tube and store in brown tubes at 4 °C.

[0057] Comparative Example 1

[0058] (1) Dialyze the antibody CD16 (purchased from Invitrogen, clone number MEM-154) overnight against 0.2 M carbonate solution at pH 9.0 - 9.5, and adjust the antibody concentration to 2 mg / mL.

[0059] (2) Dissolve FITC in DMSO at a concentration of 1 mg / mL.

[0060] (3) Set the feeding ratio of FITC to antibody CD16 as 20:1, and add FITC to CD16.

[0061] (4) Mix well, incubate at 22 °C with shaking at 600 rpm / min in the dark for 2 h.

[0062] (5) Dialyze the conjugate CD16-FITC against PBS for 24 h, changing the solution once in the middle to fully remove free FITC.

[0063] (6) Concentrate CD16-FITC, measure the concentration, scan the full wavelength, and calculate the ratio of 495 nm to 280 nm to obtain the conjugation rate of FITC.

[0064] (7) Add 0.4% BSA, 20% glycerol, and 0.1% Proclin 300 to CD16-FITC, aliquot at 1.0 mL / tube and store in brown tubes at 4 °C.

[0065] Test Example

[0066] Test the FITC-fluorescently labeled anti-CD16 antibodies prepared in Example 1 and Comparative Example 1 respectively. Scan the conjugate CD16-FITC at the full wavelength of 250 nm - 800 nm, calculate the conjugation rate, detect the emission spectrum of the emitted light OD510, record the peak value of the OD510 emission spectrum, and perform flow cytometry detection.

[0067] Figure 1 Shows the scanning results at the full wavelength of 250 nm - 800 nm.

[0068] Figure 2 Shows the emission spectrum of the emitted light OD510.

[0069] Figure 3 Shows the flow cytometry detection results of the CD16 antibody-FITC conjugate prepared in Comparative Example 1.

[0070] The flow cytometry detection results of the CD16 antibody-FITC conjugate prepared in Comparative Example 1 show that the accuracy at 2 μg / T is 1.1%, and the accuracies of other concentration gradients are all unqualified; and the fluorescence intensities at each concentration are lower than those of the control group. The positive control is a commercially available CD16 antibody-FITC conjugate (Kuangbo Biotech).

[0071] Taking the CD16 antibody-FITC (1:20) prepared in Example 1 as an example, the flow cytometry detection results are as follows Figure 4 shown

[0072] According to the scatter plot results of SSC / CD16 antibody-FITC (1:20), it shows that

[0073] For sample 78100, the accuracy at 3.56 μg / Test is -0.23%, at 2.67 μg / Test is -0.88%, at 1.78 μg / Test is 1.33%, at 0.89 μg / Test is 2.33%, at 0.445 μg / Test is 6.80%, and at 0.2225 μg / Test is 11.70%.

[0074] According to the analysis data, when the concentration of CD16 antibody-FITC (1:20) is 3.56 μg / Test, the accuracy is within the industry standard of ±2%, and its fluorescence intensity accounts for about 74.4% of the control group. The flow cytometry graph is similar to the control. Therefore, the concentration of 3.56 μg / Test is selected for multi-sample verification.

[0075] For the multi-sample (five different peripheral blood samples) verification test, according to the flow cytometry results, when the concentration of CD16 antibody-FITC (1:20) is 3.56 μg / Test, the accuracy of the five different peripheral blood samples is within the industry standard of ±2%, and the flow cytometry graph is similar to the control. Therefore, the concentration of 3.56 μg / Test is selected for reaction specificity verification.

[0076] The results of within-batch precision verification show that: in the detection results of the CD16 antibody-FITC (1:20) prepared in Example 1, the positive percentage is less than 30%, and the CV values are all not greater than 15%, meeting the industry standard.

[0077] The results of cross-reaction analysis verification are as follows Figure 5 shown. It can be seen from the analysis results that the conjugate of the CD16 antibody-FITC (1:20) prepared in Example 1 is reactive to lymphocytes, monocytes, and granulocytes, meeting the characteristics of its T cell antigen.

[0078] Summary: For the fluorescent antibody conjugate of the CD16 antibody-FITC (1:20) prepared in Example 1 of the present invention, the appropriate use concentration is 3.56 μg / Test, and the multi-sample verification, within-batch precision verification, and cross-verification are all qualified.

