Antibody Purification

Question 1

5 sources of antibody

Answer 1

1-serum (polyclonal antibody)
2-Ascites fluid
3-culture supernatant of a hybridoma cell line (monoclonal antibody)
4-egg yolk (IgY)
5-Recombinant

Question 2

3 purification methods

Answer 2

1-Crude
2-General
3-Specificity

Question 3

What is antibody purification?

Answer 3

Selective enrichment of specific isolation of antibodies

Question 4

What is the general scheme for antibody purification?

Answer 4

Sample prep—>Main purification—>Finishing (polishing)

Question 5

Sample Preparation example

Answer 5

Purpose:Removal of:

• Gross impurities
• Specific impurities
• Buffer exchange and desalting to transfer sample to the correct buffer

Question 6

What are the main approaches to sample preparation?

Answer 6

Salt precipitation
Clarification
Filtration
buffer exchange

Question 7

2 ways main purification occurs

Answer 7

1. Affinity Chromatography
2. Ligand type- Fc specific
   - Immunospecific

Question 8

Affinity Chromatography advantages

Answer 8

• High selectivity
• Target molecule is concentrated into a smaller molecule
• Purity levels as high as 99% are achievable in 1 step

Question 9

Purification Process

Answer 9

1. Apply sample in buffer
2. Wash through with buffer
3. Elute specific antibody at high or low pH
4. Collect and neutraslise

Question 10

Ion exchange Chromatography

Answer 10

Ion exchange resins contain charged groups
If groups are positively charged–> cation, positive charge
If groups are negatively charged–> anion, negative charge

Question 11

Further purification info

Answer 11

Size exclusion to separate proteins by size
Hydrophobic interaction chromatography:
-Based on the differences in hydrophobicity at high salt concentration

Question 12

What gel is usually used for Purification in HIC (hydrophobic interaction chromatography)

Answer 12

Sepharose gel

Question 13

How is HIC (hydrophobic interaction chromatography) desalted?

Answer 13

gel filration

Question 14

What is antibody purification?

Answer 14

Antibody purification is a process in which antibodies are extracted from an antiserum which contains a mixture of compounds. Once purified, the antibodies can be used in a variety of ways

Question 15

Explain crude antibody production

Answer 15

precipitation of a subset of total serum proteins that includes immunoglobulins

Question 16

Explain Specific antibody production

Answer 16

affinity purification of only those antibodies in a sample that bind to a particular antigen molecule

Question 17

Expalin general antibody production

Answer 17

affinity purification of certain antibody classes (e.g., IgG) without regard to antigen specificity