Immunoassay procedure

A similar type of biotin-dendritic multimer also was used to boost sensitivity in DNA microarray detection by 100-fold over that obtainable using traditional avidin-biotin reagent systems (Stears, 2000 Striebel et al., 2004). With this system, a polyvalent biotin dendrimer is able to bind many labeled avidin or streptavidin molecules, which may carry enzymes or fluorescent probes for assay detection. In addition, if the biotinylated dendrimer and the streptavidin detection agent is added at the same time, then at the site of a captured analyte, the biotin-dendrimer conjugates can form huge multi-dendrimer complexes wherein avidin or streptavidin detection reagents bridge between more than one dendrimer. Thus, the use of multivalent biotin-dendrimers can become universal enhancers of DNA hybridization assays or immunoassay procedures. 

Pool the fractions containing antibody and immediately mix with an amount of maleimide-activated enzyme to obtain the desired molar ratio of antibody-to-enzyme in the conjugate. Use of a 4 1 (enzymerantibody) molar ratio in the conjugation reaction usually results in high-activity conjugates suitable for use in many enzyme-linked immunoassay procedures. Higher molar ratios also have been used with success. 

Liposome conjugates may be used in various immunoassay procedures. The lipid vesicle can provide a multivalent surface to accommodate numerous antigen-antibody interactions and thus increase the sensitivity of an assay. At the same time, it can function as a vessel to carry encapsulated detection components needed for the assay system. This type of enzyme-linked immunosorbent assay (ELISA) is called a liposome immunosorbent assay or LISA. One method of using liposomes in an immunoassay is to modify the surface so that it can interact to form biotin-avidin or biotin-streptavidin complexes. The avidin-biotin interaction can be used to increase detectability or sensitivity in immunoassay tests (Chapter 23) (Savage et al., 1992). 

Table 1.10 Typical immunoassay procedure using Zymate robotic laboratory automation system... 

Johnson and Van Emon [57] have described a quantitative enzyme based immunoassay procedure for the determination of polychlorinated biphenyls in soils and sediments and compared the results with those obtained by a gas chromatographic method. The soil is extracted with methanol, or Soxhlet extracted or extracted with a supercritical fluid. In the case of the latter two extractants good agreement was obtained between immunoassay and gas chromatographic methods. Spiking recoveries from soil achieved ranged from 104% (Aroclor 1248) to 107% (Aroclor 1242). Detection limits were 9pg kg-1 (Aroclor 1245) and 10.5pg kg-1 (Aroclor 1242). Chlorinated anisoles, benzenes or phenols did not interfere. 

The enzyme immunoassay procedure [57] discussed in section 5.6.1.5 for the determination of polychlorobiphenyls in soils has also been applied to sediments. 

Although some European countries still accept the results of the four plate test as confirming the presence of antibiotic residues in samples ( ), other work indicates that FPT test is not necessarily reliable. The occurrence of natural microbial inhibitors in tissues has frequently been noted (4,9,49,82), It has also been frequently observed that the results obtained by microbial and physicochemical procedures sometimes differ considerably (9,10,45,82,86), Results obtained in our laboratory suggest that even inactivation by penicillinase may not be totally specific for B-lactam antibiotics (W), The specificity of immunoassay procedures depends on the specificity of the antibody used in the test (95), Specific antisera are not widely available at present. Physicochemical procedures are therefore essential for identification and confirmation of suspect residues detected by microbiological tests. 

Figure 4. Thermally induced precipitation immunoassay procedure.

The analysis of fish tissues for ciguatoxin by a newly developed enzyme-immunoassay procedure (26, 27) has been carried out in this study. Three areas of examinations have been attempted (1) the examination of clinically defined and documented and non-toxic consumed fish samples (2) the assessment of freshly caught fishes from the sites in the Leeward part of the island of Oahu where ciguatoxin is found and (3) competitive inhibition with suspension of purified ciguatoxin and closely related structurally similar polyether toxins. 

The comparison of clinically defined and documented toxic with non-toxic consumed fishes by EIA clearly demonstrated a statistically significant difference (p <0.001) between the two populations. This result is similar to that reported for the radioimmunoassay and enzyme-immunoassay procedures (21, 22, 26-28). The assessment of the EIA with each species of fish samples caught in the Hawaiian waters presented positive and borderline frequency values comparable to that reported earlier by Ito and Uchida (28) by the RIA, and more recently by Kimura et al. (27) by the EIA. 

The critical factors in the enzyme-immunoassay procedure as indicated previously (26, 27) are as follows (1) the size and shape of the tissue samples should be uniform, and replicate samples from each area should be tested (2) the fixation step of the fish tissue with methyl or ethyl alcohol containing is essential... 

Figure 3.29 — (A) Immunosensor scheme A Cell inlet tubing B transparent PTFE tube (1.6-mm ID x 3-mm OD C immunosorbent D frit. (B) Outline of flow-injection immunoassay procedure. The assay buffer is posphate buffered saline (PBS) at pH 7, and flow-rates and times (min) are given in the figure. Immobilized anti-mouse IgG modified sample (mouse IgG) injected at T = 0 change of the flow-rate and buffer at T = 4 injection of hydrogen peroxide in a basic medium at T = 5 then, emission monitoring and regeneration step acridinium ester-labelled antibody (emitter = N-methylacridine). (Reproduced from [218] with permission of Elsevier Science Publishers).

Fig. 1. Schematic representation of a typical competitive immunoassay procedure.

Fig. 6. Principle of a noncompetitive hapten immunoassay based on quantitative biotinylation of the target molecule (A) biotinylation of a hapten with reactive amino group(s) and subsequent (B) sandwich-type immunoassay procedure.

Fig. 12. Noncompetitive hapten immunoassay procedures (A and B) using a combination of the a-type and j6-type anti-idiotype antibodies, each recognizing the framework and paratope of the anti-hapten antibody. Anti-hap, anti-hapten antibody (primary antibody) a-Id, a-type anti-idiotype antibody /J-Id, /i-type anti-idiotype antibody S, signal-generating group B, biotin SA, streptavidin.

With the maturation of tandem mass spectrometry (MS/MS) instruments and methodologies, many commercial laboratories are switching to this technique, and high-sensitivity measurements on high volumes of samples are becoming routinely achieved. Since the future of steroid hormone analysis is MS/MS, only MS techniques will be described in this chapter. Immunoassay procedures have been well described and reviewed over the years and will cease to be mainstream methodologies within the next decade. 

King, 1980). Polysaccharides modified in this manner are effective in covalently cross-linking antibodies for use in immunoassay procedures. 

Fig. 18.5. Calibration curve obtained by the immunoassay procedure for the detection of anti-CTB using the ITO-PPB-coated optical fibers. The curve was fitted according to the equation y = A+B ln(.r), where x is the anti-CTB dilution value and y is the chemiluminescence response. The obtained correlation coefficient is R2 = 0.95.

Several methods have been described, starting with radio-immunoassay procedures (Rees 1971, Kao 1979, Krieger 1975), and proceeding to immunoradiometric methods (Hodgkinson 1984, White 1987, Zahradnik 1989, Raff 1989, Gibson 1989, Fukata 1989). 

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