Immunochemical quantification

Because of the complex and polymorphic nature of the Lp(a) lipoprotein, together with the homology of the apo(a) moiety with plasminogen, a number of specific problems arise concerning the immunochemical quantification of Lp(a). These include the selection of a suitable type of immunoassay, its specificity and sensitivity, and the type of antisera used in the assay (L2). Moreover, the selection of an appropriate standard and of the units of mass to express the amount of Lp(a) require careful consideration (L4). 

Rumbo, M., Chirdo, F.G., Fossati, C.A., and Anon, M.C. 2001. Analysis of the effects of heat treatment on gliadin immunochemical quantification using a panel of anti-prolamin antibodies. JAgric Food Chem 49(12) 5719-5726. 

Tocaj, A., Nandakumar, M. P., Holst, O., and Mattiasson, B. (1999). Flow injection analysis of intracellular /3-galactosidase in Escherichia coli cultivations, using an on-line system including cell disruption, debris separation and immunochemical quantification. Bioseparation 8, 255-267. 

Leukocyte proteases may be released if serum is allowed to sit on the clot too long after blood drawing. These proteases then form complexes with AAT, altering both electrophoretic mobility and in some cases immunochemical quantification. Bacterial contamination and release of... 

Albers JJ, Hazzard WR. Immunochemical quantification of human plasma Lp(a) lipoprotein. Lipids 1974 9 15-26. 

R12. Rowe, D, S., Radioactive single radial diffusion a method for increasing the sensitivity of immunochemical quantification of protein in agar gel. Bull. W.H.O. 40, 613-616 (1969). 

Celundcr, M., and Forlin, L. (1991). Catalytic activity and immunochemical quantification of hepatic cytochorome P450 in p-napthoflavonc and isosafrole treated rainbow trout Orchoryiichus mykiss). Fish. Physiol. Biochem. 9. 189-197. 

Levieux, D. Levieux, A. El-Hatmi, H. Rigaudiere, J-P. Immunochemical quantification of heat denaturation of camel (Camelus dromedarius) whey proteins. Journal of Dairy Research, 2006, 73, 1-9. 

Accumulation of Cyt b6/f Complex during Chloroplast Development. The time course of polypeptide accximulation for Cyt b6/f complex during light-induced development was studied by using an immunochemical quantification method. Antigens aginst Cyt f, Cyt b6, and chloroplast Rieske proteins did not cross-react... 

Table 6 Immunochemical methods developed for the detection and quantification of BPA and phthalate esters... 

Connelly PW, Vezina C, Maguire GF (1996) Quantification of apolipoprotein C-II by immunochemical and chromatographic methods. Methods Enzymol 263 188-208... 

Application of microbiological or immunochemical techniques offers the advantage of screening drug residues in foods with little or no previous sample preparation. Application, on the other hand, of physicochemical techniques, allows quantification and more tentative identification of residues in those samples found positive. The problem of analyzing for drug residues is complicated by the fact that it is not known whether residues exist, and if they exist, the type and quantity are not known. 

Microbiological or immunochemical techniques offer the advantage to screen rapidly and at low cost a large number of food samples for potential drug residues but cannot provide definitive information on the identity of the specific drug residues found. Unlike microbiological and immunochemical techniques, physicochemical techniques actually aim at the identification, quantification, and/or confirmation of the presence of violative residues in suspected samples. 

When the results obtained lie above the MRL, both qualification and quantification are becoming very important. Immunochemical tests that are often used for surveillance of registered drugs are prone to cross-reactions that may influence the result. It is therefore important to confirm and quantify the results of immunochemical methods with an independent physicochemical method. 

Protein quantification is an important step for handling protein samples for isolation and characterization, and is a prerequisite step before submitting proteins for chromatographic, electrophoretic, or immunochemical analysis and separation. The methods included in this unit are colorimetric measurements, whose procedures are faster, simpler, and less laborious than those based on estimation of total nitrogen content (vnitbi.2). 

Pentosidine is determined by HPLC with spectrofluorimetric detection (excitation and emission wavelengths of 335 and 385 nm, respectively) (S14), although immunochemical and ELISA assays for determination of various protein oxidative modification products have become increasingly popular (08). Protein-aldehyde adducts can be estimated using adduct-specific antibodies (U2, Wl). Another approach requires stabilization of adducts, producing derivatives resistant to conditions used in protein acid hydrolysis and quantification of hydrolysis products by gas chromatography-mass spectrometry (R7). 

