Glutaraldehyde method for labeling monoclonal antibody

Abstract

The invention discloses a glutaraldehyde method for labeling a monoclonal antibody, which is a glutaraldehyde two-step method, and the glutaraldehyde method for labeling the monoclonal antibody comprises the following steps: (1) dissolving HRP (horse radish peroxidase) in a glutaraldehyde solution to obtain an enzyme solution, the concentration of the HRP in the glutaraldehyde solution being 5mg / mL; and (2) adding a monoclonal antibody with the concentration of 0.24 mg / mL into the enzyme solution for labeling, wherein the molar ratio of the monoclonal antibody to the HRP is 1: 4.

Description

Technical Field

[0001] The invention belongs to immunoenzyme technology, and in particular relates to a glutaraldehyde method for labeling monoclonal antibodies. Background Art

[0002] Enzyme markers include enzyme-labeled antigens, enzyme-labeled antibodies, and enzyme-labeled SPA. The quality of enzyme markers is directly related to the success of immunoenzyme technology, so they are called key reagents. The most commonly used enzyme marker is enzyme-labeled antibody, which is formed by connecting enzymes and specific antibodies by appropriate methods. The quality of enzyme-labeled antibodies mainly depends on enzymes and antibodies with good purity, strong activity and high affinity, and secondly, a good preparation method. At present, high-quality enzymes (such as horseradish peroxidase, HRP for short) are commercially available in China. High-quality antibodies can be obtained by extraction and purification. In terms of preparation methods, it is advisable to use methods with high yield, no effect on the activity of the conjugate, no mixed interfering substances, and simple and easy operation.

[0003] Horseradish peroxidase (HRP) is the most commonly used enzyme due to its high specific activity, stability, small molecular weight, and easy preparation of pure enzyme. HRP is widely distributed in the plant kingdom and is found in high concentrations in horseradish. It is a glycoprotein composed of a colorless enzyme protein and a brown iron porphyrin, with a sugar content of 18%. HRP is composed of multiple isozymes, with a molecular weight of 40,000 and an isoelectric point of PH3-9. The optimum pH for enzyme catalysis varies slightly depending on the hydrogen donor, but is mostly around PH5. The enzyme is soluble in water and ammonium sulfate solution with a saturation of less than 58%. The maximum absorption spectra of the cofactor and enzyme protein of HRP are 403nm and 275nm, respectively. The ratio RZ of OD403nm / OD275nm is generally used to indicate the purity of the enzyme. The RZ value of a high-purity enzyme should be around 3.0 (up to 3.4). The smaller the RZ value, the more non-enzyme proteins there are. It is worth noting that purity does not indicate enzyme activity. For example, when the enzyme is denatured, the RZ value may remain unchanged. The catalytic reaction of HRP requires the substrate hydrogen peroxide (H2O2) and a hydrogen donor (DH2). Hydrogen donors are mostly colorless reduced dyes, which can generate colored oxidized dyes (D) through the reaction.

[0004] Glutaraldehyde is a bifunctional reagent that covalently binds to the amino groups on the enzyme and immunoglobulin through its aldehyde group to form an enzyme-glutaraldehyde-immunoglobulin conjugate.

[0005] Leqembi (lecanemab-irmb) is an anti-amyloid β (Aβ) monoclonal antibody that selectively binds to neutralize and eliminate soluble, toxic amyloid-β (Aβ) aggregates (fibrils) that are thought to contribute to the neurodegenerative process in AD.

[0006] However, the existing glutaraldehyde method requires an antibody concentration of 12.5 mg / mL, and the amount of HRP used is 25 mg dissolved in 1 mL of 1.25% glutaraldehyde solution (final concentration is 25 mg / ml), and the HRP-labeled antibody is an IgG antibody, that is, the existing method has large consumables and cannot determine whether the monoclonal antibody lecanemab-irmb used to treat Alzheimer's disease can be labeled. Summary of the invention

[0007] The present invention aims to overcome the deficiencies of the prior art and provides a glutaraldehyde method for labeling monoclonal antibodies.

[0008] In order to achieve the above object, the technical solution provided by the present invention is:

[0009] The glutaraldehyde method for labeling monoclonal antibodies is a two-step glutaraldehyde method: ① HRP is dissolved in a glutaraldehyde solution to obtain an enzyme solution, and the concentration of HRP in the glutaraldehyde solution is 5 mg / mL; ② a monoclonal antibody with a concentration of 0.24 mg / mL is added to the enzyme solution for labeling, and the molar ratio of the monoclonal antibody to HRP is 1:4.

[0010] Preferably, the monoclonal antibody is lecanemab-irmb.

