Immunochemical Assays

Trace contaminants such as host cell proteins (HCPs) and DNA are deterrnined by more specialized techniques. Host cell proteins are generally deterrnined using an immunochemical assay, in which an antibody preparation, raised against a mixture of the HCPs, is used to selectively detect the total level of HCPs in the product. DNA can be deterrnined using a labeled mixture, or probe, of complimentary DNA from the host cell. 

The application of PSA measurements for clinical monitoring of prostatic carcinoma requires fine tuning of PSA assays. One important aspect of this tuning is to have well-defined standards (primary calibrators). Calibrators or primary reference materials consisting of PSA complexed with ACT have been prepared and are available to sponsors of commercial immunoassays. As a result of this, some sponsors have studied calibration stability and have shown that calibration did not change within 14 to 90 days. Primary references of 90 percent PSA-ACT and 10 percent f-PS A have been shown to minimize differences in PSA measurements between different assays [NCCLS Document—Primary Reference Preparations Used to Standardize Calibration of Immunochemical Assays for Serum Prostate... 

NCCLS Document 1/L A 19-A. Primary reference preparations used to standardize calibration of immunochemical assays for serum prostate-specific antigen (PSA) Approved Guideline. NCCLS, Villanova, PA National Committee for Clinical Laboratory Standards (1996). 

The significant enhancement in emission quantum yields and lifetimes suggests that 3 can be used as a noncovalent probe in immunochemical assay and biophysical studies. This dye is quite soluble in aqueous buffer and interacts with... 

A. Dankwardt, Immunochemical assays in pesticide analysis, in Encyclopedia of Analytical Chemistry (R.A. Meyers, ed.), John Wiley Sons Ltd, Chichester (1997). 

It is at this point that the real fun begins, to visualize the material and determine whether the obtained results are scientifically useful. Instrument accessibility is an important aspect of EM immunocytochemical studies. EM facilities are expensive to acquire and maintain, which induces many universities and other organizations to centralize the EMS and require advance scheduling. This can impede developing an immunochemical assay where multiple samples must be examined in order to refine an assay protocol. It may be necessary to repeat the assays many times before obtaining a satisfactory result. It is very useful to have access to an EM that allows an assay to be conducted, followed by a quick examination of the grid, and then to conduct additional rounds of assays and examination. In most cases, it will take several assays before... 

Table 3 Some representative commercial immunochemical assay kits for the most important emerging pollutants with an industrial origin. The supplier and the contact web page are also listed... 

With the introduction of specific immunochemical assay methods for the quantitation of the individual immunoglobulins, more detailed results have become available. Tables 1 and lA show the wide range of normal values for the serum immunoglobulin concentrations for different populations. 

In Uganda, Marsden et al. (M31) and in Zambia, Lowenthal et al. (L5) reemphasized the association between tropical splenomegaly and malarial infection, and, with the introduction of specific immunochemical assay methods, interest in the association between tropical splenomegaly and malaria was reawakened. 

H3. Hayashi, H., LoGrippo, G. A., and Retry, M., Immunoglobulin levels in spinal fluid and saUva by direct immunochemical assay and microscopic measurements. Henry Ford Hosp. Med. J. 18,... 

The amount of total residues is generally determined by study with radiolabeled drugs and is expressed as the parent drug equivalent in milligrams per kilogram of the food. Bound metabolites can be measured as the difference between the total and extractable residue. Microbiological assays measure the parent molecule and its bioactive metabolites immunochemical assays measure the parent molecule and closely chemically related metabolites. 

In some instances, calibration test data may need to be subjected to some kind of mathematical transformation, prior to the regression analysis, in order to obtain linear calibration plots. In some cases, however, such as in immunochemical assays, linearity cannot be demonstrated even after any transformation. The use of nonlinear calibration curves for analysis has been discussed (27). 

