Antibody/Protein Labeling Technologies and Commonly Used Dyes

In recent years, biomacromolecule labeling technology has received great attention and achieved rapid development. This technology is widely applied in various fields such as the detection of intracellular and extracellular substances, nucleic acid hybridization sequencing, and disease diagnosis. The principle of antibody/protein labeling is that labeling reagents form connections with groups such as sulfhydryl, amino, and carboxyl groups carried by antibodies/proteins through displacement or condensation reactions. Antibody labeling technology is extensively used in medical pathology, immunohistochemistry, molecular biology, and other fields. In this issue, we will mainly introduce 3 common antibody labeling technologies: fluorochrome (FITC, PE, APC, etc.) labeling, enzyme (horseradish peroxidase, alkaline phosphatase, etc.) labeling, and biotin (Biotin) labeling.

Fluorescein Isothiocyanate (FITC) Labeling of Antibodies/Proteins

Fluorescein isothiocyanate (FITC) is obtained by introducing an isothiocyanate group (-N=C=S) into the structure of fluorescein. In an alkaline environment, the lysine R amino group of the antibody forms a covalent bond with the thiocarbamide bond of FITC, forming a stable antibody-FITC complex (Figure 1), thereby realizing the fluorescent labeling of target molecules.

Different types of fluorescent reagents have different labeling methods, but the required conditions and steps are roughly the same. Factors affecting labeling include temperature, pH value, and the ratio of fluorescent reagents to immunoglobulins in the reaction solution [1]. Commonly used methods for labeling antibodies are the stirring method and the dialysis method. Fang et al. [2] used the dialysis method to label polyclonal antibodies. The labeling method is as follows:

1.       Take 1mg of antibody protein solution, and dialyze the antibody solution with pH9.0 carbonate buffer to place the protein in an alkaline environment;

2.       Determine the optimal reaction ratio: Calculate the amount of FITC according to the ratios of FITC to protein content of 1:20, 1:10, and 3:20, add FITC dissolved in DMSO dropwise, and react at room temperature in the dark for 4h to perform FITC labeling;

3.       Determine the optimal reaction conditions for labeled antibodies: After confirming the optimal amount of FITC, perform FITC labeling under the following reaction conditions: 4°C for 2h; 20°C for 4h; 37°C for 30min;

4.       Centrifuge the reacted solution at 6000r/min for 20min using an ultrafiltration centrifuge tube, repeat several times, collect the supernatant each time, and determine the fluorescence value (F485/535) until there is no fluorescence value in the supernatant. Determine the F485/535 value after labeling, and combine with significant difference analysis of actual conditions to obtain the optimal ratio of FITC to antibody content and reaction conditions.

Horseradish Peroxidase (HRP) Labeling of Antibodies/Proteins

The principle of HRP labeling antibodies is that the sugar molecules on its surface are oxidized to aldehyde groups, which form covalent bonds with the amino groups of the antibodies to form HRP-antibody complexes. Antibodies labeled with HRP undergo color changes under specific enzymes and substrates. Qualitative and quantitative analysis of antigens can be performed by the depth of the color, which is widely used in analytical methods such as WB, ELISA, and immunohistochemistry.

The main methods for preparing horseradish peroxidase (HRP)-antibody conjugates are the glutaraldehyde two-step method and the sodium periodate method [3]. Xu et al. [4] prepared rabbit-derived M13KE polyclonal antibodies, purified the polyclonal antibodies by saturated ammonium sulfate method and Protein G Resin chromatography column, and then labeled them with horseradish peroxidase using the sodium periodate method. The labeling method is as follows:

1.       Dissolve 4mg of HRP in 1mL of deionized water;

2.       Add 400μL of freshly prepared 0.1mol/L sodium periodate and shake at room temperature for 20min;

3.       Dialyze the reactant overnight in sodium acetate buffer (0.001mol/L, pH4.4);

4.       Add 6mg of IgG, adjust the pH of the solution to 9.0~9.5 with 0.2mol/L sodium carbonate buffer (pH9.5), immediately add 200μL of freshly prepared 5mg/mL sodium borohydride (NaBH4), and react at 4°C for 2h;

5.       Purification of labeled products: Slowly add an equal volume of saturated ammonium sulfate to the prepared enzyme conjugate while stirring, precipitate at 4°C for 30min; centrifuge at 5000r/min for 15min, dissolve the precipitate with an appropriate amount of PBS, add an equal volume of glycerol, and store at -20°C for later use.

