Hydrolysis of immunoglobulin G (IgG) fragments

Summary

When a new antibody needs to be fragmented, it is often necessary to perform a touch-and-go test. When choosing the reaction time, it is preferable to keep part of the antibody from being completely degraded, so as to avoid the destruction of the antibody binding site by excessive cleavage by the protease. Author: J.E. Collier et al, Translated by Xuedao Cao et al. This experiment is from "Comprehensive Immunology Laboratory Guide".

Operation method

Hydrolysis of Immunoglobulin G (IgG) fragments

Move

Papain hydrolyzes IgG to form fabric fragmentation in a mapping assay

This method is applicable to the hydrolysis of IgG from mice (any subclass), rats, humans, goats, sheep, horses, chickens, guinea pigs, and cattle.

Materials

2 mg/ml purified IgG (Unit 1.5) in PBS (Appendix 1)

Papain (2 X crystalline suspension; Sigma)

Hydrolysis buffer: 0.02 mol/LETA (disodium salt)/0.02 mol/L cysteine/PBS (freshly prepared, stored on ice for < 10 h).

0.3 mol/L iodoacetamide dissolved in PBS (freshly prepared from crystals; Sigma)

PBS

Homemade microdialysis tanks or microdialysis cassettes (e.g., Slide-A-Iyzer dialysis cassettes; Pierce)

1. Add IOOud 2 mg/m l of purified IgG to 24 microcentrifuge tubes labeled 1 to 2 4. Mix the papain suspension well without shaking. Prepare two concentrations of papain in 5 ml of hydrolysis buffer, 0.l mg/ml and 0.02 mg/ml, respectively.

2. 100 ul of 0.1 mg/ml papain solution was added to 8 IgG centrifuge tubes (enzyme/antibody ratio 1:20), IOOmI 0.02 mg/ml papain solution was added to the second group of 8 IgG centrifuge tubes (enzyme/antibody ratio 1:100), and finally IOOmI papain-free hydrolysis buffer was added to the remaining 8 IgG centrifuge tubes (control). Finally, IOOmI papain-free hydrolysis buffer was added to the remaining 8 IgG centrifuge tubes (control) and all tubes were capped.

3. Place all tubes in a 37°C water bath and remove a group of tubes at different times to create a hydrolysis time graph. A set of tubes is defined as a hydrolysis tube with an E/A ratio of 1:20, a hydrolysis tube with an E/A ratio of 1:100 and a control hydrolysis tube. The first set of tubes is removed at Ih in the water bath, followed by the second through eighth sets of tubes at 2 h, 4 h, 6 h, 12 h, 18 h, 24 h, and 48 h, respectively. After removal of the first set of tubes, hydrolysis was stopped by adding 20M1 0.3mol/L iodoacetamide to each tube.

4. Dialyze the hydrolyzed mixture against PBS using a homemade microdialysis bath or a commercial microdialysis cassette.

5. Analyze the hydrolysis products by 150 mmXl.5 mm 10 % non-reduced SDS-polyacrylamide gel electrophoresis (Unit 12.3) with a sample volume of 80 M 1. The digestion reaction is complete when no protein bands are shown on the gel at 150 kDa (for unhydrolyzed antibody) and 120 kDa (for cysteine only). Select the optimal test conditions for hydrolyzing IgG to obtain Fab fragments and perform the test according to basic protocol 2. The Fab fragments are about 50 kDa in size, the Fc fragments are 27 kDa in size, and the thin bands at 24 kDa and 15 kDa are insignificant hydrolysis products.

Papain Hydrolysis of IgG to Obtain Fab Fragments in Large Quantities

The optimal test conditions for obtaining Fab fragments were determined by a feeler test (see Basic Scheme 1).

Materials (see Appendix 1 for items with V)

Papain (2 X recrystallized suspension; Sigma)

Hydrolysis buffer: 0.02 mol/L LEDTA (disodium salt)/0.02 mol/L cysteine/PBS (freshly prepared, stored on ice for < l0h)

IgG dissolved in PBS (Appendix 1) at a concentration > lmg/m l (total > 5 mg, individual 1.5)

Iodoacetamide crystals

P B S , p H 8. O

Egg white A cross-linked agarose gel CL-4B

Poly(propylenamide) dextran S-200 Superfine (Pharmacia Biotech)

V Borate buffer, optional

10 % (m/V) NaN3, optional

5 mmX IOOmm column (Bio-Rad)

26 mmX 900 mm column (Pharmacia Biotech)

1. Mix the papain suspension without shaking. Add a volume of hydrolysis buffer equal to the volume of antibody to be hydrolyzed to dissolve papain, and select the optimal amount of enzyme, antibody concentration, and incubation time that have been determined from a feeler test.

2. Add papain solution to IgG dissolved in PBS at a concentration of 0.05 mg/kg, mix well, and incubate in a water bath at 37°C for the desired time (as determined in Basic Protocol 1). Abort the hydrolysis reaction by adding crystalline iodoacetamide to a final concentration of 0.03 mol/L. Mix carefully to ensure complete dissolution of iodoacetamide.

3. Transfer the reaction solution to a dialysis bag (CPI Appendix 3 H) and dialyze 2LPBSpH8.0 for 12-20 h at 4°C.

4. Prepare a 5 mmX100 mm protein A-crosslinked agarose gel CL-4B column (Unit 1.5, CPI Appendix 31) and sample the dialysate. Collect unbound flow-through containing Fab fragments and enzymes. If necessary, wash the column thoroughly with PBS to collect all Fab fragments.

5. Concentrate the flow-through containing Fab fragments to ≤ 5 ml (CPI Appendix 3 H).

6. Spike the concentrate onto a 26 mmX 900 mm polyacrylamide dextran S-200 Superfine column and collect fractions with a molecular mass size of 50 kDa for identification of molecular mass size by SDS-PAGE or use a pre-equilibrated gel filtration column.

7. The purity of the final product was determined by electrophoresis of 1 to 80 M1 of the final product on a 10 % non-reduced SDS-polyacrylamide gel. The concentration of Fab fragments is determined by measuring the _A280 value (Table 1.5.1). The Fab fragments are stored in boric acid buffer at 4°C or in PBS containing 0-02% NaN3 at 70°C.

Pepsin Hydrolysis of IgG to F (ab') ₂ Fractions in a Tactile Assay

Pepsin Hydrolysis of IgG to Obtain Bulk Preparation of F (al/)2 Fragments


Alternative option: Preparation of F(ab)2 by hydrolysis of IgG with pre-activated papain.

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