Immunoaffinity matrix

For immunoaffinity matrix, to affinity-purify the antibodies generated... 

A typical protocol for purifying an antigen using an immunoaffinity matrix packed in a Bio-Rad Econocolumn, with a low pH or chaotrope elution method. 

Another subset of SPE is immunoaffinity extraction, in which an antibody specific to the analyte is incorporated into the SPE sorbent. This technique is very selective to the analyte and would be very effective in separating the marker residue from tissue-related matrix components. Disadvantages of immunoaffinity extraction are the need to develop a specific antibody-based SPE for each analyte. This approach holds promise for the future as the development of antibody-based methods becomes more commonplace. 

Suresh Babu CV, Lee J, Lho DS, Yoo YS. 2004. Analysis of substance P in rat brain by means of immunoaffinity capture and matrix-assisted laser desorption/ionization time-of-flight mass-spectrometry. J Chromatogr B Analyt Technol Biomed Life Sci 807 307. 

A technique called on-line immunoaffinity CE has been presented (45) that was also coupled to MS. However, in this setup the affinity principle is used to extract the analyte from a complex matrix in a microchamber affinity device prior to CE separation. Therefore, it cannot be considered ACE. 

Selectivity in lAC depends on the specificity of the immobilized antibody and, thus, monoclonal antibodies are preferentially used. In that case, a large amount of sample can be subjected to immunoaffinity cleanup without any retention of matrix components. This opens the possibility to determine very low concentrations of drug residues in edible animal products. For example, 20 ng chloramphenicol in 1 L milk can be determined with a recovery of 99% when 1 L of defatted milk is submitted to immunoaffinity cleanup. The chromatograms obtained after LC analysis were as clean as those obtained when 10 ml milk containing the same amount of chloramphenicol was also submitted to immunoaffinity cleanup (170). 

Following initial sample extraction, the primary extract must frequently be subjected to some kind of further cleanup including liquid-liquid partitioning, diphasic dialysis, solid-phase extraction, matrix solid-phase dispersion, immunoaffinity chromatography cleanup, liquid chromatography cleanup, or online trace enrichment. In some instances, some of these procedures are used in combination in order to attain higher purification levels. 

The aqueous or organic extract obtained at this step of analysis may be a very dilute solution of the analyte(s) of interest. It may also contain coextractives, which if allowed in the final extract will increase the background noise of the detector, making it impossible to determine trace level concentrations of the analyte(s). To reduce interferences and concentrate the analyte(s), the primary sample extract must be subjected to cleanup procedures such as liquid-liquid partitioning, solid-phase extraction, matrix solid-phase dispersion, ultrafiltration, immunoaffinity chromatography, and online trace enrichment. In many instances. 

Ultrafiltration (278, 279) and immunoaffinity chromatography (282) have also been described for removal of matrix components from milk extracts, while online trace enrichment has been reported for isolation/purification of tetracycline, oxytetracycline, demeclocycline, and chlortetracycline residues from animal tissues and egg constituents (305). The latter technique involves trapping of the analytes onto a metal chelate affinity preconcentration column (Anagel-TSK Chelate-5PW), rinsing of coextracted materials to waste, and finally flushing of the concentrated analytes onto the analytical column. 

Immunoaffinity chromatography cleanup has also been applied as an ideal and reliable strategy for residue analysis. Immunoaffinity columns prepared by coupling the antibodies to a cyanogen bromide-activated support were used to analyze avermectin BI residues in cattle tissues (359) and ivermectin in sheep serum (376). An immunoaffinity column prepared by an alternative activation/ coupling procedure with carbonyl diimidazole was also employed to analyze ivermectin residues in swine liver (361) since the earlier-reported methods did not work well in the analysis of this matrix. This recent work demonstrated the high specificity of tire antibody-mediated cleanup, but also showed that the immunoaffinity procedures could not always or completely eliminate matrix interference of samples. Therefore, application of additional cleanup steps before or after these procedures is often inevitable. 

Starting with a description of the analytical challenge in Chapter 19, the third part, which is devoted to analytical attitudes, proceeds with a detailed description in Chapter 20 of modern sample preparation procedures including solid-phase extraction, matrix solid-phase dispersion, use of restricted-access media, supercritical fluid extraction, and immunoaffinity cleanup. Flexible derivatization techniques including fluorescence, ultraviolet-visible, enzymatic, and photochemical derivatization procedures are presented in Chapter 21. 

