AURION IMMUNOGOLD REAGENTS EDITION VI FREQUENTLY ASKED QUESTIONS


What about sensitivity, signal-to-noise ratios and detectability?


Sensitivity can be considered at different levels in the total preparation and incubations. Ideally during preparation one would like to preserve all antigens present. In many cases this is not possible. But at least a representative fraction should be preserved and be available for immuno labeling. It all depends on the preparation procedure (fixation, embedding, temperature, etc.), which leaves you with a specimen or section with a given number of available and recognizable antigens. The ensuing detection protocol has 100% sensitivity if all the remaining antigens are detected, i.e. are represented by at least one gold particle or marker molecule. Again, due to masking and steric hindrance by the specimen composition this will only in exceptional cases by fully attained.


The immuno labeling sensitivity thus expresses the degree to which available antigens can be detected by the employed combination of primary antibody and secondary conjugate.


The quality of the primary antibody is the next important item. Theoretically the Kd-value of an antibody/antigen reaction is a measure for the dilution at which the incubations should be performed and for the stability of the ensuing bond. Sensitivity will go up with more concentrated antibody solutions up to a maximum level. However, when the primary antibody shows cross-reactivity there is not necessarily an improved signal-to-noise ratio. The reliability of the detection by the primary antibody improves in such cases with higher dilutions, probably leading to a smaller amount of antigens detected, but to an improved signal-to-noise ratio.


Thus, sensitivity at the level of the primary antibody has to be balanced against the signal-to-noise ratio.


The last step is the quality of the secondary reagent. In fact you will be looking at a number of gold particles which represents a number of secondary antibodies which have detected a number of primary antibodies. For the interaction between the secondary reagent and the primary antibody the same rules apply as indicated for the antigen/primary antibody reaction.


Detectability reflects the degrees to which the final result of all the reactions involved can actually be seen. This is depending on the right match between particle size and magnification. Ultra-Small particle-based conjugates for instances are among the most efficient detection systems, but you will only detect them after silver enhancement (in most applications).


What is epi-polarization microscopy?


Epi-polarization is a technique used for the very sensitive light microscopical observation of metal particles. Where bright field microscopy depends on contrast levels in discriminating signals, epi-polarization works differently: provided particles are large enough individual particles will be observed. So in fact you are evaluating your labeling results on the same basis as with an electron microscope by looking at individual particles. This makes this technique so valuable as it builds a bridge between the light level and the electron microscopical observation.


What you need to do is this: a high-quality light microscope equipped with an epi-illumination source, preferably a high pressure Hg-lamp (although a halogen source may also do). Many laboratories have an epi-fluorescence microscope at their disposition with a 40X (or higher) oil objective. Such microscope equipment forms the correct basis. You only need to implement an epi-polarization filter (the so-called epi-block or IGSS filter) in the filter housing. The epi-block contains two polarizers, differing 90 degrees in orientation with respect to each other.


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How does it work (in short)? High intensity light passes the first polarizer in the epi-block and becomes polarized. The polarized incident light passes the objective lens and interacts with the specimen. The biological material hardly gives any reflection, and the reflected light is unmodified. The metal particles mirror the polarized light, thereby randomizing the polarization angle.


Reflected light passes up through the objective lens. On its way to the eye pieces or the photo camera the light passes the second polarizer in the epi- block. While doing so, light with the original polarization angle (the way it was polarized in the first place before ever hitting the specimen) is extinguished, whereas light that has become randomly polarized (and which comes from the silver metal particles) passes the epi-block. As a result you will see individual bright stars (the gold/silver particles) against a dark background.


Epi-polarization observation can be combined in real time with bright field imaging, providing for a very sensitive detection of even extremely low amounts of antigen while still having the advantage of full morphological details in the specimen.


My specimens for pre-embedding have a lot more antigens than a corresponding ultrathin section. Should I use more concentrated reagents?


The increased amount of antigens should be balanced by a larger amount of reagent volume at an appropriate dilution (the same as used on sections with low amounts of antigen), and not by more concentrated reagents. The reason is that with increased concentration more cross-reactions may occur and signal-to-noise ratios will decrease.


Incubating specimens for pre-embedding in larger volume quantities is best performed on a rocking table for a prolonged time to warrant penetration to antigenic sites in the specimen.


What is the advantage of gold nanoparticle conjugation via adsorption?


This is an easy and straight forward method for the conjugation of high molecular weight macromolecules. The flocculation test that tests the effect of a high salt concentration on the stability of the complex makes it easy to check if adsorption to the gold nanoparticle surface is successful or not.


Can you give me more information on gold nanoparticle conjugation strategy?


First check if conjugation via adsorption is feasible. If yes, check bio activity e.g., via a dot spot test. Biomolecules that are too small to be conjugated via the classic adsorption method can be covalently linked to functionalized gold nanoparticles as an alternative. A prerequisite for successful conjugation to Carboxyl-functionalized gold nanoparticles is the presence of primary amine(s) in the target molecule. As a rule of thumb conjugation via classic adsorption to conventional gold nanoparticles is possible with macromolecules having a MW > 40 kDa.


What is the conjugation principle behind covalent conjugation using carboxyl-functionalized gold nanoparticles?


The conjugation relies on the well known and proven EDC/sulfo-NHS chemistry. EDC/sulfo-NHS activation at pH 5 results in an amine reactive ester, immediately followed by binding to free amine on the target molecule.


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