- Polyclonal Antibody Overview
- Polyclonal Antibody Production Methods (Rabbit Polyclonal Antibody)
Polyclonal antibody Overview
There are two different immune responses: humoral and cell mediated response. Humoral response involves activation of B cell that is converted to plasma cells, which secretes antibodies. During a humoral response, the antigens are separated into small pieces due to antigen presenting cells (APC) and are detected by B cells. During this activation process, B cells differentiate into plasma cells massively producing and secreting a single type of antibody.
There are many different types of antibodies produced because antigens in general possess a variety of epitopes and plasma cells. The various types of antibodies secreted from a plasma cell are called a polyclonal antibody. On the other hand, monoclonal antibody is a single type of antibody that is produced from a single type of plasma cell.
The first step of polyclonal antibody production is to immunize mouse, rabbit, or goat with antigen to produce antibody against that antigen. The polyclonal antibody that is produced is then drawn from the blood of the animal. During immunization, prepare the antigen in a similar fashion as when producing monoclonal antibody. Here, we will discuss production of rabbit polyclonal antibody.
Polyclonal Antibody Production Methods (Rabbit Polyclonal Antibody)
* This method is a general method, and the steps may vary depending on the experiementer.
- (1) Acquire a pre-immune serum by bleeding the ear of a rabbit.
(This serum will be used as a negative control.) - (2) Mix the antigen (Protein or peptide conjugated with KLH) with CFA (Complete Freund’s adjuvant to produce
an emulsion. - (3) Inject the emulsion into various parts of the rabbit (1st immunization).
- (4) After 4 weeks, produce a second emulsion by mixing the antigen with IFA (Incomplete Freund’s adjuvant).
Inject this into the rabbit (2nd immunization). - (5) After 1 week, collect blood from the rabbit and measure the level of immunization.
- (6) 2 weeks after the second immunization, create an emulsion by mixing the antigen with IFA
(Incomplete Freund’s adjuvant) and inject into the rabbit (3rd immunization). - (7) After 1 week, collect blood from the rabbit and measure the immunization level.
- (8) 2 weeks after the third immunization, create an emulsion by mixing the antigen with IFA
(Incomplete Freund’s adjuvant) and inject into the rabbit. - (9) 1 week after the fourth immunization, draw the whole blood.
* Whole blood drawing can be conducted before the 4th immunization if the titer is high.