Labels secondary

For indirect immunoassay methods, the antigen (analyte) is bound to support materials and excess binding sites are blocked. Analyte and primary antibody are then added simultaneously, followed by the addition of enzyme-labeled secondary antibody and color reagent. The bound analyte (coating antigen) and free analyte (in... 

Figure 1 Schematic sequence of the direct and indirect competitive ELISA. The principle difference is that for direct competitive immunoassay, the well is coated with primary antibody directly, and for indirect competitive immunoassay, the well is coated with antigen. Primary antibody (Y), blocking protein (Y), analyte (T), analyte-tracer ( ), enzyme labeled secondary antibody ), color development ( J)...

In some cases, the preparation of a fluorescently labeled antibody is not even necessary. Particularly, if indirect methods are used to detect antibody binding to antigen, then preparing a fluorescently labeled primary antibody is not needed. Instead, the selection from a commercial source of a labeled secondary antibody having specificity for the species and class of primary antibody to be used is all that is required. However, if the primary antibody needs to be labeled and it is not manufactured commercially, then a custom labeling procedure will have to be done. 

FIGURE 14.5 Multiprotein electrical detection protocol based on different inorganic colloid nanocrystal tracers, (a) Introduction of antibody-modified magnetic beads (b) binding of the antigens to the antibodies on the magnetic beads (c) capture of the nanocrystal-labeled secondary antibodies (d) dissolution of nanocrystals and electrochemical stripping detection (reproduced from [29] with permission). 

Washes and dilutions wash sections in PBS or TBS for 2x3 min. PBS can be used for all washes and dilutions. When working with AP-labeled secondary antibodies, TBS must be used. 

Secondary antibodies incubate sections for 30 60 min at room temperature with a HRP- or AP-labeled secondary antibody raised against the corresponding IgG of the primary antibody. Wash sections in a buffer recommended for the corresponding enzyme substrate development for 2x3 min. 

Notes. When using biotin-labeled secondary antibodies instead of enzyme-labeled antibodies, you have first to detect biotin with enzyme-labeled (strept) avidin and proceed further with the Substrate Step (9). Do not add normal serum, non-fat dried milk, culture media or other potential sources of biotin to (strept)avidin-containing reagents. This may result in reduced sensitivity. Solutions containing sodium azide or other inhibitors of peroxidase activity should not be used in diluting the peroxidase substrate. 

Never block with normal serum from the host species of the primary antibody. If immunoglobulins in the normal serum from the host species of the primary antibody bind to the specimen of interest, they will be recognized by the labeled secondary antibody, resulting in higher background. 

When using biotin-labeled secondary antibody, you have first to visualize biotin with a fluorophore-labeled (strept)avidin employing ABC technique (see Sect. 6.2.1), before proceeding to counterstaining (step 7). 

Labeling with fluorescent phalloidins may be combined with immunostaining. In this case, the phalloidin-staining solution can be applied in a mixture with fluores-cently labeled secondary antibodies. Combination of immunostaining with fluorescent phalloidins and fluorescently counterstained nuclei are extremely useful in multiple labeling strategies to locate antigens of interest with specific components of the cell. 

Tyramide signal amplification This procedure, designated as a catalyzed reporter deposition (CARD) or tyramide signal amplification (TSA), takes advantage of horseradish peroxidase (HRP) from an HRP-labeled secondary antibody to catalyze in the presence of hydrogen peroxide the oxidation of the phenol moiety of labeled tyramine. On oxidation by HRP, activated tyramine molecules rapidly bind covalently to electron-rich amino acids of proteins immediately surrounding the site of the immunoreaction. This allows an increase in the detection of an antigenic site up to 100-fold compared with the conventional indirect method with no loss in resolution. 

Figure 7.8 Some examples of antibody detection techniques, (a) Direct labelling of the primary antibody, (b) indirect using labelled secondary antibody, (c) indirect using biotinylated secondary antibody and labelled streptavidin.

For many years, due to the availability and low cost of radioisotope-labeled secondary antibodies, radioactive detection was the method of choice in Western blotting. Newer methods that are less hazardous and easier to use, while maintaining comparable sensitivity, have been developed. Today, Western blotting detection methods can be light-based, (chemiluminescence, bioluminescence, chemifluorescence, and fluorescence), radioactivity-based, or color-based. It is important to note that the detection sensitivity depends on the affinity of the primary antibody for the antigen and on the affinity of the secondary antibody for the primary antibody and can therefore vary considerably from one protein sample to another and from one antibody batch to another. 

