Nano-Secondary antibody are a recombinant novel class of secondary antibodies for ELISA,WB,IHC, higher resolution & cleaner images to substitute for conventioal secondary antibodies.They consist of Nanobodies/ VHHs that bind to primary antibodies with high affinity in a species and subtype specific manner. Nano-Secondaries are conjugated to HRP or Alexa Fluor^® dyes etc.

Pre-mixing overcomes the species limitation for multiplexing microscopy 

In standard immunoassays, normally the primary Abs are first incubated with the sample followed by washes to eliminate the non-bound excess of primary Abs. Only at this point conventioal secondary antibodies are incubated for a period of time with the sample followed by washes to eliminate the non-bound excess of secondary antibodies before imaging the specimen. Pre-mixing the primary Ab with the sencondary antibodies prior to incubating them with the sample would shorten protocols and save considerable amount of time and costs (e.g. in clinical pathology laboratories). However, this is currently not possible due to the polyclonality and the bivalency of secondary antibodies that result in agglutination (aggregation or clustering) of the 1.Abs-2.Abs complex and thus in a failure to stain the intended target in the sample .Nano-Secondary antibody probe binds to the primary Ab in a monovalent fashion,allow direct pre-mixing with first.Abs before staining , and enabling the use of multiple primary antibody from the same species.

 In multiplexed immunostaining, i.e. when multiple targets are stained in the same sample, one is constrained to the use of primary Abs coming from different species. This is because the standard immunostaining needs to be done in a sequential manner: first 1.Abs are incubated on the sample, washed off and then secondary antibodies are incubated. Therefore, 1.Abs should be raised in different species and secondary antibodies should recognize one species specifically, limiting the choice of antibodies for the different targets. It has been shown that by pre-mixing 1.Abs with Nano-Secondary antibodies in a tube prior staining, one could circumvent this species limitation and use on a sample 1.Abs raised in the same species.  

Pre-mixing shortens experimental time for WB

Nano-Secondary antibodies allow direct pre-mixing with first Abs before WB reducing experimental time.

Nano-Secondary antibody provide higher staining accuracy than secondary antibodies

The primary antibody-conventioal secondary antibodies can measure up to 30 nm, leading to a large distance between the targeted molecule and the detection element, causing the so called “linkage” or “displacement” error. While this might not influence the results in some applications (e.g. epifluorescence, ELISA or FACS), it is of major relevance for super-resolution microscopy techniques where the localization precision can be as high as 1 nm. The linkage error can be reduced by using directly labeled small affinity probes like Nano-Secondary antibody, which all have sizes below 3 nm.