Cleanup An additional separation of the mycotoxin from lipids and other components of the matrix is accomplished through the cleanup step. Most procedures include solid-phase extraction on stationary phases such as silica, C,8, florisil, and phenyl. Prepacked columns are largely used, with the variations between lots being recently ameliorated. Alternatively, the use of cleanup by immunoaffinity, based on the formation of mycotoxin-protein conjugate, is on the increase, since this is very rapid, selective, and usefully employed in various food matrices. One disadvantage is that the cost is still rather high, and cross-contamination phenomena (false-positive) can occur (30). 

The general principle of immunoaffinity chromatography is illustrated in Fig. 1. The analyte in the sample matrix is loaded onto the column, the column is washed to remove interfering substances, and the analyte is eluted from the column for subsequent use. The column is the heart of the purification system and must bind the analyte specifically enough to allow other substances to be rinsed off the column, allow the elution of the analyte under conditions that do not elute interferences, and permit the column to be regenerated multiple times for subsequent use. 

The analyte binding efficiency is matrix dependent. Some matrices, such as urine and tissue extracts, can be directly loaded onto the column, other matrices such as milk may need sample processing prior to loading onto an immunoaffinity column. The simplest sample preparation method is dilution this method has been applied to serum, liver, and kidney extracts after removal of particulates. Sometimes dilution alone is not sufficient to eliminate the matrix effect and classical sample preparation techniques (solvent/solvent extraction, solid phase extraction, etc.) will be necessary prior to immunoaffinity chromatography. We found milk often needs this type of treatment. 

Immunoaffinity chromatography can be used to purify protein antigens by immobilizing the relevant antibodies on an inert matrix such as polysaccharide beads. When exposed to a protein mixture, only the protein recognized by that antibody will bind to the beads and can be eluted later in pure or almost pure form. Cells bearing the antigen on their surface can also be purified using a similar procedure. 

Holtzapple et al. developed an immunoassay method for determination of four fluoroquinolone compounds (including ciprofloxacin) in liver extracts [64]. In this method, an immunoaffinity capture SPE column was used, that contained anti-sarafloxacin antibodies covalently cross-linked to protein G. After interfering liver matrix compounds had been washed away, the bound ciprofloxacin was eluted directly onto the HPLC column. The HPLC system used a 5 pm Inertsil phenyl column (15 cm x 4.6 mm i.d.), with 0.1 M-glycine hydrochloride/acetonitrile (17 3) as the mobile phase (eluted at a rate of 0.7 mL/min). Fluorimetric detection at 444 nm was used after excitation at 280 nm. The recovery of ciprofloxacin ranged from 85.7 to 93.5%, and the detection limit was... 

Ferguson et al. [16] developed a method, based on immunoaffinity extraction coupled to LC-ESI-MS, for the determination of P-estradiol, estrone, and oc-ethinyl estradiol in wastewater. The immunoaffinity extraction not only removed interfering sample matrix components, but also lowered the isobaric noise in SIM traces. Detection limits in the range of 0.07-0.18 ng/1 were achieved with recoveries better than 90% and a precision better than 5%. 

Interferring compounds from the sample matrix can become a major problem in the analysis of cytokinins that normally occur at trace levels. Immunoaffinity purification methods that are based on polyclonal or monoclonal antibodies enable a selective single-step clean-up and concentration of a certain group of cytokinins. These methods are usually combined with various techniques of final analysis such as HPLC-UV, HPLC-ELTSA, HPLC-MS [277-279]. However, the antibodies prepared so far do not show suitable affinity to certain metabolites (O-glucosides, N -glucosides, nucleotides). 

Enzyme-antibody complex formation represents the simplest among the immunoaffinity immobilization procedures and the immunocomplexes can be readily formed simply by mixing of the enzyme solution with the antibody or even antiserum. Interestingly neither pure enzyme nor pure antibody may be required for the formation of immunocomplexes. Several early [14,36,39] and some recent studies [22,24,28] indicate high retention of catalytic activities by various enzymes in the immunocomplexes and marked stability enhancement against various forms of inactivation. The small particle dimensions of the enzyme-antibody complexes may however lead to their compact packing and consequently to slow flow-rates in the column reactors. Their usefulness can be however remarkably enhanced by entrapping the complexes in a polymeric matrix [60,61]. 

---