Antibody Conjugation Protocol

Antibody labeling (also, bioconjugation) is a fundamental technique in biomedical research and diagnostics, enabling the precise attachment of molecules to antibodies for a wide range of in vitro and in vivo applications. There are several methods for attaching functional ligands to antibodies, categorized into three groups based on the site of conjugation and the techniques employed.

• Natural strategy: Conjugation occurs on a site native to the antibody structure.
• Non-natural strategy: Conjugation occurs on sites added to the antibody backbone through genetic manipulation.
• Specialty strategy: All other types of conjugation, including protein-protein interactions, engineered tags, or antibodies that possess catalytic activity.

Amine, sulfhydryl, and carbohydrate reactive moieties in the antibodies are commonly favored as sites of conjugation. The basic approach for antibody conjugation is to modify the reactive moiety with the choice fluorescent dye, probe, or biotin. Antibody conjugation is simple to perform, and the conjugates retain high levels of biological activity. The selection of conjugation technique is dependent on a number of factors including the intended target, the desired level of conjugation, and the ability to conjugate without negatively impacting antibody specificity.

Classifications of Labeling Reactions

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On the basis of the functional group involved in the conjugation process, the labeling reactions are categorized into following classes:

1. NHS/Amine Reaction: This type of reaction utilizes NHS (N-hydroxysuccinimide) esters that react with primary amines (-NH₂) present on antibodies, forming stable amide bonds. It is commonly used for attaching fluorescent dyes (e.g., FITC), enzymes (e.g., HRP), and biotin.
2. Maleimide/Thiol Reaction: This involves maleimide groups that reacts with sulfhydryl groups (-SH) on cysteine residues of antibodies or other proteins forming stable thioether bonds. This type of reaction is suitable for attaching fluorescent dyes, biotin, or nanoparticles and for antibody-drug conjugates (ADCs) for targeted therapy.
3. EDC Reaction: EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide) activates carboxyl groups (-COOH) to react with primary amines or other nucleophiles, thereby forming amide bonds, commonly used for coupling peptides or proteins to antibodies.

Materials and Reagents needed:

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• Reaction buffer (1M Sodium Bicarbonate solution or 1M Phosphate Buffer pH 8.5 - 9.0)
• Target protein (>2 mg/mL)
• 1X Phosphate Buffered Saline (PBS) pH 7.2 - 7.4
• Anhydrous DMSO
• iFluor® 488 succinimidyl ester
• Goat anti-mouse IgG secondary antibodies
• Microcentrifuge
• Sephadex G-25 column

