Enzyme-linked immunosorbent assay, or ELISA, is a widely used laboratory procedure for identifying proteins or other antigens present at concentrations as low as one part in a billion within complex mixtures such as serum and urine. As such, the assay has been employed to detect viral antigens, tumor antigens, and many other protein moieties for years. Two basic methods are commonly used: (1) the specimen containing the suspected target antigen is coated on the substrate, then detected by enzyme-linked specific antibody, or (2), in the sandwich ELISA assay, the plate is coated with specific antibody and washed, the serum, urine, or similar complex protein mixture is added then eluted, and finally an enzyme-linked version of the same antibody is added and subsequently detected by chromogenic or fluorescent methods. The latter method presumes the existence of more than one epitope on the antigen; otherwise, the second antibody will not bind and no antigen is detected. Thus, either multiples of the same epitope must be present on the antigen, or two different antibodies directed against two epitopes must be used in the assay. Quantitation is readily performed when dilutions of the target mixture are performed and assayed, as the optical density of the mixture following enzymatic chro-mogen generation (as with horseradish peroxidase, a commonly used enzyme) increases linearly with increasing concentration of the target antigen. When suitable positive controls are performed in parallel, the concentration of the antigen in the serum or urine can be readily calculated from a simple y = mx + b graph of optical density (OD) versus dilution. A typical result performed in a 96-well format is illustrated in Figure 21.4.
ELISA assays are probably the most common laboratory procedure performed for immunodetection of protein antigens. However, the confluence of several factors has lent a new life to this venerable assay: as genomic methods increasingly identify gene targets, their protein end product can be predicted with accuracy and a suitable ELISA assay established. This method is of particular value in serum-based diagnostics, where the presence of enormous concentrations
of albumin and immunoglobulins vastly overshadows the relatively rare tumor cell products. However, by virtue of the remarkable ability of solid-phase immunoabsorption to retain even rare proteins on an immobilized immunoglobulin-coated solid substrate, it is possible to both detect and quan-titate such proteins. The presence in serum and urine of many tumor-associated proteins or peptides makes detection and quantitation of these antigens by ELISA an attractive means of early detection and monitoring of tumor persistence or recurrence. The marked sensitivity of the assay, coupled with its low cost, proven sensitivity and specificity, and amenability to rigorous laboratory quality control procedures, has made it the method of choice for such assays. However, successful use presumes both knowledge of the antigen, as well as the availability of a suitably specific antibody. Initially, these requirements are rarely satisfied, and tumor antigens must be identified by other methods. This process has been markedly enhanced in the past decade by rapid progress in mass spectrometry and related methods, well suited to detection, characterization, and even quantitation of unknown proteins and peptides.
Although antibody-based protein detection methods are preferred when the antigen is known and a suitable antibody to detect it is available, such methods are of no value with unknown antigens, which is the more common situation when searching for novel tumor antigens. Although a high index of suspicion may derive from preliminary gene expression analyses on expressed mRNA, the ultimate identification requires detection and at least partial sequence identification. By far the most common method of doing so currently is mass spectrometry. However, mass spectrometry is not a single method; rather, there are many variations, too many to discuss here. Suffice it to say that increasingly, sample preparation methods that mimic ELISA or affinity capture methods are coming to dominate clinical proteomics, while rather more demanding, tedious, expensive, but ultimately unequivocal methods, such as tandem mass spec-trometry are finding wide acceptance as research tools. Here, we focus on mass spectrometry with suitable interface for specimen complexity reduction coupled with high throughput.
Clinical proteomics, as opposed to research proteomics, necessitates high throughput, reproducibility, and reasonable cost. Most research methods that utilize preparative columns for sample cleanup before mass spectrometry are not suitable for clinical use. The columns are expensive and must be replaced frequently. However, raw serum or urine cannot be successfully analyzed by mass spectrometry because of the vast difference in concentration between target protein or peptide compared to contaminating proteins such as albumin. However, depletion methods that selectively remove albumin and serum globulins may have an untoward effect: a very high percentage of serum proteins, likely including tumor antigens, are in fact bound to albumin. Removal of albumin may thus remove a large amount of the target antigen. Thus, methods which selectively immobilize target proteins or peptides from complex mixtures are to be preferred, not unlike the solid-phase adsorption of ELISA assays discussed previously.
SELDI TOF is a currently popular method of preisolating a vast variety of proteins or peptides before mass spectrome-try. This method relies on the selective elution with laser desorption and ionization of proteins, followed by time of flight mass spectrometry. Marketed by Ciphergen, this technology has been used for the majority of clinical proteomics publications to date.22-32 In essence, this method utilizes a specimen target immobilization using a "protein chip," the characteristics of which vary widely, from strong anion or cation exchangers to immobilized antibody bases. Each spot is repeated several times in a row, and the adsorbed target is selectively eluted under varying elution conditions, from weak to strong. Each step is ionized and separated on the basis of the mass to charge (m/z) ratio. The resultant peaks are recorded and conditions optimized to identify the peak or peaks of interest. A typical example is illustrated in Figure 21.5. For identification, the same protein chip can be incubated with one or more proteases, such as trypsin and the resultant peptides spectrographed. The peptide pattern is highly reproducible and by definition identifies partial sequences within the intact protein. When compared to the vast online libraries of peptide fragments that result from any protein treated with any common protease, it is possible to make an identification from peptide mapping more than 90% of the time. If any doubt remains, the same specimen can be subjected to tandem mass spectrometry with quadrapole-based, collision-induced dissociation of individual amino acids, which can then be readily identified and the amino acid sequence deduced. This finding is then easily compared to genomic or protein library data for a positive identification, regardless of whether prior knowledge exists.
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