Enzymatic labeling methods

The pyrimidine nucleosides dUTP or dCTP can be modified at their C-5 position with a spacer arm containing a tag, such as a biotin group, and still remain good substrates for DNA polymerase. Enzymatic labeling with a biotin-modified pyrimidine nucleoside triphosphate is one of the most common methods of adding a detectable group to an existing DNA strand. 

Enzymatic labeling using any of these polymerase methods results in derivatized nucleoside triphosphates being incorporated at numerous locations within an oli-... 

Other methods of detection of hybridisation event consist of the use of labels. These labels can be divided into electroactive and enzymatic labels. The schemes commonly used for detection are scheme b or c. The scheme selected to detect the hybridisation event depends on whether the target is labelled with a marker by PCR amplification or using a labelling kit, or whether a labelled synthetic detector oligonucleotide is used. These methods are usually the most sensitive, but the need of a labelling step makes them more complex, tedious and... 

The methods based on the use of enzymatic labels are without doubt among the most sensitive. Moreover, because routine laboratories are usually working with enzymatic labels, this might make them the most... 

The enzymatic radioassay method for the analysis of acetylcholine and choline in brain tissue has been reported by Reid et al. [210]. The method describes the determination of nanogram amounts of acetylcholine and choline in as little as 10 mg of brain tissue, involves isolation of acetylcholine by high-voltage paper electrophoresis, alkaline hydrolysis of acetylcholine to choline, and conversion of this into [32P]-phosphoryl choline in the presence of choline kinase and [y32P] ATP. The labeled derivative is isolated by column chromatography on Bio-Rad AG1-X8 resin, using Tris buffer solution as the eluent. Cerenkov radiation from 32P is counted (at 33% efficiency) in a liquid scintillation spectrometer. The amount of phosphorylcholine is proportional to the amount of choline over the range of 0.08-8.25 nmol. 

Ladinsky and Consolo used an enzymatic radioassay method for the determination of acetylcholine and choline [218], The method was based on the electrophoretic separation of acetylcholine and choline, hydrolysis of acetylcholine to form choline, and acetylation of the choline with labeled AcCoA and choline acetyltransferase. The labeled acetylcholine formed was isolated and quantitated. The method was sensitive and specific, and permitted the routine handling of a large number of samples in a single experiment. The standard curves were linear up to at least 42.5 ng (0.4 nmol) choline and 45 ng (0.3 nmol) acetylcholine. The lower limit of sensitivity was 2ng, and the recovery of acetylcholine was 95% when carried through the entire procedure. 

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. 

There are two alternative methods based on (i) enzymatic labels and (ii) luminogenic labels. The sensitivity and the principles on which these methods are based have been outlined below. 

Profile Analysis of Oligosaccharides from Glycoproteins by PMP Labeling. Comparison of Chemical and Enzymatic Release Methods Using RP-HPLC and Mass Spectrometry... 

Enzymatic digestion of proteins in 1 1 0/ 0 water has been applied to facilitate de novo sequencing [136]. The labelling results in split peaks with a 2 Da difference for the y-ions, enabling easy discrimination between peaks due to b- and y-ions. Other stable isotope labelling methods (Ch. 18.4.2) involving modification of either the N- or the C-terminal can assist in de novo sequencing as well. 

Enzymes are currently the most widely used and investigated labels for immunoassays, because a single enzyme label can provide multiple copies of detectable species. This catalytic amplification results in immunoassay detection limits that rival those of radioimmunoassay without the storage and disposal problems associated with radioisotopes. Enzyme immunoassays label either ligands or antibodies with enzyme, and enzyme activity in bound or free fractions is measured. Heterogeneous immunoassays employing enzymatic labels have been named enzyme-linked immunosorbent assays (ELISAs). ELISA methods usually employ antibody immobilized onto the wells of polystyrene microtiter plates, and may be... 

Acridinium-NHS (AT-hydroxysuccinimide) ester labeling reagent is synthesized as described by Weeks et al. (1983) and probes containing an allylamine linker arm (Fig. 7.2.1) are prepared either as described for nucleotides and for enzymatically labeled probes (Section 7.6.2.2), by transamination (Section 7.8.1) or during oligomer synthesis (Section 6.4). The acridinium-NHS ester is then reacted with the linker arm by standard methods (Section 7.4.1). 

In the second step, the purified, transaminated DNA is brought to a higher pH (8.5-10) in order to decrease the protonation of the reactive amino group. Viscidi et al. (1986) labeled the N -substituted cytosine residue with biotin using the NHS-biotin ester, whereas, e.g., Hurskainen et al. (1991) labeled the same intermediate with a europium chelate (for time-resolved fluorescence). The Cq/qj increases about 1 log but this is compensated by the high probe concentration. This method yields probes with a similar detectability (10 molecules) as enzymatically labeled biotin probes, but the reagents are inexpensive and easily available. 

Methods based on radiolabels continue to hold an important place in routine analysis and in research related to clinical testing. The main techniques included in this group are radioimmunoassy (RIA), immunoradiometric assay (IRMA), and scintillation proximity assay (SPA). Many researchers in this field use short-lived radioisotopes and chelating agents in antibody labeling.139 The most popular types of immunoassay are methods that use enzymatic labels the enzyme-linked immunosorbent assay (ELISA), the enzyme-monitored immunotest (EMIT), the competitive binding enzyme immunoassay (EIA), and the immunoenzymometric assay (IEMA). 

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