Immunometric methods

Using a direct immunometric method usually comprises the following stages (Rittenburg, 1990) ... 

Immunometric methods find increasingly wide applications owing to their superiority and continuous improvements. One of the major advantages of immunometric methods is that they provide consumers with early information (prior to food consumption) on possible health hazards (allergenic characteristics) of specific food products. The sensitivity of immunoassays is largely dependent on the type of a compound subject to analysis (up to lOpg/mL of the analyte). 

Observing recent advances in applying immunometrical methods in medical science and their increasing role in food industry and quality control, we have to take into account equally dynamic application of immunometric methods in food analysis. 

Jgdrychowski, L. 2003b. Examination of immunoreactive and immunomodulative properties of food components with the application of immunometric methods. Pol J Food Nutr Sci 12/53(SI 2) 32-39. 

Albumin can be measured quantitatively by the bromcresol green method in most species (Evans and Duncan 2003). Other proteins (described below) are measured by immunometric methods. Newer methods based on proteomics technology (concentration of proteins by acetone precipitation or ultracentrifugation, separation by 2-d gel electrophoresis or chromatographic techniques with subsequent identification and quantitation by mass spectrometry) have been used experimentally (Bandara and Kennedy 2002 Chapman 2002 Thongboonkerd et al. 2002a, b). 

The majority of assay methods for excreted low molecular weight proteins are based in immunometric methods (gel immunodiffusion, nephelometry or ELISA) and the antibodies do not cross-react with homologous proteins in animal urine (the exception is p2 -microglobulin, for which a rat-specific latex... 

It should also be emphasized that although the precision of the automated particle-enhanced immunometric methods is higher than that of the enzyme-amplified single radial immunodiffusion first used to quantitate serum cystatin C, it is still lower than that for most methods for determination of serum creatinine. Moreover, the data for the intraindividual variation of serum cystatin C strongly indicate that a higher precision of the method would markedly improve the clinical usefulness of serum cystatin C determinations according to the criterion of Cotlove (N6). 

Available evidence indicates that serum cystatin C is a better marker for GFR than serum creatinine, particularly for the identification of an initial small decrease in GFR, i.e., in the so-called creatinine-blind GFR range. The most efficient use of this knowledge in clinical practice requires that quantitative methods of good precision, undisturbed by sample turbidity, are used. At least some of the presently available particle-enhanced immunometric methods seem to fulfil the first criterion of acceptable precision, but nonturbid fasting samples should preferably be used until new methods undisturbed by turbidity are developed. 

Most immunometric methods use solid phase separation by immobilizing the IF binder on beads or magnetic particles. The free vitamin B12 then remains in the supernatant, and the bound analytes become part of the solid phase suspension. For simultaneous folate/vitamin B12 measurement, a gamma-scintiUation counter that discriminates between the energy levels of Co (for vitamin B12) and I (for folate) must be used. 

Noncompetitive (also known as two-site, sandwich, labeled antibody, or immunometric methods) (see Chapter 9) require two antibodies capable of simultaneously binding PTH (Figure 49-14) (1) a capture antibody immobilized to a solid phase, and (2) a signal or reporter antibody labeled with a measurable substance or an enzyme changing the concentration (substrate or product) of a measurable substance. Unlike competitive immunoassays, with noncompetitive methods, both antibodies are added in excess, ensuring that aU analyte is measured. After formation of the ternary complex or sandwich, excess labeled antibody is removed by washing before quantification of complexes. Noncompetitive immunoassays provide increased sensitivity, specificity, reproducibility, and convenience. 

Immunometric methods based on chemiluminescence are capable of measuring TSH at concentrations required to accurately detect the low concentrations of serum TSH found in patients with true hyperthyroidism or the suppressed concentrations fmmd in patients with nonthyroidal illnesses. These sensitive methods resulted from the appHca-tion of the immunometric sandwich configuration, in which a serum TSH molecule forms a bridge between two or more... 

Generally, immunoassays can be classified into limited reagent immunoassay systems (FIA) and excess-reagent immunometric methods (immunofluoro-metric assay, IFMA). 