IgA, with these variant albumins varies with storage conditions, pH, and concentration of low molecular weight —SH compounds, such as glutathione hence the same serum sample may appear different on electrophoresis at different times. Perhaps the most important laboratory consequence of these variants is the formation of complexes with AAT, resulting in apparent deficiency of this protein on electrophoretic patterns. However, quantification of AAT by immunochemical methods is normal. 

Because CRP is normally present in plasma at a concentration of less than 5mg/L (50(lg/dL), highly sensitive immunochemical methods are required for its quantification, Current assays include particle-enhanced immunotur-bidimetry or nephelometry, immunofluorescence, and hnmunochemiluminescence. CRP migrates on cellulose acetate or agarose gel electrophoresis anywhere from the slow-y to mid-P regions, depending upon the calcium ion content of the buffer. 

Tayab Z R, Balthasar J P (2004). Development and validation of enzyme-linked immunosorbent assays for quantification of anti-methotrexate IgG and Fab in mouse and rat plasma. J. Immunoassay Immunochem. 25 335-344. 

Immunochemical methods have their origin in the medical field. The first lA, a RIA for the quantification of insulin in serum, was described by Yalow and Berson. Later, radiolabels were replaced by enzymes in EIAs by Engvall and Perlmann and Van Weeman and Schuurs. ) Since then radiolabels have obtained broad application in medical diagnostics and environmental analysis. 

At the present time, there are several commercial kits for the quantification of CDT based on the fractionation of CDT and non-CDT serum variants by anion-exchange chromatography with immunochemical detection [176], The main characteristic of almost all of these micro columns is that they do not separate CDT sialoforms and they are quantified as a whole. Therefore, the CDT forms may contain an undetermined amount of trisialo-Tf, which is under debate as to whether it should be considered a CDT form, and that introduces an error in the determination [185,186], Another drawback of the anion-exchange chromatographic methods is that they fail to detect genetic variants, which may give rise to incorrect determination of CDT or even false-positive results when sera from individuals with genetic Tf variants are analyzed [177,186]. Some authors have claimed that the separation of individual Tf sialoforms is a more accurate procedure than CDT quantification as a whole [68,69,185,186], One of these commercial tests was compared with HPLC and CZE methods to conclude that the data obtained needed to be systematically confirmed by HPLC or CZE [187], In spite of these drawbacks, these kits are still convenient for routine analysis due to their speed. 

The quantitative aspects of changes in various intracellular pools of NADPH protochlorophyllide oxidoreductase (POR) are important for understanding the principles of operation of the multienzyme system of chlorophyll biosynthesis which proteins are encoded by nuclear DBA and synthesized in the cytoplasm. The application of immunochemical methods /I,2/ allowed to detect loss of POR in the system of intraplastid membranes of etiolated leaves under illumination. In this work the enzyme-linked immunosorbent assay (ELISA) was employed for quantification of POR in different intracellular compartments of postetio-lated and green barley seedlings. 

All protein determination methods described are not absolute and demand some form of calibration. The Kjeldahl s method remains the only official method currently available for calibration purposes and maintains its position as the most frequently used technique for the determination of organic nitrogen in food products. CE and immunochemical (enzyme-linked immunosorbent assay) techniques are most suitable for rapid separation and quantification of individual food proteins and are promising for widespread use in food protein analysis due to their high sensitivity, specificity, and simplicity of operation. There are numerous methods for the evaluation of the nutritional quality of food proteins. 

Schenk et al. reported the quantification of cytokines in cell extracts using micro-HPLC packed microcolumns coupled with immunochemical detection. Various types of chromatographies were associated with the immunochemical detection system in order to perform the separation of cytokines. These included cation-exchange, size exclusion, and re-versed-phase chromatography, with the latter being the most successful. 

Other detection methods include radioactivity for labeled compounds (a non-destructive physical method) and biological methods (e.g., immunochemical or enzymatic reactions). Coupled detection methods such as TLC/ infrared spectrometry (TLC/IR) or TLC/mass spectrometry (TLC/MS) can be used for confirmation of zone identity as well as quantification in some cases. 

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