Any antibody preparation has some potential to produce non-specific reaction in the assay. This originates from:
- non-specific antibodies that are present in some proportion in any polyclonal antibody preparation, including affinity purified ones (often “affinity purified” means only isolation of IgG fraction on Prot A/G column, not the purification on the antigen column under very stringent conditions)
- low specificity antibodies among specific ones in polyclonal
- fragments of fallen apart IgGs in stored preparations, including monoclonal
- separate heavy and light chains of specific antibodies, produced by most hybridomas
All these are capable of binding non-specifically to molecules on tissue sections, blots, fixed cells and other objects for immune detection. In case of retrieved formalin sections the risk of non-specific reaction is even increased, since to active the epitope recovery the proteins comprising the tissue sections are denatured during HIER, thus making accessible many domains that are charged and are capable of binding the test immunoglobulins on non-specific manner.
The standard means to block non-specific binding of specific antibody preparation is to add some irrelevant protein, such as BSA, other serum, casein, etc. However, everyone who has attempted to do this knows that increasing (for effective blocking) concentration of such blocking agents leads to a great reduction of specific reaction as well. This is due to large blocking molecules binding to accessible sites on sections and thus sterically blocking access of specific antibodies to epitopes of interest (schematically represented in the figure on the left, top). Instead of these proteins, all of our buffers developed for immune assays contain short (0.6–2 kD) peptides that are capable of effectively blocking non-specific reactions while not affecting the specific binding of antibody.
Low background and strong specific reaction. Standardized staining.
Aptum's collection of Immunohistology buffers also has some other benefits (see below) and allows you to achieve the best quality IHC result without compromising the antigen detection. The buffers can also be used in other immune assays, such as immunofluorescence on sections, flow cytometry on fixed cells, hybridization of sections with antibody detection. The Retriever IHC buffers empower you to control non-specific staining on every step of immunohistochemistry. They are especially highly recommended for research pathology where, in contrast to diagnostics, many polyclonal and/or low-affinity antibodies are used.
All buffers available in 50 ml, 125 ml and 500 ml package. Ready to use. Certified for use in diagnostic applications.
A new class of blocking solutions are based on chemically modified and fragmented ultra-pure casein. This effectively reduces unwanted binding of primary antibody and conjugates you use to charged surfaces of the slide and tissue section, and it greatly reduces non-specific binding while preserving the specific reaction, by saturating potential non-specific protein-protein interactions. Moreover, in contrast to BSA-based, IgGm casein or serum -based blocking solutions there is no interaction of specific antibody and blocking protein itself. It is not comparable to other commercilally available or home-made blocking solutions. Recommended for research and diagnostic pathology, especially for retrieved sections and polyclonal antibodies.
This diluent is specifically designed for preparing solutions of your HRP-conjugate used as the detection reagent. It is the Antibody-diluent buffer with additional components for stabilising your HRP-conjugate. It allows you to further standardise the assay by preparing ready-to-use conjugate solutions in advance ans store them in a refrigerator without loss of activity.
Buffer for diluting your primary and secondary antibodies, especially if they were stored for longer periods of time, even at –20°C in glycerol, or in refrigerator. Nonspecific binding of the antibodies, negative effects of disturbing substances and low or medium affinity cross-reactivities of the antibodies will be minimised, making your result more reliable. Excellent for IHC (frozen and formalin sections), flow cytometry on fixed cells, Western Blot and other immune assays.
When used in pathology, in also greatly reduces non-specific reactivity of human serum components and immunoglobilins in tissue, vessels and cells with mouse antibodies used on section.
For especially “troublesome“ antibodies, as well as for in situ PCR applications, this diluent may also be used as a washing buffer, preventing secondary binding of your analystes during washing.