Indirect labeling method

In the indirect labeling method, amine modified cDNA is first synthesised by incorporating aminoallyl-modified nucleotides in first strand cDNA by a reverse transcriptase. After hydrolysis of the RNA template, and purification of the amine-modified cDNA, chemical labeling with N-hydroxyl succinimidyl-ester derivative of the Cy dye is performed. A high excess of Cy dye NHS-ester is needed for an efficient reaction. The Cy dye-cDNA is then purified to remove Cy dye that is not incorporated into labeled cDNA. 

Compared to direct labeling, the indirect labeling method described takes longer and requires more steps. However, the added advantage of brightness, lack of enzymatic bias, and lower cost makes this method worthwhile. 

Alternately, the activity of conjugates to be used with the indirect labeling method can be tested by determining the conjugate concentration required to saturate antibody-sensitized cells with latex spheres. The number of markers bound per cells can be determined by SIM or by radioactivity if the spheres have been labeled with a radioactive isotope. 

Astatine-211 is a promising radionuclide for systemic therapy1 3 due to its decay properties with a half-life of 7.2 hours and an effective emission of one a-particle per decay. However, the weakness of the astatine-protein bond formed after direct astatination1 4 has so far limited its clinical use. To overcome these problems indirect labelling methods have been tried such as the use of ALsuccinimidyl-(trialkylstannyl) benzoate as an intermediate for the astatination of antibodies using conjugation procedures.5 8... 

Fig. 13. Schematic representation of direct labelling methods using (A) primary antibody coupled to gold or (B) Fab fragment coupled to gold and indirect labelling methods using (C) protein A-gold, (D) secondary antibody coupled to gold or (E) the streptavidin-biotin system, showing clearly the difference between resolution and sensitivity.

There are two common forms of heterogeneous assays used to detect and quantify disease markers the direct-labeling method, in which the proteins in a sample are labeled with a detectable tag and isolated from a complex sample by means of a bed of immobilized antibodies, and the indirect-labeling method, in which two antibodies are used for each marker. Sandwich-type immunoassays, such as the enzyme-linked immunosorbent assay (ELISA), require two antibodies an immobilized antibody used to capture a molecule of interest - a protein, a virus, or a small molecule (e.g., a hormone. 

Many antibodies have been labeled in one or the other way and kits for patient studies are commercially available. The method is difficult to apply for smaller molecules. Despite the fact that some important (cyclic) peptides have at least one disulfide bridge in their structure, the coordination affects the structure of this small molecules resulting frequently in the loss of bioactivity. The indirect labeling method is essentially limited to antibodies. 

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... 

Primary antibodies blot excess blocking solution from sections and incubate for 60 min at room temperature or overnight at +4°C with a mixture of correspondingly diluted unlabeled primary antibodies raised in two different host species (e.g., mouse and rabbit). When using fluorophore-labeled primary antibodies as in direct immunostaining method (one antibody layer), you may skip step (6) with secondary antibodies for indirect immunostaining method (two antibodies layers). Wash sections in PBS for 3 x 3 min. 

Direct and indirect immunostaining methods The direct method is a one-step staining method, and involves a labeled antibody reacting directly with the antigen in tissue sections. In this method, the primary antibody can be labeled with a fluorophore or biotin. In indirect immunostaining, the bound unlabeled primary antibody (first layer) is visualized with a secondary antibody (second layer) bearing label, such as a fluorophore, biotin or an enzyme. 

Colloidal gold. This is a useful label for both direct and indirect staining methods since it requires no further reagent additions. Gold probes may be readily seen by light microscopy when coupled with silver enhancement and in addition, being electron dense, offer excellent sensitivity for the electron microscope. 

Fluorescent labeling of cDNA can be a potential source of technical variability. In a typical two-color experiment, fluorescently labeled cDNA probes are transcribed from separate mRNA populations (e.g., cerebral ischemia versus sham). One set of cDNA probes is labeled with one fluorescent dye (typically Cy5) and the second set with a different fluorescent dye (Cy3). A number of methods for making labeled cDNA from the RNA samples have been tested and reviewed (Stears et al., 2000 Vernon et al., 2000 Li et al., 2002) and a number of potential sources for variation must be appreciated. First, the molecular structure of the fluorescent dyes used in making labeled cDNA can affect efficiency of dye incorporation. Second the mode of dye incorporation (direct verses indirect labeling) can affect subsequent hybridization kinetics (Stears... 

Double labeling with IGSS and IPO may be followed by the detection of a third antigen consecntively with an indirect lAP method as detailed above. Recommended combinations for seqnential triple antigen detection are ... 

AP- and/or horseradish peroxidase (HRP)-conjugated antibodies for the haptens used for labeling probes (a direct detection method) or unconjugated antibodies for the haptens and AP- and/or HRP-labeled anti-species antibodies (an indirect detection method) (Vector Laboratories, Inc., Bnrlingame, CA, USA) (see Note 8). 

Multi-step technique (3) This is an indirect/direct method combining unlabeled primary antibodies with directly-conjugated antibodies. The method starts with staining the unlabeled antibody/antibodies with the appropriate detection system, but without performing the final enzymatic staining reaction. The tissue is blocked with normal serum from the host of the first primary antibody before the second, directly-labeled primary antibody is added. The staining ends with the two enzymatic reactions being performed sequentially. 

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