Antigens, immunochemical detection

Immunochemical Detection of Antigens After Electrotransfer (Immunoblotting)... 

Immunochemical detection is possible in one step, if the detecting antibody carries the signal-forming principle. But also cascades of steps are used to identify the first-bound antibody and by it the antigen immobilized by blotting. Antibodies form these cascades, i.e., if the first bound antibody is from species A, e.g., rabbit, a second antibody from other species, e.g., goat, directed against the primary, is used. 

Leduc, V., Demeulemester, C., Polack, B., Guizard, C., Le-Guern, L., Peltre, G. 1999. Immunochemical detection of egg-white antigens and allergens in meat products. Allergy 54 464-472. 

Figure 3. Immunochemical detection of lactose-protein Maillard products in several market and powdered milk samples. ELISA plates were coated with appropriately diluted milk samples and the lactose-derived antigens were detected with LlOl. Apparent antigen amount is shown as ELISA value (absorbance at 405 nm).

Va.ria.tions in Methods. The various immunochemical methods can differ in a number of ways. For example, the analytical reagent may be cmde antisemm, monoclonal antibodies, isolated immunoglobulin fractions, etc. The conditions under which the method is mn, detection of the antigen—antibody complex, and the techniques used to increase sensitivity or specificity of the reaction all maybe varied. 

Very recently, a sandwich assay for prostatic acid phosphatase antigen was carried out using two cascaded enzyme reactions to provide amplification of the immunochemical event. In one format, an optical readout was used whereby a forma-zan dye was generated by reaction of a dye precursor and NADH generated from the second enzyme cycle. In the electrochemical format, the NADH generated in the second enzyme cycle was used to reduce Fe(CN) to FeCCN) " which was then detected amperometrically. While the use of Fe(CN) in ECIA has appeared in the... 

Immunochemical techniques are based on the immunological reaction derived from the binding of the antibody to the corresponding antigen. This reaction is reversible and is stabilized by electrostatic forces, hydrogen bonds, and Van der Waals interactions. The formed complex has an affinity constant (k j that can achieve values around the order of 1010 M. This great affinity and specificity between the specific antibody and the antigen (or the analyte) have turned these techniques into powerful analytical tools to detect and quantify... 

After total delipidation of the Lp (a)-lipoprotein, only apo VLDL is soluble in 0.9% NaCl and detectable by immunochemical means (S28). It therefore seems unlikely that the specific antigenic determinant of the Lp (a)-lipoprotein is located on or is part of the apo VLDL protein moiety. At present, the nature of the antigenic determinant of Lp(a)-lipoprotein is not known. 

It has been proposed to use a Si/Si02/electrolyte structure for the detection of immunochemical reaction. The antibodies are immobilized at the surface of the Si02 and their interaction with the antigen is monitored by observing the shift of the inflex point on the C-V curve, using 150 mV p-p, 1 KHz modulation. 

The development of immunoassays for the detection of food components and contaminants has progressed rapidly in the last few years [7]. Antibodies against almost all the important food residues compounds are currently available. Classical immunochemical methods such as immunodiffusion and agglutination methods for food analyses generally involve no labeled antigen or antibody. Concentration of the antigen-antibody complex is estimated from the secondary reaction that leads to precipitation or agglutination. These methods are not sensitive, are subject to... 

Immunosensors are affinity biosensors and are defined as analytical devices that detect the binding of an antigen to its specific antibody by coupling the immunochemical reaction to the surface of a device... 

A direct detection method was recently developed for these adducts in stratum comeum of human skin based on immunofluorescence microscopy (30). Three partial sequences of keratins containing glutamine or asparagine, adducted with a 2-hydroxyethyl-thioethyl group at the omega-amide function, were synthesized and used as antigens for raising antibodies. After immunization, monoclonal antibodies were obtained with affinity for keratin isolated from human callus exposed to 50 xM sulfur mustard (see Plate 1). In contrast to the immunochemical... 

Because a library can contain thousands of different clones, it can be difficult to isolate a clone with DNA of specific interest. This is because the majority of cloned DNAs do not contain a readily selectable genetic marker, such as antibiotic resistance, or lead (as discussed earlier) to the production of a foreign protein. Methods to achieve this have been developed and utilize hybridization, immunochemical, and structural techniques. A specific DNA sequence of only several kilobases can be isolated from a genome containing in excess of 106 kilobases. Hybridization requires a radioactive DNA or RNA molecule (a probe) that is complementary (or partially so) to the sequence of the cloned DNA. Immunologic techniques require that the polypeptides coded by the DNAs of interest are available and have specific antigenic properties that allow detection. Structural analysis can also be used when the other techniques are inapplicable. 

Immunoassays, electrochemical — A quantitative or qualitative assay based on the highly selective antibody-antigen binding and electrochemical detection. Poten-tiometric, capacitive, and voltammetric methods are used to detect the immunoreaction, either directly without a label or indirectly with a label compound. The majority of electrochemical immunoassays are based on -> voltammetry (-> amperometry) and detection of redox-active or enzyme labels of one of the immunochemical reaction partners. The assay formats are competitive and noncompetitive (see also -> ELISA). 

Western blotting is one of the most widely used immunochemical techniques in modern biochemistry. In general, it is used to detect the presence of a particular protein antigen in an impure mixture. Western blots can provide information that is (1) qualitative Is the protein present (2) quantitative How much of the protein is present and (3) structural What is the size of the protein The general protocol for Western blotting is as follows (see Fig. 18-1) ... 

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