For haptens

Fig. 13a-e. The increase of the signal intensities by the addition of the dendritic complexes composed of IgGs and protein A. The hapten was immobilized to the surface of the SPR sensor chip. The increase of the signal intensities on the complex formation of hapten with the antibodies were monitored. The addition of mouse IgG specific for hapten (Abl) (a), the complex of the Abl with protein A (b), one to one complex of Abl with anti-mouse IgG (Fc) antibody (Ab2) (c), two to one complex of Abl with Ab2 (d), and two to one complex of Abl with Ab2 in the presence of protein A (e)... 

Raising the pi of macromolecules also can significantly alter the immune response toward them upon in vivo administration. Cationized proteins (those modified with diamines to increase their net charge or pi) are known to generate an increased immune response compared to their native forms (Muckerheide et al., 1987a, b Apple et al., 1988 Domen et al., 1987 Domen and Hermanson, 1992). The use of cationized BSA as a carrier protein for hapten conjugation can result in a dramatically higher antibody response toward a coupled hapten (Chapter 19). 

The non-competitive FI immunoassay for haptens recently reported by Gunaratna and Wilson [219] offers rather an interesting means for developing flow-through sensors for this type of compound. 

Fig. 4. Classification of reported noncompetitive immunoassays for haptens based on the assay principle. (A) Assays that include a chemical modification of hapten to allow sandwich-type detection. (B1) Improved single-antibody immunometric assays that separate immune complex and excess labeled antibody, either by using a hapten-immobilized affinity column or based on differences in their physical properties. (B2) A variation of single-antibody immunometric assays based on masking of unoccupied antibody by an immunoreactive macromolecule followed by selective capture and detection of the hapten-occupied antibody. (C) Assays employing a probe molecule specific to a hapten-antibody complex.

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. 

Self (S4) first proposed the concept of noncompetitive assay for haptens utilizing an adequate combination of an a-type and a jS-type anti-idiotype antibody, in which he used the term, selective antibody for the a-type antibodies. Then, Barnard and Cohen (Bl) applied this assay principle for the determination of serum E2, naming the assay system an idiometric assay. Figure 12A illustrates the assay procedure of the idiometric assay of E2. The target hapten is captured by excess anti-E2 antibody immobilized on microtiter strips by incubation at room temperature for 1 h (step i). After washing the strips, the /3-type anti-idiotype antibody was added in order to saturate (or block) the unoccupied paratope of the anti-E2 antibody (incubation, room temperature for 30 min) (step ii). The a-type anti-idiotype antibody, which has been labeled with a europium chelate (H4), was then added to the plate and incubated at room temperature for a further 2 h (step iii). Finally, fluorescence intensity due to bound europium was measured with a time-resolved fluorometer. Because of large steric hindrance around the bound jS-type antibody (MW 150,000), the labeled a-type antibody would. 

Ishikawa, E., Tanaka, K., and Hashida, S., Novel and sensitive noncompetitive (two-site) immunoassay for haptens with emphasis on peptides. Clin. Biochem. 23, 445 53 (1990). 

The first antibody-catalyzed asymmetric 1,3-dipolar cycloaddition was reported recently by Janda and co-workers (382). The reaction of the relatively stable nitrile oxide 280 and dimethyl acrylamide 281 was catalyzed by antibody 29G12 having turnover numbers >50, and the product 282 was obtained in up to >98% ee (Scheme 12.89). The antibody 29G12 was formed for hapten 283 and coupled to a carrier protein by standard protocols. The hapten 283 contains no chiral center and therefore the immune system elicited a stereochemical environment capable of stabilizing the enantiomeric transition state leading to 282. 

Since, the broad protein and carbohydrate band overlapped each other in the 42 - 200 kDa range of SDS-PAGE and in the pH 3.0 - 4.3 range of IEF-PAGE (data not shown), MALDI MS of ARS2 was performed as previously reported for hapten-carrier protein conjugates For more detail MW determination [29-31],... 

Grant, S., A. Porter, and W. Harris (1999). Comparative sensitivity of immunoassays for haptens using monomeric and dimeric antibody fragments. J. Agric. Food Chem., 47 340-345. 

Conditions and abbreviations pH 7.5 antiserum, As43 RO, percentage of phenoxide species Klff, affinity constant of the antibody-hapten-BSA interaction relative to the affinity of the antibody for hapten 5-BSA conjugate n.a., not available [891. 

There are many factors to be taken into consideration for assay standardization. The first factor is the immunoreagents themselves. It is known that the antibodies are pleomorphic over much of their molecular structure even in uniform populations [128]. The analyte should be stable, available in a pure form, the structure should be well understood and invariable with time, and this is generally the case for hapten analytes [129]. More problematic are analytes where the molecular size is large and biologically variable (e.g., certain hormones and enzymes) [128]. Standardization is thus almost impossible because the analyte itself cannot be obtained in an homogeneous, pure form, or the standard is not identical to the corresponding substance in the sample to be measured [130-132]. 

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