Question 1

Why can't B-cells be cultured directly to produce monoclonal antibodies?

Accepted Answer

B-cells do not survive well in long-term culture, making it impossible to maintain them indefinitely for antibody production. To overcome this limitation, scientists fuse B-cells with immortal myeloma cells to create hybridomas, which combine the antibody-producing properties of B-cells with the indefinite growth capacity of myeloma cells.

Question 2

What is the difference between polyclonal and monoclonal antibodies?

Accepted Answer

Polyclonal antibodies are a mixture of antibodies, each recognizing different epitopes on an antigen, produced by multiple B-cell clones. Monoclonal antibodies are identical antibodies produced by a single hybridoma cell line, recognizing only one specific epitope. This makes monoclonal antibodies more specific and useful for distinguishing between similar antigens.

Question 3

How does limiting dilution ensure monoclonal antibody production?

Accepted Answer

Limiting dilution is a serial dilution process that statistically ensures single cells are seeded into screening plate wells. Wells containing one cell grow into monoclonal populations producing identical antibodies. This cloning method guarantees that the resulting hybridoma line releases only the desired antibody of known specificity.

Question 4

What role does ammonium sulfate play in antibody purification?

Accepted Answer

Ammonium sulfate precipitation removes antibodies from culture medium by competing with proteins for water molecules through hydrophilic interactions. The highly charged ammonium and sulfate ions decrease antibody solubility, causing them to precipitate out of solution. This salting-out method is a standard technique for protein purification from culture supernatants.

Question 5

Why is dialysis tubing used after ammonium sulfate precipitation?

Accepted Answer

Dialysis tubing removes residual ammonium sulfate from the antibody solution through osmotic exchange. The antibody solution is placed in dialysis tubing and immersed in PBS buffer overnight. Water and small salt ions pass through the tubing membrane while antibodies remain inside, resulting in purified antibody free of ammonium sulfate.

Question 6

How is antibody concentration determined after purification?

Accepted Answer

Antibody concentration is measured using a spectrophotometer at 280 nanometers, which detects protein absorbance. A 20-fold dilution of the antibody sample is prepared in PBS, then analyzed using the spectrophotometer. The concentration is calculated using a standard formula and recorded for documentation and storage of the purified monoclonal antibody.

Question 7

What screening method is used to identify hybridomas producing the desired antibody?

Accepted Answer

ELISA assays indirect sandwich and competitive methods are used to screen hybridoma culture media for the presence of the target antibody. After hybridoma fusion and limiting dilution cloning, ELISA detects which wells contain cells producing the desired monoclonal antibody. This screening step ensures only hybridomas with the correct antibody specificity are selected for expansion and antibody production.

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