[0079] The flow cytometry detection results of the CD16 antibody-FITC (1:50) and CD16 antibody-FITC (1:100) of the present invention are similar to those of the CD16 antibody-FITC (1:20), and will not be shown one by one here.

[0080] The summary of the coupling rate, peak emission spectrum, and flow cytometry test results of the tested CD16 antibody-FITC conjugate is as follows:

[0081]

[0082] By comparison: For the CD16 antibody-FITC fluorescent antibodies with three feeding ratios in the present invention, the coupling rate and the peak OD510 of the emission spectrum are significantly higher than those of the comparative example. The flow cytometry test results further show that the CD16 antibody-FITC conjugate prepared in Example 1 is significantly superior to Comparative Example 1, and there are differences in the flow cytometry test results among the three feeding ratios in the examples, indicating that it is necessary to set three feeding ratios.

[0083] Example 2

[0084] The operating steps for preparing the FITC-labeled anti-CD16 antibody in this example are as follows:

[0085] (1) Dialyze the antibody CD16 overnight in a 0.2M carbonate solution at pH 9.0 - 9.5, with an antibody concentration of 1.5 mg / mL;

[0086] (2) Dissolve FITC in DMSO at a concentration of 3.5 mg / mL;

[0087] (3) Set three feeding ratios for FITC and antibody CD16, that is, the molar ratios of FITC:CD16 are 20:1, 50:1, and 100:1 respectively. According to the three feeding ratios, add FITC to CD16 (setting three feeding ratios can basically ensure that more than 98% of the labeled antibodies meet the detection requirements);

[0088] (4) Mix well, incubate at 20°C, 600 rpm / min, in the dark with shaking for 3 h, and then transfer to an 8°C vertical mixer to incubate overnight to further increase the coupling rate of FITC;

[0089] (5) Dialyze the conjugate CD16 antibody-FITC with PBS for 24 h, changing the solution once in the middle to fully remove free FITC;

[0090] (6) Concentrate the CD16 antibody-FITC, measure the concentration, scan the full wavelength, calculate the ratio of 495 nm to 280 nm to obtain the coupling rate of FITC. The coupling rate is between 0.5 - 2.0. Dilute the conjugate to 10 μg / mL, use PBS as a blank control, and measure the emission spectrum at the emission wavelength of OD510. The peak fluorescence intensity of the CD16 antibody-FITC is more than 100,000 higher than that of the blank;

[0091] In (7) CD16 antibody-FITC, add 0.4% BSA, 20% glycerol, and 0.1% Proclin 300. Aliquot at 1.0 mL / tube and store in brown tubes at 4°C.

[0092] Example 3

[0093] The operating steps for preparing FITC-labeled anti-CD16 antibody in this example are as follows:

[0094] (1) Dialyze antibody CD16 overnight against 0.2 M carbonate solution at pH 9.0 - 9.5 with an antibody concentration of 0.5 mg / mL.

[0095] (2) Dissolve FITC in DMSO at a concentration of 4 mg / mL.

[0096] (3) Set three feeding ratios of FITC to antibody CD16, i.e., the molar ratios of FITC:CD16 are 20:1, 50:1, and 100:1 respectively. According to the three feeding ratios, add FITC to CD16 (setting three feeding ratios can basically ensure that more than 98% of the labeled antibody meets the detection requirements).

[0097] (4) Mix well, incubate at 25°C with shaking at 600 rpm / min in the dark for 1.5 h, and then transfer to a 2°C vertical mixer for overnight incubation to further improve the coupling rate of FITC.

[0098] (5) Dialyze the conjugate CD16 antibody-FITC against PBS for 24 h and change the solution once in the middle to fully remove free FITC.

[0099] (6) Concentrate CD16 antibody-FITC, measure the concentration, scan the full wavelength, calculate the ratio of 495 nm to 280 nm to obtain the coupling rate of FITC. The coupling rate is between 0.5 - 2.0. Dilute the conjugate to 10 μg / mL, use PBS as a blank control, and measure the emission spectrum at the emission wavelength of OD510. The fluorescence intensity peak of CD16 antibody-FITC is more than 100,000 higher than that of the blank.