Another issue of relevant importance to the interpretation of analytical results is the analytical specificity of the test, particularly when in an immunobased assay. Specificity is exquisite in immunochemical assays but, at the same time, it can be exquisitely troublesome. For example, when an immunochemical assay for the penicilloyl group is used to monitor the pharmacokinetics of penicillin elimination from the serum of treated animals, the measured levels remain high for at least several weeks, although the antibacterial activity was all eliminated from bovine serum within 24 h after injection. This is because the immunochemical assay measured not only the free drug but also the penicilloyl groups covalently bound to proteins in serum. The half-life of these bound residues is roughly equal to the half-life of the proteins in the circulation. 

The ratio of the hapten to the carrier molecules is also very important for the success of an immunochemical assay. Too few or too many hapten molecules linked to a carrier molecule will inevitably lead to poor immunogenicity. Desirable ratios of hapten to carrier molecules are in the range 10-100 1 (3). 

Although both polyclonal and monoclonal antibodies have been effectively used in immunochemical assays, only the latter can provide the high specificity required in some applications. Antibody specificity, on the other hand, is both a major advantage and disadvantage for immunochemical methods. It allows for highly selective detection of analytes but at the same time may complicate the development of multiresidue methods. Moreover, production of monoclonal antibodies requires special expertise and it is much more expensive than polyclonal antibodies. Thus, in cases where a range of analytes similar in molecular structure are required to be determined, a polyclonal may be more suitable than a monoclonal antibody. 

Although immunochemical assays can employ crude antisera, purification helps in improving assay sensitivity and specificity, reduces analysis time, and aids in standardization of the assay. Various degrees of antibody purification can be performed prior to incorporation of an antibody into the assay format. 

Common immunochemical assay formats to select from include the 96-well microtiter plates, dipsticks, coated test tubes, and membrane-based flow through devices. If the end-user is a trained technician working in a well-equipped laboratory and needs to detect and tentatively identify, for example, antimicrobial residues in hundreds of meat samples per day, a multiwell or other high-through-put format should be chosen. If, on the other hand, the end user is a quality control inspector at a milk factory who has limited time to find out whether the penicillin residues in the milk waiting to be unloaded exceed a certain level, the same reagents used in the first instance may require a more user-friendly format such as dipstick or membrane-based flow through device. 

Penicillin G is by far the most common -lactam antibiotic used in veterinary medicine however, cloxacillin, ampicillin, amoxicillin, cephalexin, and cefuro-xime also share a high proportion of the market. As a result of this rather wide use, a generic -lactam immunochemical assay that can detect several different... 

Microbiological assays can be used to identify and rank samples that contain biologically relevant levels of drug residues. Immunochemical assays can also help screen out negative samples and prioritize samples for mass spectrometry. Both microbiological and immunochemical assays can play important roles in the analytical laboratory of the future and have the potential to reduce overall costs and expedite studies that may not otherwise be possible. 

Despite the fact that biological and predominantly immunoenzymatic methods have received increasing attention during the last decade for the determination of aflatoxins, chemical and immunochemical assays have to be preferred for their characteristics, including a lower limit of detection and high specificity. 

NHS esters of D-biotin have been used in many applications, including the biotinylation of rat IgE to study receptors on murine lymphocytes (Lee and Conrad, 1984), in the development of an immunochemical assay for a postsynaptic protein and its receptor (LaRochelle and Froehner, 1986a), in the study of plasma membrane domains by biotinylation of cell surface proteins in Dictyostelium disoideum amoebas (Ingalls et al., 1986), and for the detection of blotted proteins on nitrocellulose membranes after transfer from polyacrylamide electrophoresis gels (LaRochelle and Froehner, 1986b). 

The spectral changes observed by pulse radiolysis have been attributed to unimolecular transformation reactions and the suppression of these reactions by cysteamine has been studied (Hankiewicz et al. 1992 Hankiewicz 1996,1998). Among the products the formation of 8-oxo-A, FAPY-A (Alexander et al. 1987) and cA were noticed, whereby at pH 7 the R isomer dominates over the S isomer by a factor of 2.5 (G(total) = 0.09 x 10 7 mol J Fuciarelli et al. 1986). This low yield is in contrast to a value of 1.4 x 10 7 mol J 1 obtained by an immunochemical assay (Fuciarelli et al. 1985). 

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