Biotin (Biotin-NHS) Labeling of Antibodies/Proteins

Biotin (D-Biotin) is a small molecule with a molecular weight of only 244.31Da. After activation (Biotin-NHS), it can bind to the amino groups of biomacromolecules such as proteins/antibodies. It not only maintains the biological activity of macromolecules well but also has a multi-stage amplification effect when used in combination with avidin, making it widely used in qualitative and quantitative detection of trace antigens and antibodies as well as localization observation research.

Studies by Wang et al. [5] have shown that there should be an appropriate ratio for biotin labeling of human immunoglobulin IgG heavy chains, so that as much biotin as possible is labeled, while avoiding excessive biotin causing steric hindrance. In this study, biotin was reacted with human immunoglobulin IgG heavy chains at various ratios, and then reacted with microsphere particles connected with anti-human immunoglobulin IgG heavy chain antibodies to evaluate the titer of the reaction between biotin and the human immunoglobulin IgG heavy chains to be labeled. The labeling method is as follows:

1.       Dissolve 4mg of purified human immunoglobulin IgG heavy chain in 1mL of NaHCO3 (0.1mol/L);

2.       Dissolve 5mg of biotin in 0.5mL of DMSO to obtain a 10mg/mL biotin solution;

3.       Slowly add 100μL of the prepared biotin DMSO solution to the solution containing dissolved human immunoglobulin IgG heavy chain, react human immunoglobulin IgG heavy chain with biotin at room temperature for 1h, and then pass the reactant through a Sephadex G column to terminate the reaction.

Fluorochrome-labeled antibodies can be used in labeling and tracing experiments such as flow cytometry (FACS), immunofluorescence (IF), and in vivo imaging; enzyme-labeled antibodies can directly react with detection substrates and are used in experiments such as ELISA, WB, and IHC; biotin-labeled antibodies can react with enzyme-linked avidin or enzyme-linked anti-biotin antibodies, and are used in antibody purification, determination of trace antigens/antibodies, etc. ANT BIO PTE. LTD. can provide a variety of antibody products and antibody labeling dyes, as well as protein/antibody labeling services with a mature system. Currently, the cycle for conventional dye labeling is 1-2 weeks.

References

[1] Chen Z J, Zhang Y L, Huang J Y. Fluorescently labeled biomacromolecules and their applications[J]. Foreign Medical Sciences. Section of Biomedical Engineering, 2004, 27(6): 348-352.

[2] Fang Z, Ju W, Qin Y N, et al. Preparation and evaluation of fluorescein isothiocyanate-labeled antibody against Escherichia coli O157:H7[J]. Journal of Jilin University (Medical Edition), 2013, 39(1).

[3] Sun L S, Qi Y, Wang B, et al. Purification of duck egg yolk immunoglobulin and preparation of rabbit anti-duck IgY (H+L) HRP-labeled antibody[J]. Chinese Journal of Veterinary Medicine, 2007(1):10-12.

[4] Xu J J, Li D, Cao Y S, et al. Preparation and preliminary application of horseradish peroxidase-labeled rabbit anti-filamentous phage coat protein antibody[J]. Heilongjiang Animal Science and Veterinary Medicine, 2013(3):3.

[5] Wang Y Y, Zhang Y D. Effect evaluation of biotin-labeled human immunoglobulin IgG heavy chain[J]. China Journal of Modern Medicine, 2012, 22(12):3.

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