[0100] (7) In CD16 antibody-FITC, add 0.4% BSA, 20% glycerol, and 0.1% Proclin 300. Aliquot at 1.0 mL / tube and store in brown tubes at 4°C.

[0101] In this patent, the concentration of FITC dissolved in DMSO is 3 mg / mL, which reduces the content of DMSO in the subsequent coupling system and avoids reducing the coupling rate of FITC due to the high content of DMSO. In the prior art, the coupling system was prepared according to the feeding amount with a molar ratio of FITC to antibody CD16 of 20:1. However, in this patent, three feeding ratios of the molar ratio of FITC to antibody CD16 are set, namely 20:1, 50:1, and 100:1, which solves the problem that the coupling rates of different antibodies are different in the same feeding ratio, resulting in unqualified subsequent detections. At the same time, in order to further improve the coupling rate, this patent makes improvements on the basis of the prior art. After incubating at room temperature for 2 hours, it is transferred to 4 degrees and incubated overnight.

[0102] In this patent, the detection of the fluorescence intensity of CD16 antibody-FITC is increased. That is, CD16 antibody-FITC is diluted to 10 ug / mL, and PBS is used as a blank control to measure the emission spectrum at the emission wavelength of OD510. If the peak value of the fluorescence intensity of CD16 antibody-FITC is more than 100,000 higher than that of the blank, it meets the standard. The purpose of adding this detection is to prevent fluorescence quenching of FITC during the coupling process, thereby affecting subsequent detections.

[0103] All the documents mentioned in this invention are cited in this application as references, just as if each document was cited separately as a reference. In addition, it should be understood that after reading the above teachings of this invention, those skilled in the art can make various changes or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims of this application.

Claims

1. A method for fluorescein isothiocyanate (FITC) labeling antibody CD16, characterized in that, The method includes the steps of: S1. Providing an anti-CD16 antibody solution with an antibody concentration of 0.5 - 1.5 mg / mL in the anti-CD16 antibody solution; S2. Providing a FITC solution dissolved in DMSO with a FITC concentration of 2 - 4 mg / mL in the FITC solution; and S3. Mixing the FITC solution and the anti-CD16 antibody solution according to a set feeding ratio to carry out a coupling reaction to obtain a coupling product.

2. The method according to claim 1, characterized in that, The anti-CD16 antibody solution is subjected to dialysis treatment.

3. The method according to claim 2, characterized in that, The anti-CD16 antibody solution is dialyzed with a 0.1 - 0.3 M carbonate solution at pH 9.0 - 9.

5.

4. The method according to claim 1, characterized in that, The FITC concentration in the FITC solution is 3 - 4 mg / mL.

5. The method according to claim 1, characterized in that, In step S3, the molar feeding ratio of FITC to the anti-CD16 antibody is: 10 - 200:1; preferably 20 - 100:

1.

6. The method according to claim 1, characterized in that, In step S3, multiple sets of feeding ratios are set and coupled respectively.

7. The method according to claim 6, characterized in that, In step S3, three sets of feeding ratios are set and coupled respectively; preferably, in step S3, the molar feeding ratios of FITC to the anti-CD16 antibody are: 20:1, 50:1, and 100:

1.

8. The method according to claim 1, characterized in that, In step S3, the coupling reaction conditions are: reaction temperature 15 - 35 °C, incubating with light avoidance and shaking for 1 - 3 h; then transferring to 0 - 10 °C for incubation for 6 - 24 h to complete the coupling reaction.

9. The method according to claim 1, characterized in that, The method further includes the steps of: S4. Dialyzing the coupling product with PBS to remove free FITC.

10. The method according to claim 9, characterized in that, The method further includes the steps of: S5. Performing a full wavelength scan on the coupling product after the dialysis treatment in step S4, calculating the ratio of 495 nm to 280 nm to obtain the coupling rate of FITC, and controlling the coupling rate to be 0.5 - 2.0; and / or In step S5, it further includes the steps of: adjusting the concentration of the coupling product to 10 μg / mL, using PBS as a blank control, measuring the emission spectrum at the emission wavelength of OD510, and controlling the fluorescence intensity peak value of the coupling product to be more than 100,000 higher than that of the blank.

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