Radioisotopes such as P, and I can be used to label secondary antibodies that are detected... 

Noncompetitive ELISA methods are based on sandwich assays in which an excess supply of immobilized primary antibody, the capture antibody, quantitatively binds the antigen of interest and an enzyme-labeled secondary antibody is then allowed to react with the bound antigen forming a sandwich. A color reaction product produced by the enzyme is then used to measure the enzyme activity that is bound to the surface of the microtiter plate. Sandwich ELISA (noncompetitive) methods yield calibration curves in which enzyme activity increases with increasing free antigen concentration. 

Figure 3.25 Scheme of a SWNT immunosensor using secondary antibody labeled with HRP enzyme. HRP catalyses H2O2 and generates electrons that can be amperometrically detected, (a) Treatment with a conventional HRP-labeled secondary antibody providing one label per binding event and (b) treatment with an HRP-... 

CNT labeled secondary antibody for signal amplification with numerous enzyme levels per bindingevent.AFM imageofthe nanotube forest immunosensor. Reprinted with permission from Ref [169]. Copyright, 2006, American Chemical Society. 

Incubate with Rhodamine and/or FITC labeled secondary antibody for 1 hr at RT. 

In case of fluorescent labelled secondary antibody mount the slide and visualise under fluorescent or Confocal microscope. For peroxide visualisation incubate with a DAB solution till brown color is seen followed by stopping the reaction under running tap water. 

The use of specific antibodies labeled with a fluorescent dye to localize substances in tissues was first devised by A. H. Coons and his associates. At first, the specific antibody itself was labeled and applied to the tissue section to identify the antigenic sites (direct method) (1). Later, the more sensitive and versatile indirect method (2) was introduced. The primary, unlabeled, antibody is applied to the tissue section, and the excess is washed off with buffer. A second, labeled antibody from another species, raised against the IgG of the animal donating the first antibody, is then applied. The primary antigenic site is thus revealed. A major advantage of the indirect method is the enhanced sensitivity. In addition, a labeled secondary antibody can be used to locate any number of primary antibodies raised in the same animal species without the necessity of labeling each primary antibody. 

Amplification can be attained with this type of method by employing two indirect techniques simultaneously. After the specimen is thoroughly rinsed, following the labeled secondary antibody incubation, a third antibody may be used, which is enzyme-labeled and reactive against the species immunoglobulin responsible for the secondary antibody. 

As previously mentioned, the principles of staining are identical to those used in paraffin sections. The techniques used may be direct or indirect as described in Chapters 15-18. Indirect techniques are generally more sensitive and, therefore, preferable. Indirect techniques can be broken down into three steps. In the first step, an antibody directed against the antigen of interest is applied to the tissue section. In the second step, a labeled secondary antibody directed against the first antibody is applied. The last step consists of a detection step that is composed of linking the secondary antibody to a detection system and... 

In this method, the primary antibodies are combined into a single cocktail and applied simultaneously in a single-reaction step. The same is done for the labeled secondary antibodies. Antibodies of different animal species or immunoglobulin isotypes (noncrossreactive) or autologous antibodies that are directly coupled to different enzymes/fluorochromes are required. Immuno-fluorescent methods are particularly suited for this method. 

Apply appropriate, labeled secondary antibodies together (e.g., goat antimouse and antirabbit immnnoglobnlins), labeled with either colloidal gold, horseradish peroxidase, or alkaline phosphatase, respectively. Alternatively, use secondary antibodies labeled with separate fluorescent dyes. Incubate paraffin sections for 1-2 h and frozen sections for less than 30 min. 

Detection reagent (substrate) for enzyme-labeled antibody 2,2-azino-di(3-ethyl-benzthiazohne sulfonate-6) (ABTS) 0.3 g/L in 0.15 M sodium citrate with 0.1% H2O2 for the detection of horseradish peroxidase-labeled secondary antibody or p-nitrophenyl phosphate (pNPP) 1 g/L in 1M diethanolamine in water for the detection of an alkaline phosphatase-labeled antibody (Kirkegaard and Perry Laboratories). 

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