Several noncompetitive assays for haptens reported so far can be regarded as variations of conventional single-antibody immunometric assays, the majority of which (the assays in Sections 3.1 and 3.2) are based on principle B1 in Fig. 4. In many cases, these methods employ an automated flow system to simplify the assay procedure and minimize the time and labor required for the analysis. 

Traditionally, TSH activity was determined by bioassays. There is now sufficient evidence for the correlation of biological activity and results of immunoassays. Reference standards are available for rat TSH, dog TSH and human TSH (DSL Diagnostic Systems 2005). Immunometric measurements of TSH are available (Utiger 1979, Spencer 1994, Meinhold et al. 1994), for rat radio-immunoassay methods see section I.0.4.3 of this Chapter. Large scale synthesis of recombinant human thyrotropin has been reported (Cole et al. 1993, Hussain et al. 1996). The role of... 

Cystatin C has been measured by immunodiffusion or rocket electroimmunoassay, but the methods are too insensitive, and any form of labeled immunometric assay is too cumbersome and time consuming for the response time required. The most practical approaches described are to use a latex particle-enhanced turbidimetric or nephelometric immunoassay. An intraassay precision of less than 3% can be expected at the upper limit of the reference interval ( 1.00mg/L), with less than 4% for the between-day value. Further, cystatin C measurement appears unaffected by the spectral interferences affecting creatinine assays. ... 

Immunoassays employing antibodies are widely used to quantify hormones (see Chapter 9). Currently, labeled-antibody (immunometric) assays with nonisotopic labels are the method of choice for measuring most hormones, especially peptides and proteins. Immunometric assays use saturating concentrations of two or more antibodies (often... 

Two-site immunometric or sandwich assays that made use of two or more antibodies directed at different parts of the PRL molecule were next to be developed. As with other two-site IRMA assays, the capture antibody is attached to a solid phase separation system and the second or signal antibody is labeled with a detection molecule (e.g., radio-isotope, enzyme,fluorophor, or chemiluminescence tag ). In some assays, the capture antibody is attached to the wall of test tubes, plastic beads, microtiter plates, ferromagnetic particles, or glass-fiber paper. Other assays have used the strep-avidin approach that couples biotin to the signal antibody with avidin linked to a solid phase. Most of the current immunometric assays for PRL have been adapted to fully automated immunoassay systems. Compared with the older traditional RIA methods, these automated immunometric assays for PRL generally achieve lower detection limits (0.2 to 1.0 ig/L) and improved precision (interlaboratory coefficients of variation of <8% at all concentrations), and have superior specificity (<0.05% crossreactivity with GH). 

The introduction of two-site immunometric assays significantly improved the method for gonadotropin measure-... 

A number of commercial kits are available for determining PRA " the basic elements of these procedures are summarized in the following sections. With continued improvements and availability of the immunometric assay methods for determining renin mass, renin activity assays will likely be replaced by the direct mass assays for the routine assessment of plasma renin. 

Immunometric assays for TSH are available commercially as manual kit procedures or for use on automated systems. For practical reasons, nonisotopic methods dominate the market and have replaced radioactive tracer methods in most routine laboratories. The majority of immunometric TSH assays label the detection antibody with chemiluminescent labelled molecules other labels include peroxidase or alkaline phosphatase and sensitive photo-metric and fluorescenri molecules. Other assays are based on the use of fluorescent labels using europium chelates chemiluminescent compounds such as acri-dinium esters or ruthenium or bioluminescent molecules such as recombinant aequorin. ... 

The assay format for C-peptide was based on the two-step method and the two-site immunometric principle using two MoAbs, which recognize different epitopes. 

Scott-Moncrieff, J. C., M. A. Kosho, J. A. Brown, K. Hill, and K. R. Refsal. 2003. Validation of a chemiluminescent method enzyme immunometric assay for plasma adrenocorti-cotrophic hormone in the dog. Veterinary Clinical Pathology 32 180-187. 

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