Immunoassay separation methods used

Fraction-separation methods used in immunoassay can be divided into two major groups liquid-phase and immunoreagent modified solid-phase separations. 

It may be important to consider the variability of the matrix due to the physiological nature of the sample. In the case of LC-M/MS-based procedures, appropriate steps should be taken to ensure the lack of matrix effects throughout application of the method, especially if the nature of the matrix changes from the matrix used during method validation. For Microbiological and immunoassay, if separation is used prior to assay for study samples but not for standards, it is important to establish recovery and use it in determining results. In this case, possible approaches to assess efficiency and reproducibility of recovery are ... 

Y4. Yamamoto, R., Hattori, S., Inukai, T., Matsuura, A., Yamashita, K., Kosaka, A., and Kato, K., Enzyme immunoassay for thyroxine and triiodothyronine in human serum, with use of a covalent chromatographic separation method. Clin. Chem. 27, 1721-1723 (1981). 

Much effort has been made to detect steroids in biological fluids. Even simple TLC methods have been used for qualitative analysis [38], One method that been used for quantification involves an immunoassay, but several problems exist with that method, most notably cross-reactions and interference with other substances [39], On the other hand, a number of chromatographic methods have been developed to overcome these problems. The majority of analytical methods involved GC, which has good detection limits, but requires previous derivatization [40] of the steroids to accomplish volatilization. Many methods have also been reported using HPLC with UV detection or LC-MS [40, 41], Previously used stationary phases for LC was e.g., Sephadex LH-20, Celite and Lipidex, but they could not be operated with high pressure [42], These columns were therefore slow to run and the separation of steroids was very time-consuming [43], Nowadays applications mainly use HPLC as a separation method with both normal-phase and re-versed-phase chromatography. 

Unlike direct reference assays, indirect index methods use two separate tests to estimate the free hormone concentration a total serum T4 (or T3) measurement and an assessment of either the serum TBG concentration or the fraction of T4 (or T3) that is free m serum the latter is traditionally derived using equilibrium tracer dialysis or T uptake methods. Results of these tests are then combined mathematically to give estimates of the free hormone concentration. Indirect index-based methods have essentially fallen out of favor in deference to the direct immunoassay methods. 

A number of nonisotopic immunoassays for estradiol have been developed and adapted for use on fuHy automated immunoassay systems. All are heterogeneous assays (separation step needed), but most are direct assays and do not require prehminary extraction. Most procedures offer the convenience of solid-phase separation methods. For routine clinical applications, the greatest experience is with enzyme immunoassays. Most commercial enzyme immunoassays use horseradish peroxidase or alkaline phosphatase to label estradiol antigens enzyme activity is determined using a variety of photometric,fluorescent,or chemiluminescent substrates. ... 

Heterogenous fluorescence immunoassays can be carried out with the aid of the same separation procedures used in radioimmunoassay. The more expedient homogenous fluorescence immunoassays require quenching, enhancement, polarization, or shifting of the fluorescence of the label upon binding of the labeled analyte to its antibody. Occasionally, a second antibody, directed at the anti analyte antibody, will be used in a double-antibody method to precipitate the bound labeled and unlabeled analyte or to alter the optical properties of the label in such a way as to make the analysis more sensitive. 

Microdialysate samples have been analyzed using a variety of nonseparation-based analytical techniques including immunoassay, biosensors, and MS [1-4]. The main limitation to the use of these methods is that they are typically restricted to the measurement of a single analyte. For more complex samples, the detection of multiple substances is usually necessary. In this case, the dialysate sample is normally analyzed by conventional chromatographic or electrophoretic separation methods employing optical, electrochemical, or mass spectrometric modes of detection [5]. 

Another application that microfiuidic bioreactors have been used for is in immunoassays. These are based on antigen-antibody interactions and are a powerful technique because these interactions have very high selectivity and are extremely sensitive. Typically, an immunoassay is performed by immobilizing one of the binding partners placed within the system. The corresponding pair is then incubated within the device. The advantage of this approach is the ability to very specifically concentrate molecules on the active surface of the device. This enables enhanced reaction efficiencies, simplified analytical procedures, and reduced consumption of reagents. These immunoassay methods are often coupled with separation methods such as capillary electrophoresis in order to further increase the utility of the method [10]. 

Although the use of CE in food analysis is limited compared with LC, many applications have been reported in recent years in the study of food proteins and peptides. It has been shown that the sensitivity provided by CE using LIE detection for bovine whey proteins is twice as good as that provided by UV detection, under the same separation conditions. LIF detection has also been used together with affinity interactions to enhance the detection sensitivity in CE. For instance, an immunoassay detection method has been described using a polyclonal antibody marked with fluorescein to determine lactoferrin. CE-LIF has also been applied to the determination of amines and amino acids in several samples such as wine and milk products. 

Two immunoassays of this type have been described with differing methods to separate the free and bound fractions of the tracer. In the assay described by Matsunaga, the method used is based on the difference in the isoelectric point (pi) values of the free and bound antibodies, permitting separation on an ion exchange capillary column [84—86]. The bound fraction of the antibodies (pi = 5.6) is not retained on the column and is quantified by continuous amperometric flow analysis, while the free fraction (pi = 7) is subsequently eluted using a buffer (pH 6.0 with 50 mM malonate) and can be reused up to 8 times (Fig. 8.16). 

In principle, the same isolation and separation methods and mass spectrometric techniques are used for the unambiguous identification of veterinary medicines as for pesticides (cf. 9.4.3). Enzyme immunoassays are also used (cf. 2.6.3). 

LC—MS/MS assays for 25-OHD [27] are now commonplace in clinical laboratories, especially larger reference laboratories. Often, the first step in the analysis is a liquid—liquid extraction of nonpolar 25-OHD and a deuterated internal standard from serum into an organic solvent, followed by evaporative concentration and injection into the LC—MS/MS. Additional reported metirods for specimen preparation include manual and online solid-phase extraction and simple protein precipitation with an organic solvent. The advantages of LC—MS/MS assays over traditional radioimmimoassays and more current chemiluminescent immunoassays include the use of an isotope dilution for precise quantitation, as well as the ability to distinguish and separately quantitate 25-hydroxy vitamin D2 and 25-hydroxy vitamin D3. There is considerable controversy as to which method of 25-OHD quantitation is the best, however, since many large scale population studies on vitamin D, such as the Women s Health Initiative (WHl) and the National Health and Nutrition Examination Survey (NHANES), utilized radioimmunoassays [28]. 

The methylxanthines can be determined in foods and biological systems by the chromatographic methods of TLC, GC, HPLC, or CE. Ultraviolet spectroscopy following a separation procedure can also be used. More recently, immunoassay methods have been developed. There is no single best method the analyst must balance the features of each assay with the final requirements for data precision and reproducibility. 

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