The coupling modification of gABA

gABA（Gamma-Aminobutyric Acid）is the main inhibitory neurotransmitter of central nervous system in mammal, which mainly reduces the excitability of neurons. Besides the ability of directly adjusting the muscle tensity, gABA also has other functions, for instance, people will feel anxious and tired when he/she lacks gABA in the body. Consequently, gABA has great influences on normal physiological function of human being.

gABA is a natural non-protein amino acid, which shares same molecular weight in different species. Aiming at this, Cloud-Clone Corp. developed a series of detection tools on gABA which includes coupling molecules, antibodies and Elisa kits. The development and market access of gABA products bring researchers great access on gABA research tools.

1. The coupling modification of gABA

gABA is a small molecule that has no immunogenicity, which needs to couple with other proteins to acquire immunogenicity. Cloud-Clone Corp. adopts glutaraldehyde to couple gABA with BSA, OVA and HAS to obtain BSA Conjugated gABA (CPA900Ge11), OVA Conjugated gABA (CPA900Ge21) and HSA Conjugated gABA (CPA900Ge41). The process is as followed. Dissolving gABA and any of above vector proteins in PBS, and then adding glutaraldehyde and reacting for 1 h, followed by separating with G-100 column, antigen with immunogenicity was obtained.

2. Preparation of anti-gABA antibody and its coupling modification

The OVA-gABA was used as the antigen to immunize rabbit to prepare antiserum, after purification, specific anti-gABA polyclonal antibodies were obtained (PAA900Ge01).

And then, the antibody was labeled by biotin to prepare biotin-linked antibody to gABA (PAA900Ge71).

Biotin labeling method:

 There are two cyclic structures in biotin (Fig. 3), the valeric acid side chain end is the binding site of antibody or other biomacromolecules. The biotin is activated by chemical modification, and then the activated biotin could be coupled with biomacromolecules by one kind of protein cross-linking agent, the biomacromolecules include protein, nucleic acid, polysaccharide, lipid, etc.

Dissolving gABA with carbonate buffer solution, adding the same mole of activated biotin-ester, mixing and depositing overnight at 37℃. After getting rid of the uncombined gABA by dialysis tube with 0.01M PBS, the conjugated products were collected, concentrated and freeze-dried for later use.

The FITC labeling method:

In alkaline conditions, The sulfur producyion chemical bound of FITC will react with gamma amino groups of lysine in protein to get FITC labeled fluorescent compounds. The basic principle is as followed:

FITC-N=C=S +N-H2-protein            FITC-NS-C-N-H2-protein

Cloud-Clone Corp. adopts the Marsshall method to label FITC on gABA.

3. The gABA Elisa kits

Based on the development of coupled molecules and specific antibodies, Cloud-Clone Corp. develops gABA Elisa kit (CEA900Ge). As gABA is a small molecule and exists as the same form in different species, so the antibodies and kits could be used in the detection of gABA from samples collected from different species.

For more information, please visit http://www.cloud-clone.us/.