Signature Immunologics, Inc. Product Users
How can antibodies detect such small molecules?
Antibody binding targets a distinctive pattern of atoms. This pattern can be as small as a single amino acid residue. A non-immunogenic small molecule (a hapten) can be presented to the immune system by coupling it to a carrier protein with a bifunctional link such as glutaraldehyde (GA). This creates a highly immunogenic hapten-GA-carrier complex that elicits production of IgGs highly specific for the hapten. Read more & references.
How can an antibody selectively detect free amino acids but not those in proteins?
Anti-amino acid IgGs target the α-carboxyl groups of free amino acids. In proteins, α- carboxyl groups are occluded by peptide bonds. Cells containing 0.1-10 mM glutamate are readily detected by SII E100 IgG, but glutamate in proteins is invisible to E100, even though the molar equivalent of glutamate in protein ranges from 50-150 mM.
Why does SII sell polyclonal antibodies? Aren't monoclonal antibodies better?
Polyclonal antibody methods allow for rapid production of the highest-affinity and highest-titer anti-hapten IgGs. There is no advantage to more expensive monoclonal methods because our immunogens are effectively monoepitopic.
Everything is made in rabbit? Don't you have any other hosts?
Yes. Chicken IgY anti-GABA, anti-glutamate and anti-AGB will be available in early 2008. More will be produced soon.
Do I have to use glutaraldehyde?
Yes and no. Most SII IgGs are generated using hapten-glutaraldehyde-carrier techniques and the affinity of the IgGs is highest for the hapten-glutaraldehyde product. Further, radiotracer studies show that over 95% of free amino acids are lost when only formaldehyde is used. So if you want the highest selectivity and sensitivity, some glutaraldehyde is necessary see table. However, there are slow (and poorly understood) formaldehyde reactions that can lead to small amounts of cross-linking. This products are sometimes detectable with high-amplification methods. SII does not guarantee that you will be able to do this in your tissue.
How much do I glutaraldehyde do I need?
Amino acid trapping is a function of time and glutaraldehyde concentration. Optimal trapping is achieved with 0.1-2.5% glutaraldehyde,1% formaldehyde using High Performance Immunocytochemistry (HPI).
Why do I also have to use formaldehyde?
FA tremendously reduces non-specific background signals. The mechanism is complex, but it is a very powerful effect and we recommend that you always use FA in fixatives.
What is the best histologic method for visualizing small molecules?
Thin epoxy resin sections with resin-etching and conventional transmission or fluorescence imaging provides extremely high sensitivity and reproducibility for small molecule detection. The optimal method is High Performance Immunocytochemistry (HPI).
I want to label frozen sections. Will SII antibodies work for my experiment?
Yes, but it is technically challenging and we do not recommend it for quantitative analysis. The best method developed so far is that of Sun and Kalloniatis, J Comp Neurol 494:686 -703, 2006. Pubmed
I want to double label my sections? Will SII antibodies work for my experiment?
Yes, but with the same caveats as above for combining small molecule and macromolecule labeling (see Sun and Kalloniatis, J Comp Neurol 494:686 -703, 2006 Pubmed). For double, triple and higher multiplexing of small molecules, we recommend HPI and computational molecular phenotyping (CMP) for high-sensitivity and reproducibility (see Marc and Jones, Molecular phenotyping of retinal ganglion cells. Journal of Neuroscience, 2002. 22: p. 412-427 Pubmed)
What is HPI?
HPI is High-Performance Immunocytochemistry, based on sensitive silver-based detection of small molecules on thin sections. The key features of HPI are:
- efficient capture of small molecules
- absence of any diffusion barriers to IgG binding
- high speed, reproducibility and sensitivity
Do I have to use silver-immunogold detection?
No. Though silver is archival, any fluor will do. Cy3tm remains an exceptional detection method in our experience. Though expensive, quantum dot detection is also compatible with our IgGs.
What is CMP?
CMP is Computational Molecular Phenotyping. It is a fusion of HPI with digital imaging, image registration and modern cluster analysis methods to produce multiplexed images. See Marc and Jones, Molecular phenotyping of retinal ganglion cells. Journal of Neuroscience, 2002. 22: p. 412-427 Pubmed
Where can I buy SII products?
Other Questions about Signature Immunologics, Inc.
Can my company distribute SII products?
Yes. for a copy of our distribution policy.
Will SII distribute my antibody?
No. SII only handles antibodies that we make.
Will SII make an anti-hapten antibody for me?
Yes. Please for a copy of our antibody production policy