Production of anti-peptide antibodies
Anti-peptide antibodies (APAs) are extremely important tools for biomedical research. Many important techniques, such as immunoblots, ELISA immunoassays, immunocytochemistry, and protein microarrays are intrinsically linked to APA function and completely dependent on APA quality. Unfortunately, not all commercially-available APAs have good antigen binding characteristics; as a result, researchers are often unable to perform high quality protein analysis experiments. This disclosure describes a new method for the scalable production of polyclonal APAs using recombinant antigens. These recombinant peptide antigens have several advantages over traditional peptide antigens which improve the ease and speed of antibody production. The recombinant antigens can be scalably produced and purified much faster than traditional synthetic peptide-conjugates. These recombinant antigen-carriers are designed to specifically aggregate in vivo after administration into the host; this aggregation greatly enhances immunogenicity and may eliminate the need for the use of chemical adjuvants which cause physical irritation and discomfort to the host.
1. A method of producing an antibody through immunization using a antigen compound, the method comprising:
obtaining an antigen compound comprising a peptide antigen fused to a carrier protein, wherein the carrier protein is GAPDH, GroEL, or KLH, fused to a thermally-responsive polypeptide sequence containing elastin-like VPGXG (SEQ ID NO. 4) polymers capable of causing the compound to aggregate when heated to temperatures above about 30° C.;
introducing the antigen compound into a living organism having an internal temperature of greater than 30° C.; and
collecting antibodies produced by the living organism in response to the antigen compound.
2. The method of claim 1 , wherein the antigen compound is introduced into the living organism in the absence of any immunostimulatory adjuvants which enhance the formation of antibodies.
3. The method of claim 1 , wherein the peptide antigen has a length of up to about 40 amino acids.
4. The method of claim 1 , wherein the carrier protein is GroEL protein fused to a thermally-responsive aggregation polypeptide sequence.
5. The method of claim 1 , wherein the carrier protein is GAPDH protein fused to the thermally-responsive aggregation polypeptide sequence.
6. The method of claim 1 , wherein the antigen compound further comprises an acceptor sequence which is heptosylated when expressed in an E. coli strain expressing the aah (bacterial heptosyl-transferase) enzyme.
7. The method of claim 1 , wherein the antigen compound further comprises T cell activating sequences.
8. The method of claim 1 , wherein the antigen compound further comprises B cell activating sequences.
9. The method of claim 1 , wherein the thermally-responsive polypeptide sequence is capable of causing the compound to aggregate when heated to temperatures above about 37° C.
10. The method of claim 1 , wherein the thermally-responsive polypeptide sequence comprises VPGVG (SEQ. ID. NO. 2) and VPGFG (SEQ. ID. NO. 3) pentapeptide subunits.