General Protocol for conjugating fluorescent dyes to antibodies

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1. Protein Preparation
   1. Dissolve the protein in 1X PBS at a pH 7.2 - 7.4.
      Note: If the protein is dissolved in Tris or glycine buffer, it must be dialyzed against 1X PBS or undergo desalting. This will remove free amines or ammonium salts (e.g., ammonium sulfate and ammonium acetate) that will interfere with efficient conjugation. Also, impure antibodies or those stabilized with bovine serum albumin (BSA) or gelatin will not be labeled well. Even trace amounts of sodium azide or thimerosal might interfere with the conjugation reaction, and should be removed by dialysis or spin column techniques.
   2. The final protein concentration in the range of 2-10 mg/mL is recommended for optimal labeling efficiency.
      Note: Conjugation efficiency is significantly reduced if the protein concentration is less than 2 mg/mL.
2. Protein Stock Solution
   1. Mix reaction buffer (e.g., 1M Sodium Bicarbonate solution or 1M Phosphate Buffer with pH ~8.5 to 9.0) with the target protein solution to produce1 mL of the protein stock solution in such a way that the concentration is greater than 2mg/mL.
      Note: The pH of the protein stock solution should be 8.5 ± 0.5. If the pH is too low, adjust the pH of the protein stock solution by using 1 M Sodium Bicarbonate solution or 1 M pH 9.0 Phosphate Buffer.
3. Fluorescent Conjugate Stock Solution
   1. Prepare the stock solution immediately before starting conjugation.
   2. Add required amount of anhydrous DMSO into the vial of fluorescent conjugate to make the suggested stock solution.
   3. Gently mix the solution by pipetting while avoiding bubbles; briefly centrifuge the vial.
   4. Fluorescent stock solutions can be stored at -20°C for up to two weeks protected from light and moisture, but extended storage of the dye stock solution may reduce the dye activity. Avoid freeze-thaw cycles.
4. Conjugation Reaction
   1. First, determine the Dye:Protein ratio.
      The optimal Dye:Protein ratio is dependent on inherent properties of the protein and the dye. Over-labeling of a protein could detrimentally affect its binding affinity, while under-labeling may not provide an adequate fluorescent output.
      Note: Each protein and label combinations may require optimization. Use a 10:1 molar ratio of Dye:Protein solution as the starting point.
   2. Add 5 µL of the dye stock solution (10 mM) into 95 µL of the protein stock solution. Pipet or centrifuge briefly to mix.
   3. Assuming the protein concentration of 10 mg/mL and molecular weight of ~200KD, the amount of the protein in the sample is ~0.05 mM.
   4. If results are not optimal, alternate dye:protein ratio (e.g., 5:1, 15:1, and 20:1) may be used.
   5. Cap the vial and place the sample on a gentle rotator or shaker, and mix reaction mixture at room temperature for 30-60 minutes. Aluminum foil may be wrapped around the vial to protect the sample from light.
      Note: Excessive agitation of the sample may result in protein denaturation, and must be avoided.
5. Purification of the Conjugated Proteins
   Note: The following protocol is an example of dye-protein conjugate purification by using a column containing a Sephadex G-25 medium.
   1. Prepare a Sephadex G-25 column according to the manufacture instruction.
   2. Load 100 µL of the dye-conjugate sample into the center of the column.
   3. Add PBS (pH 7.2-7.4) as soon as the sample starts to run, just below the top resin surface.
   4. As the sample absorbs into the resin bed, place the column into an empty collection tube and
   5. Transfer the Sephadex G-25 column to a new collection tube, and add 100 µL PBS to the column. Centrifuge the sample at 1000 g for 5 minutes.
   6. Collect the fractions that contain the desired dye-protein conjugate.
      Note: For longer-term storage, the dye-protein conjugate solution should be concentrated or freeze-dried.
6. Characterize the Desired Dye-Protein Conjugate
   The Degree of Substitution (DOS) or Degree of Labeling (DOL) is important to calculate for characterizing dye-labeled protein. Proteins of lower DOS usually have weaker fluorescence intensity, but proteins of higher DOS (e.g., > 6) tend to have reduced fluorescence. The optimal DOS for most antibodies is recommended between 2 -10, depending on the properties of dye and protein.
   1. Measure absorption
      Dilute a small amount of the dye-protein conjugate in PBS and measure the absorbance at 280 nm (A280) and the absorbance maximum for the respective dye at its expected wavelength. For most spectrophotometers, the sample (from the column fractions) may need to be diluted with deionized water or PBS so that the OD values are in the range of 0.1 - 0.9. It is recommended to keep the sample concentration between 1-10 µM depending on the extinction coefficient of the dye.
   2. Calculate the protein concentration
      Note: A nanodrop or similar equipment may also be used to measure the protein concentration.
   3. Use the equation below to calculate the protein concentration, where CF if the correction factor of the fluorophore and Ɛ is the extinction coefficient of IgG, at 280 nm.
      Protein concentration (M) = ([A280 - (Adye x CF)] x dilution factor) / Ɛ
   4. Calculate the degree of labeling
      Use the equation below to calculate the degree of labeling, where Ɛdye is the molar extinction coefficient of the specific dye.
      Mol dye per Mol protein = (Adye x dilution factor) / (Ɛdye x protein concentration)
      
      You can calculate DOS using our tool by following this link:
      https://www.aatbio.com/tools/degree-of-labeling-calculator

Dive into our Antibody & Protein Labeling Selection Guide for extensive range of products, or reach out to us for Personalized Solutions tailored to your unique labeling needs!

Also explore AAT Bioquest's Array of Antibody and Protein Labeling Kits, meticulously designed with easy protocols to support scientists at every stage of their research journey.

Further reading: A Practical Guide for Labeling Antibodies

References

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1. Antibody conjugates for cell biology
2. Antibody conjugation and formulation
3. Bioconjugation Protocols