[0011] The present invention sets different molar ratios of antibody to HRP, which are 1:2; 1:4; 1:10 respectively. The most suitable molar ratio is found (antibody molecular weight 150KD, HRP molecular weight 40KD). The amount of HRP required for HRP labeling lecanemab-irmb is tried at different concentrations of 25mg / mL, 10mg / mL, and 5mg / mL; the glutaraldehyde concentration required to dissolve HRP is 1.25%, and the glutaraldehyde diluent is double distilled water. After HRP labeling the antibody, TMB color developing solution is used to observe the color change and determine the amount of HRP; the concentration of the labeled antibody is detected by ultraviolet spectrophotometer.

[0012] The invention reduces the amount of HRP by changing the glutaraldehyde diluent, and reduces the concentration and amount of the antibody by setting the molar ratio of the antibody to the HRP, and can be well used for labeling monoclonal antibodies and HRP. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 :25mg / mL HRP excess caused precipitation, the result after centrifugation;

[0014] Figure 2 :Electrophoresis of lecanemab-irmb (left) and LEC-HRP (right);

[0015] Figure 3:Detection results after lecanemab-irmb was labeled with HRP (5 mg / mL HRP);

[0016] Figure 4 :Detection results after lecanemab-irmb was labeled with HRP (10 mg / mL HRP);

[0017] Figure 5 :Detection results after lecanemab-irmb was labeled with HRP (25 mg / mL HRP); DETAILED DESCRIPTION

[0018] Existing glutaraldehyde two-step method:

[0019] Weigh 25 mg of HRP (RZ = 3) and dissolve it in 0.2 mL of 1.25% glutaraldehyde solution (0.1 M PBS pH 6.8), and let it stand overnight at room temperature; the enzyme solution after the reaction is chromatographed on a Sephadex G-25 column and eluted with physiological saline. The flow rate is controlled at 1 mL / min, and the brown effluent is collected;

[0020] 12.5 mg of the antibody to be labeled was diluted to 5 mL with physiological saline, and added dropwise to the enzyme solution under stirring; 0.25 mL of 1 mol / L pH 9.5 carbonate buffer was added, and the pH was adjusted to 9.0-9.5, and stirring was continued for 3-4 hours; 0.25 mL of 0.2 mol / L lysine was added, and the mixture was mixed and placed at room temperature for 2 hours; an equal volume of saturated ammonium sulfate was added dropwise under stirring, and the mixture was placed at 4°C for 1 hour; centrifuged at 3000 r / min for 30 minutes, the supernatant was discarded, and the precipitate was washed twice with half-saturated ammonium sulfate, and finally the precipitate was dissolved in a small amount of 0.15 mol / L pH 7.4 PBS;

[0021] The above solution was placed in a dialysis bag and dialyzed against 0.15 mol / L pH 7.4 PBS buffer to remove ammonium ions (detected by Nessler's reagent). The solution was centrifuged at 10,000 rpm for 30 min to remove the precipitate. The supernatant was the enzyme conjugate (see results). Figure 1 and Table 1).

[0022] Table 1: Concentration of 25 mg / mL HRP collected after centrifugation of the supernatant (volume 1 mL) and passed through the column

[0023] 403nm 403nm*0.4 No. 2 0.078 0.031 No. 3 0.113 0.045 No. 4 0.080 0.032 No. 5 0.065 0.026 No. 6 0.045 0.018 .

[0024] Calculate the recovery rate after HRP hydroformylation (Table 2):

[0025] (0.031*10+0.045*10+0.032*10+0.026*10+0.018*10) / 25*100%≈6.1%

[0026] Table 2: Antibody quantity detection after antibody labeling with HRP

[0027]

[0028] Design the molar ratio of antibody to HRP 抗体 :n HRP =1:2 and 1:4, 1:10; determine the volume of the antibody to be 5 mL, use a UV spectrophotometer to detect the concentration of HRP collected after column purification, and calculate the concentration of the antibody and HRP concentration and volume (n 抗体 :n HRP =C 抗体 *5mL / 150KD:C HRP *V HRP / 40KD). After diluting the labeled lecanemab-irmb to 100 mg / mL with saline, add it dropwise to the enzyme solution, inverting and mixing once every 100 μL. 抗体 :n HRP =1:2=0.12mg / mL*5mL / 150KD: 0.4mg / mL*0.8mL / 40KD; n 抗体 :n HRP =1:4=0.24mg / mL*5mL / 150KD:0.4mg / mL*0.8mL / 40KD; n 抗体 :n HRP =1:10=0.60mg / mL*5mL / 150KD:0.4mg / mL*0.8mL / 40KD. Add 0.25mL of 1M pH 9.5 carbonate buffer, mix by inversion for 3h in a dark environment. Add 0.25mL of 0.2M lysine, mix well, and mix by inversion for 1h in a dark environment. Add an equal volume of saturated ammonium sulfate (without pH adjustment) dropwise while stirring, and place at 4℃ for 1 hour. (Make sure the saturated ammonium sulfate is around 4℃) Centrifuge at 10000rpm for 10 minutes at 4℃, and discard the supernatant. Wash the precipitate once with 50% saturated ammonium sulfate, centrifuge at 10000rpm for 10 minutes at 4℃, and discard the supernatant. Finally, dissolve the precipitate in a small amount of 0.15M pH 7.5 PBS (1mL). The above solution was placed in a dialysis bag and dialyzed against 0.15M pH 7.5 PBS buffered saline. 1L PBS buffer was dialyzed overnight. The solution was changed once in the morning, once at noon, and once in the afternoon. After removing the ammonium ions, the solution was centrifuged at 10,000 rpm for 30 minutes to remove the precipitate. The supernatant was the enzyme conjugate (see the results). Figure 2 and Table 3). The enzyme conjugates labeled with three different concentrations of antibodies were collected and the antibody concentrations were tested respectively. Antibody concentration = (OD280nm-OD403nm*0.42)*0.94*0.62.

