It is not difficult to immobilize a protein on a solid support using any classic activation method such as the cyanogen bromide or N-hydroxysuccinimidylcarboxylate... However, if the protein of interest is a specific Ig (IgG, IgY...), its biological activity will be affected if not entirely lost after the covalent linkage between the solid support with the terminal H₂N of the Ig (IgG, IgY...).

AFFILAND has developed the Activated Protein A like-Matrix useful for the immobilization of IgG on the F_c portion, leaving free the Fab: the biological properties of the specific Ig (IgG, IgY...) will be conserved.

CHARACTERISTICS OF THE GEL

Matrix: Sepharose^TM CL-4B
and/or Sepharose^TM 4 Fast Flow
Ligand: PROTEIN A LIKE which has a specific binding for the F_c of immunoglobulins. It does not recognize serum albumin or serum proteins other than immunoglobulins.
Rapid formation of a stable complex between the ligand and the F_c portion of Ig (IgG, IgY...) from all animal species, in the binding & coupling buffer, followed by an immediate covalent linkage between the Activated matrix and the F_c portion, leaving free the F_ab of Ig (IgG, IgY...).
Easily realized in a column or in a batch.
Sample: can be either the antisera, ascites fluid, extract containing antibodies or purified Ig (IgG, IgY...)...
Stability of the matrix: pH 6-9.
Shelf life of the matrix: one year after delivery when stored at 4°C.
Storage: 4°C in PBS 50mM pH 7.4; NaN₃ 0.02% (w/v).

Results

Easy recovery of the antibody immobilized on the gel in a record time of 30 minutes maximum. The capacity of the obtained affinity matrix may be 5-6 mg of mouse monoclonal Ig (IgG, IgY...) or 10-12 mg of polyclonal Ig (IgG, IgY...) of other animal species, per ml of matrix.

APPLICATIONS

• Purification of antigens by immunoaffinity chromatography.
• Easy preparation of serum free of antigen.
• Radio or enzyme immunoassay.

INSTRUCTION FOR USE

Reagents ready for use:

• Activated Protein A like-Agarose (Code: APAL-M1).
• Binding & coupling buffer (Code: BPAL).
• Saturation reagent (Code: SRP).

Washing Buffer (WB) to be prepared:
by mixing 500 ml of distilled water with 500 ml of Binding & Coupling Buffer (BPAL) of the kit.

1. Load the gel into a column with a frit of porosity P.1.
2. Equilibrate the column with 2x volume of Washing Buffer (WB) as prepared previously.
3. Make up the sample.
   - Either 1 vol. of antisera, ascites fluid, extract containing antibodies... + 1 vol. of binding & coupling buffer.
   - Or dissolve the solid purified Ig (IgG, IgY...) in the Washing Buffer (WB) as prepared previously, at a concentration of 1-2 mg of Ig/ml.
4. Apply the sample into the column at a flow rate of 20-30 ml/cm²/hour.
5. Wash the column with the Washing Buffer (WB) at a flow rate of 50 ml/cm²/hour until the O.D at 280 nm of the eluent falls to the baseline level.
6. Wash the column successively with the PBS 50 mM pH 8.0 buffer and the NaOAc 50 mM pH 4.0 buffer, at a flow rate of 50ml/cm²/hour until the O.D at 280 nm of the eluent reaches the baseline level.
7. Load onto the column 3 volumes of saturation reagent, at a flow rate of 20 ml/cm²/hour.
8. Wash the column with 3x volumes of PBS 50 mM pH 7.4 buffer or the TBS 20 mM pH 7.4 buffer, at a flow rate of 50 ml/cm²/hour, until the O.D at 280 nm of the eluent reaches the baseline level.
9. The obtained immunoaffinity gel is ready-to-use.
10. Storage: 4°C in PBS or TBS buffer pH 7 .4, in the presence of NaN₃ 0.1% (w/v).

1. Filter the gel on a buchner funnel.
2. Wash the gel with 3x volume of distilled water and then with 3x volume of Washing Buffer (WB) as prepared previously.
3. Mix gently (using a rotor) a suspension of Activated PROTEIN A LIKE-AGAROSE either in 1 vol. of antisera, ascites fluid, extract containing antibodies... plus 1 vol. of binding & coupling buffer or in a solution of solid purified Ig (IgG, IgY...) dissolved in the Washing Buffer (WB) as prepared previously (1-2 mg of Ig (IgG, IgY...) per ml), for 20-25 minutes.
4. Filter the suspension on a buchner funnel.
5. Wash the gel with the Washing Buffer (WB) (3 times of 2 volumes).
6. Wash the gel successively with the PBS 50 mM pH 8.0 buffer and the NaAcO 50 mM pH 4.0 buffer (3 times of 2 volumes for each).
7. Mix gently (using a rotor) the gel with 3x volumes of saturation reagent, at a flow rate of 20 ml/cm²/hour, for 15 minutes.
8. Load the suspension into a column with a porosity P.1 and wash the column with 3x volumes of PBS 50 mM pH 7.4 buffer or the TBS 20 mM pH 7.4 buffer, at a flow rate of 50 ml/cm²/hour, until the O.D at 280 nm of the eluent reaches the baseline level.
9. The obtained imunoaffinity gel is ready-to-use.
10. Storage: 4°C in PBS or TBS buffer pH 7 .4, in the presence of NaN₃ 0.1% (w/v).

Immunoaffinity gel preparation: 4 ml APAL-M1 + (2 ml rabbit antisera anti hTBG + 4 ml binding & coupling buffer) (see instruction for use).
Sample: 15 ml human pregnancy serum.
Binding & washing buffer: K₂HPO₄ 50mM, NaCl 0.9% (w/v) pH 7.4.
Elution buffer: Glycine 0.1M pH 2.8 (with HCl 6N).
Flow rate: 20ml/cm²/hour for all operations.
Regeneration buffer: Acetic acid 1M.

Results

1. 190 µg of hTBG in 2 mg of total proteins.
   hTBG was determined by RIA and total proteins by Lowry method.
2. Life time of the immunoaffinity gel with routine use: Minimum 6 cycles with no loss of binding capacity.

Similar experiments were realized with rabbit antisera to hCG, rabbit antisera to hSA, mouse anti hTg ascites fluid and mouse anti pSA ascites fluid. The antigen binding ratio was determined to be approx. 1:1 (Ab:Ag) in all cases.