The Detailed Preparation Method for Polyclonal Antibody

Polyclonal antibodies (pAbs) are secreted by different B cell lineages within the bodies. They are a collection of immunoglobulin molecules that react against  specific antigen, each identifying a specific epitope. Polyclonal antibodies can be used in WB, IHC, Elisa, IP and so on, it is indispensable research partners in biomedical research. Here we introduce the polyclonal antibody preparation.

1 The preparation of antigen

For ordinary protein, it can be obtained after the following steps: 

A: combinate with molecular cloning vector

B: inducible expression of recombinant proteins in E. coli

C. purification. 

Then the purified protein can be directly used as the immunogen.

Small molecular proteins or peptides, their weight is very light, they can not be used for antigen directly. Hence, they need for coupling to a carrier molecule . In this way small molecular proteins or small compounds can be transformed to immunogenic antigen, common carriers such as BSA, OVA, HAS.

2 Immunized animals

When choose an immunized animal, researcher should consider the species differences between antigens and immune animal, the similarity will affect the immunization. Generally, animal-age, health, weight need to meet the requirements. 

The commonly used anti-serum animals are guinea pigs and rabbits and chickens, rats and mice, etc. If for mass production, the dogs, sheep, goats were the better choices. Immunizing dose:  mice 1.0 ~ 2.0 ml, rabbits is 2 ~ 4 ml. 

Adding adjuvant may help sustain release of antigen at the injection site, increased immune effect, Freund's incomplete adjuvant is widely used adjuvant .

After the primary immunization , it still need more than 2 to 3 times boosters to generate a higher level of IgG antibody formation. The interval between the primary immunization and revaccination, generally every 10 to 14 days for small animal( mouse, rat and so on),  for the large animal(goat, dogs as above) it will be around 60 days large animals. After each booster, the anti-serum titer determination method was used for detecting antibody titer , only the titer is high enough , the anti-serum can be collected .

3 Collection of Anti-serum

Collection methods:

	Rabbit	Sheep	goat	Chicken	Dog	Rat	Guinea Pig	Mouse
Artery blood	♦	♦	♦
Heart blood	♦			♦	♦	♦	♦
Venous blood	♦	♦	♦
Orbital blood/Tail							♦	♦

The blood serum precipitation: 3000rpm centrifugal 15 min, the supernatant. 

This process should pay attention to animal welfare, the anti-serum still needs further purification.

4 Purification and Characterization of Anti-serum

After collection, the anti-serum needs further purification. Now, conjugated antigen affinity column is the common tool for purification. The purified antibody is provided with high titer, high specificity, high purity. 

Next step is the identification of the purified antibody, in terms of relative molecular weight, purity and specificity.

5 Preservation of Anti-serum

It is recommended to aliquot preservation. Generally the antibody is more stable at lower temperature, it can be stored at  -20 ℃ ~ -80 ℃  for 5 years without degradation. The period will be longer if stored in form of the vacuum drained . 

Kind notice: Sterilization and add preservatives before preservation.

For the polyclonal antibody has a low cost, short preparation period and the preparation step is relatively simple and can recognize multiple epitopes advantages , it is widely used in the field of scientific research. 

Above is the basic preparation process of Polyclonal antibody .

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