[0029] Table 3: Antibody quantity detection after different concentrations of antibodies labeled with HRP

[0030] Concentration before antibody labeling (mg / mL) Concentration detected after antibody labeling (mg / mL) <![CDATA[n 抗体 :n HRP =1:2]]> 0.12 0.06 <![CDATA[n 抗体 :n HRP =1:4]]> 0.24 0.13 <![CDATA[n 抗体 :n HRP =1:10]]> 0.60 0.25

[0031] Conclusion: Based on the amount of antibody labeled with the original scheme, the amount of antibody detected after antibody labeling with HRP is low, and the antibody concentration required for labeling is high. The concentration and electrophoresis of the antibody after the molar ratio is changed, and the recovery rate of HRP is 抗体 :n HRP =1:4 is the most suitable molar ratio, the required antibody concentration is 0.24 mg / mL, and the antibody concentration is reduced.

[0033] Weigh 10mg and 5mg of HRP and dissolve them in 1mL of 1.25% glutaraldehyde solution (diluted with water); leave it at room temperature overnight. The enzyme solution after the reaction is passed through a (PD-10) Sephadex G-25 chromatography column and eluted with physiological saline. Collect the brown effluent at 1m / tube (Table 4, Table 5).

[0034] Table 4: Concentration of 5 mg / mL HRP (volume 1 mL) collected after column

[0035] 403nm 403nm*0.4 No. 2 0.032 0.013 No. 3 0.099 0.040 No. 4 0.074 0.029 No. 5 0.030 0.012 No. 6 0.016 0.006

[0036] Calculate the recovery rate after HRP hydroformylation: (0.013*10+0.04*10+0.029*10+0.012*10) / 5*100%≈8.2%.

[0037] Table 5: Concentration of 10 mg / mL HRP (volume 1 mL) collected after column

[0038] 403nm 403nm*0.4 No. 2 0.059 0.023 No. 3 0.089 0.036 No. 4 0.035 0.014 No. 5 0.018 0.007

[0039] Calculate the recovery rate after HRP hydroformylation: (0.023*10+0.036*10+0.014*10) / 10*100%≈7.3%

[0040] Conclusion: From the recovery rate, the original scheme of 25mg HRP was excessive and precipitated, and the recovery rate was low. After changing the glutaraldehyde diluent, no precipitation occurred after HRP was dissolved, and the recovery rate was improved. 5mg / mL is the optimal concentration of HRP and will not cause excess HRP waste.

[0041] Detect the labeling of antibody and HRP; dilute the labeled antibody-HRP at different times and add TMB colorimetric solution (see the results). Figures 3 to 5 ).

[0042] The dilution range of HRP-streptavidin ELISA detection is 4000-20000 times; antibody-HRP turns blue after dilution and exceeds the detection range. The dilution multiples of lecanemab-irmb labeled with HRP are all within the ELISA detection range, but from the recovery rate and the color development multiple after dilution, 5mg / mL HRP can save dosage and meet the experimental standards.

Claims

1. A glutaraldehyde method for labeling monoclonal antibodies, characterized in that: The glutaraldehyde method for labeling monoclonal antibodies is a two-step glutaraldehyde method: ① HRP is dissolved in a glutaraldehyde solution to obtain an enzyme solution, and the concentration of HRP in the glutaraldehyde solution is 5 mg / mL; ② a monoclonal antibody with a concentration of 0.24 mg / mL is added to the enzyme solution for labeling, and the molar ratio of the monoclonal antibody to HRP is 1:

4.

2. The glutaraldehyde method for labeling monoclonal antibodies according to claim 1, characterized in that: The monoclonal antibody is lecanemab-irmb.

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