Affinity Purification of Immunoglobulin

Aim: Affinity Purification of Immunoglobulin

BACKGROUND

Numerous in vivo and in vitro applications of antibodies include drug targeting, the development of immunodiagnostics, immunoprophylactic, and immunotherapeutic, the detection of foodborne pathogens, adulterants, toxins, and residues in food samples, environmental analysis/monitoring, and the isolation of a variety of proteins and other targets for basic research and industrial processes. Homogeneous antibody preparations are excellent for the majority of the aforementioned applications. In most cases, antibodies are extracted from bacterial cultures, plasma, serum, ascites fluid, or cell culture medium. Effective techniques for the purification of antibodies are also essential because all of these sources also contain a variety of possible contaminants.

There have been many documented chromatographic procedures that make it easier to isolate antibodies, for example, based on various biochemical traits (such size, solubility, charge, hydrophobicity, and binding affinity). Affinity chromatography, however, is the most effective and popular method for antibody purification.

The sole method in purification technology that allows the purification of a biomolecule based on its specific chemical makeup or biological function is affinity chromatography. The approach was created by Meir Wilchek, Meir Wilchek, and P. Cuatrecasas, for which they received the Wolf Prize in Medicine in 1987.

By using a reversible association between a protein (or collection of proteins) and a particular ligand attached to a chromatographic matrix, affinity chromatography separates proteins. Affinity chromatography frequently makes purification possible that would otherwise be time-consuming, challenging, or even impossible using other techniques. The method can be used to isolate pure chemicals present in huge quantities of crude sample at low concentrations as well as to remove particular impurities. It can also be used to separate active biomolecules from denatured or functionally different versions. The method delivers high resolution, good selectivity, and often high capacity for the target protein. Recovery of active material is often quite high, and purification rates can reach several thousand-fold.

In immunoaffinity chromatography (IAC), the basis for separation is the binding affinity of an antigen to a parent antibody. Antigens and antibodies bind strongly and specifically, allowing for a variety of applications in IAC employing either immobilized antigens or antibodies. IAC was identified back in the 1930s. With the advancement of chromatographic materials and immobilization techniques over the past 20 to 30 years, it has steadily turned into a crucial instrument in the life sciences.

Immobilized antibodies and antigens have been used by researchers as affinity reagents for the purification of antibodies. However, these methods were constrained by the caliber of the antigen and/or antibodies, which resulted in harsh elution conditions, low binding capacity, and/or low specificity. On the other hand, affinity chromatography techniques using Proteins A, G, A/G, and L and thiophilic ligands have expanded in their range of applications, particularly in the context of industrial-scale purifications where Protein A is a highly favoured and widely utilized ligand. Each of these ligands, however, comes with a set of benefits and drawbacks.

REQUIREMENTS

S. no.
1	Ascites fluid or monoclonal/polyclonal antibodies supernatant
2	PBS Buffer
3	Tris base Buffer
4	Citric acid
5	Column
6	Dialysis tubing and clamps
7	Magnetic stirrer and stirring rod
8	Absorbent paper
9	Forceps
10	Glass pipette
11	Pump
12	Refrigerated centrifuge
13	Centrifuge tube
14	0.45µm filter
15	Syringe
16	Spectrophotometer
17	Pasteur pipette
18	Test tubes

PROCEDURE

1. Centrifuge the supernatant of a monoclonal antibody at 10,000 rpm and 4 °C.
2. Pass a 0.45-m filter through the supernatant to filter it.⁵
3. Dialysis against PBS and use of dialysis tubing to bring pH of monoclonal supernatant to 8.0.
4. Protein A-Sepharose column preparation and attachment to fraction collector.⁴
5. Equilibrate the column at 4 °C or room temperature using PBS, pH 8.0.
6. The resin bed with an antibody solution layer.
7. Wash the column with a lot of PBS that has a pH of 8.0.³
8. Elute at a suitable pH of 3 (raise to the proper pH with 1 M NaOH) with 0.1 M citric acid for mouse IgG1, 4.5 M for IgG2a, and 3.0 M for IgG2b and IgG3.
9. Dialyze the elutes against PBS, pH 7.3, after collecting the eluent in vials.
10. Two times through the dialysis buffer. Samples should be kept at 4°C in PBS or borate-buffered saline.³

CONCLUSION

Immunoglobulins purified successfully by using affinity chromatography method and store sample up to 2 to 3 months at 4˚c.

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REFERENCES

1. Shuai Sheng and Fansheng Kong, “Separation of antigens and antibodies by immunoaffinity chromatography”, Pharmaceutical biology,2012, 50(8): 1038-1044.
2. Aiying Xue and Saijun Fan, “Matrices and affinity ligands for antibody purification and corresponding application in radiotherapy”, Biomolecules, 2022, 12, 821.
3. Sushrut Arora, B. Vijayalakshmi Ayyar and Richard O’Kennedy, “Affinity Chromatography for antibody purification”, Methods in molecular biology (Clifton, N.J.), March 2014.
4. Yuichi Imura, Toshiaki Tagawa, Yuya Miyamoto, Satoshi Nonoyama, Hiroshi Sumichika, Yasuhiro Fujino, Masaya Yamanouchi and Hideo Miki, “Washing with alkaline solution in Protein A purification improves physiochemical properties of monoclonal antibodies”, Scientific reports, 19 January 2021.
5. https://vlab.amrita.edu/?sub=3&brch=70&sim=1099&cnt=2

Also read:

• Preparation of enzyme conjugated antibodies
• Purification of Immunoglobulin by Precipitation
• Microplate based Alamar Blue Assays
• Isolation of Genomic DNA from E. coli
• Estimation of DNA and its purity check using UV spectrophotometer (A260 measurement)
• Study of Polyploidy in Onion Root Tip Bycolchicine Treatment

FAQs

1. Can we reuse column resin?

 Ans: Yes, resin can be reuse up to 10 without losing its binding capacity.

2. How can we confirm whether our immunoglobulin is purified or not?

 Ans: For immunoglobulin purity conformation use SDS-PAGE or Spectrophotometric analysis.

 

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