5 Methods of Antibody Purification

After making and harvesting antibodies, you will need to purify them and get rid of unwanted material from the serum. Antibody purification is a mandatory stage before fragmentation. There are many methods you can employ to purify the antibodies and achieve your target. Of the many methods, we delve into five of the most-relevant ways you can use in your lab.

1. Ammonium Sulfate Precipitation

Ammonium sulfate is a common precipitation agent when purifying antibodies from serum or supernatant from cell culture. It also works well with ascites. Ammonium sulfate is a salt that causes proteins to become less soluble. When adding it to your sample, a process called salting out, it causes antibodies and other molecules to precipitate from serum.

Complete saturation of ammonium sulfate is 4.32M, and at between 40 and 50% of the concentration, immunoglobulin starts precipitating, leaving other serum components dissolved. To achieve adequate precipitation, you start by adding a small amount of the salt into the antibody-containing sample. Then give your mixture some hours of incubation to settle at around 4°C.

Afterwards, you can use centrifugation to remove the supernatant. You can then dissolve the antibody-pellet in a buffer. The yield of antibodies and their purity, selectivity, and reproducibility depends on the pH, temperature, and time of incubation. The rate at which you add ammonium sulfate also matters.

2. Melon Gel Chromatography

Melon Gel is a chemical buffer system that purifies antibodies through fractionation. The buffer binds the non-IgG components in the sample and leaves the IgG antibodies dissolved. You can collect the antibodies from the flow-through fraction.

Antibody production companies use Melon Gel as an effective method of bovine serum albumin removal. The buffer also removes gelatin from stock antibody solutions. This step ensures that the antibodies labeling process goes smoothly.

When working with monoclonal antibodies, you will need a two-step purification. A cell culture supernatant needs to undergo ammonium sulfate purification before you use Melon Gel. A conditioning reagent is necessary before you purify ascites fluid with Melon Gel. This reagent will reduce the chances of purifying transferrin along with the antibodies.

3. Ion Exchange Chromatography

Apart from being more convenient and easy-to-implement, ion exchange is also a cost-effective method of antibody purification. The method utilizes the charge on the molecules and compounds to separate antibodies from other proteins and substances dissolved in your sample.

The chromatography employs a column with resins of either positive or negative charges. When the proteins pass through the column, they bind to the resins according to the corresponding charge. Proteins (antibodies) with a positive charge bind to negatively-charged resins and vice-versa.

Using this method, you can charge the antibodies you need to purify so that they bind as other substances pass through with the buffer. Another method to employ is to design your chromatograph to bind to other proteins except for the antibodies. This alternative method helps you when you have a specific target of antibodies to purify. The system will then bind to everything else except the target antibodies, which you can process further.

4. Antigen-specific Affinity Purification

One fact about antibodies is that each specific antibody targets a specific antigen. With this characteristic, you can purify specific antibodies from other forms of proteins (including antibodies you do not need) from the serum. This method is most-effective when you want specific types of polyclonal antibodies.

In this method, you will need an antigen that is specific to the antibody you want to purify. Known as ligand immobilization, the process entails immobilizing an antigen that attracts the antibodies you want to purify. You would need about 1mg of the antigen, and use agarose beads to immobilize it. Then add the antiserum to the affinity column.

The time taken for the reaction (affinity) depends on a number of factors, but the concentration and rate of diffusion matter the most. After the binding, you elute the antibodies from the antigen to obtain pure antibodies as per your specific target. An ELISA of both the crude serum and the purified antibodies will help you determine your success in the process.

5. Chromatography by Size Exclusion

Various compounds and proteins dissolved in the serum have a distinct weight and size. Size exclusion helps you to purify antibodies according to their size. The chromatography column uses agarose, polyacrylamide, or dextran beads. The separation of the antibodies relies on the size of the beads used in your chromatography.

Through dialysis, desalting, and diafiltration, you can separate the smaller molecules from the solution. Resin can then separate immunoglobulins of 140 kDa or less from the remaining solution of heavier molecules. This method allows you to get your polyclonal antibodies as per their exact weight and size.

In Summary

Although antibodies are proteins like many other constituents of blood serum, you can use specific methods to purify them from the rest of the content. The method you choose to purify the antibodies may vary from another depending on your goals. You will need to study the type of antibodies you are working with and their chemical, biological, and physical properties to determine the best antibody purification method for your scenario.

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