Immunometric assays sites

A two-site immunometric assay of undecapeptide substance P (SP) has been developed. This assay is based on the use of two different antibodies specifically directed against the N- and C-terminal parts of the peptide (95). Affinity-purified polyclonal antibodies raised against the six amino-terminal residues of the molecule were used as capture antibodies. A monoclonal antibody directed against the carboxy terminal part of substance P (SP), covalently coupled to the enzyme acetylcholinesterase, was used as the tracer antibody. The assay is very sensitive, having a detection limit close to 3 pg/mL. The assay is fiiUy specific for SP because cross-reactivity coefficients between 0.01% were observed with other tachykinins, SP derivatives, and SP fragments. The assay can be used to measure the SP content of rat brain extracts. 

AOSD, adult onset Still disease AS, ankylosing spondylitis CA, crystal-induced arthritis ERA, enthesitis-related arthritis JA, juvenile arthritis PA, psoriatic arthritis RA, rheumatoid arthritis SE, synovium explants SLE, systemic lupus erythematosus SPCIA, solid phase 2 site chemiluminescent immunometric assay RP, relapsing polychondritis. 

Figure 14.4. Diagram of three basic immunoassay formats, (a) Common competitive format (b) competitive assay with immobilized antigens bound to a carrier protein (immunometric assay) (c) two-site immunometric assays. Haptenic analytes are indicated as triangles, whereas larger-molecular-weight analytes are shown as teardrop shapes. Conjugated fluorescent probes are denoted by the letter "F. ...

Immunometric Assays Based on Masking Unoccupied Antibody Binding Sites. . 155... 

Fig. 3. Schematic representation of noncompetitive immunoassays. (A) Two-site immunometric assays (sandwich assays) and (B) single-antibody (single-epitope) immunometric assays.

The modified single-antibody immunometric assays discussed below are based on principle B2 in Fig. 4. In these assays, unoccupied antibody-binding sites are masked by reaction with a multiply hapten-labeled macromolecule to permit subsequent selective determination of hapten-occupied antibodies. 

Using an antibody specifically recognizing the antigen-antibody complex, more direct noncompetitive hapten immunoassays, which can be regarded as semi two-site immunometric assay, could be established (S3). Figure 14 depicts two typical procedures of noncompetitive assays using anti-metatype antibodies, which are based on principle C in Fig. 4. 

In double-site immunometric assays (i.e., sandwich assays [29], see Fig. 9.9) the sample containing the analyte is incubated with both a capture antibody, usually immobilized on a solid phase, and a labelled detecting antibody. After the excess of labelled antibody is removed, the measured signal can be correlated directly to the analyte concentration. 

Fig. 9.8. Single-site immunometric assay The sample containing the anal5d e, previously incuhated with a surplus of labelled Einti-anafyte antibodies (1), is passed through a column with immobilized antigen (2), able to trap the excess of antibodies (3). The anafyte—Euitibody complex is eluted from the column (4) uid can be detected downstream.

Fig. 9.9. Double-site immunometric assay The sample containing the anedyte is incubated with the capture antibodies immobilized on a solid support (1), until equilibrium (2) is established. Then, labelled detection antibodies (3), recognizing a different epitope on the antigen than capture antibodies, are added onto the support, and the excess of detection antibodies is removed by washing (4). The amount of the label bound to the support is detected (5).

In a one-site immunometric assay, the sample is first incubated with a known excess of labeled antibodies (or Fab fragments) that are specific for the analyte of interest. After this binding has occurred, the mixture is applied to a column that contains an immobilized analog... 

The technique of postcolumn immunodetection involves the use of an lAC column that is attached to the exit of an analytical HPLC system. The lAC column in this approach serves to collect and retain a specific analyte from the HPLC column eluent for later detection. Both the on-off mode and immunoassay formats of I AC have been used as strategies for postcolumn immunodetection. The most common of these approaches is the one-site immunometric assay. One reason for this is that the immobilized analog columns in one-site immunometric assays often have a much more flexible selection of elution conditions than immobilized antibody columns. Another reason is that one-site immunometric columns can usually be used for many sample injections before they are eluted and regenerated, which helps to decrease the overall analysis time associated with their use in postcolumn detection. 

Seth R, Motte P, Kehely A, Wimalawansa SJ, Wimalawansa S, Self CH, et al. The development of a two-site enzyme immunometric assay (EIA) for calcitonin and its application in the measurement of hormone in normal subjects, MTC patients and postmenopausal women. Horm Metab Res 1989 21 3-5. 

Zink A, Blind E> Raue F. Determination of serum calcitonin by immunometric two-site assays in normal subjects and patients with medullary thyroid carcinoma. Eur J Clin Chem Clin Biochem 1992 30 831-5. 

Sensitive immunometric assays that use excess labeled antibodies in noncompetitive formats have been developed for measuring Several of these two-site sandwich ... 

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-... 

C Creminon, O Dery, Y Frobert, J-Y Couraud, P Pradefles, J Grassi. Two-site immunometric assay for substance P with increased sensitivity and specificity. Anal Chem 67 1617, 1995. 

M9. Mashiter, K., Love, C., Loizou, M., Aslam, M., Allen, G. J., and Holian, J., Development of a simultaneous incubation two-site immunometric assay for follicle stimulating hormone (FSH) based on enhanced luminescence. Clin. Chem. (Winston-Salem, N.C.) 33, 892 (abstr.) (1987). 

Negroni L, Bernhard H, Clement G, etal. (1998). Two-site enzyme immunometric assays for determination of native and denatured P-lactoglobuUn. J. Immunol Methods, 220(1-2) 25-37. 

In contrast to competitive immunoassays, the number of theoretical models describing the kinetics and thermodynamics of non-competitive immunoassays is considerably lower and most of them are derived from double-site immunometric configurations. The dose-response curve for such assays is sigmoid the signal increasing with the analyte concentration with a plateau value reached when the capture antibody becomes saturated. 

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. 

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