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Editorials |
1 Department of Clinical Chemistry, University Hospital Ghent (2P8), De Pintelaan 185, B9000 Ghent, Belgium
aAuthor for correspondence. E-mail Joris.delanghe{at}ugent.be.
In this issue, Levy and Levy (1) report on the development of an ELISA using single-chain antibodies (ScAbs) from a phage library for the determination of the major haptoglobin (Hp) phenotype in serum. This technical achievement is remarkable as the structural differences between the major Hp phenotypes 1-1, 2-1, and 2-2 are only minor (2). Apart from a single junction at the site of duplication of exon 3, there are no differences in primary amino acid sequence between the Hp alleles Hp1 and Hp2. The authors have taken advantage of the unique polymeric differences among Hp phenotypes to develop their ScAb-based ELISA.
ScAbs have been used for several years as a tool in the biomedical research laboratory. They have been applied in areas such as antibody and protein engineering, enzyme technology, vaccine development, and ligand-receptor studies with applications in oncology and immunology (3). The cloned antibodies can also be used as "intrabodies" to study function (4) or to act therapeutically at the intracellular level (5). By contrast, their diagnostic applications to date have been scarce.
ScAbs allow an easier and more cost-effective scale-up for manufacturing compared with monoclonal antibodies. Furthermore, the enhanced screening capabilities of the repertoire of libraries allow for the more rapid assessment of ScAb proteins of desired specificity by use of high-throughput screening methods. Their design maintains the complete and intact antigen-binding region of an antibody, but at a fraction of the size of conventional antibodies. ScAbs can be screened efficiently by phage, yeast, ribosome, and bacterial displays. A major strength of these platforms is the ability not only to identify such antibodies but also that such antibody pairs work well in ELISA-type "sandwich" assays (6). Antibodies against self-antigens and poorly immunogenic targets can be more easily selected. However, antibody fragments recovered from libraries generally show only a moderate binding strength: binders can be obtained with dissociation constants between 10–5 and 10–8 mol/L (6). This concern is of limited importance for abundant proteins such as Hp. Nevertheless, antibody fragments can be further modified to improve affinity (to the nanomolar range) or avidity, respectively, by mutating crucial residues of complementarity-determining regions or by increasing the number of binding sites by making dimeric, trimeric, or multimeric molecules. ScAbs can also be used as tools to visualize structural diversity in strongly related compounds (5).
The report by Levy and Levy (1) nicely illustrates the large potential of highly specific ScAb-based ELISAs in the field of clinical laboratory medicine. Similar applications can be expected in the near future in a broader variety of domains, including plasma protein chemistry, tumor markers, serology, and coagulation testing.
Traditionally, the determination of Hp phenotypes has been based on time-consuming electrophoretic or chromatographic techniques (2)(7), which are poorly suited to mass screening purposes. These techniques can, however, be helpful in the case of rare Hp mutants (2). Raising monoclonal antibodies against human Hp phenotypes did not allow the development of reliable Hp phenotyping methods (8). Genotyping of Hp based on PCR is another option that became available recently (9). Genotyping methods offer the advantage of remaining applicable in clinical conditions associated with very low Hp concentrations.
The existence of the Hp polymorphism is a major caveat in the interpretation of Hp concentrations in serum or plasma because the reference intervals for serum or plasma Hp are strongly depending on the phenotype (2). In the clinical laboratory, determination of Hp concentration in serum or plasma is most commonly used for detecting in vivo hemolysis (2).
The new Sc-based Hp phenotyping method has other broad implications. Notwithstanding the fact that the function of Hp traditionally has been considered to be the binding of hemoglobin, recent findings point toward additional, clinically important functions. Hp phenotypes differ in their immunologic properties (10). The Hp 2-2 phenotype has also been associated with iron overload (11)(12) and a lowering of vitamin C status (13). Hp 2-2 is a major determinant in the outcome of infectious diseases [e.g., HIV (14), tuberculosis (15), and malaria (16)] and is regarded as an important independent cardiovascular risk factor, especially in diabetes (17). The ScAb-based Hp phenotyping method proposed by Levy and Levy (1) allows mass screening, which may be a prerequisite for better-tailored, patient-specific, individualized prevention of cardiovascular disease (18).
References
ocha A, Dobryszycka W. Monoclonal antibodies against human haptoglobin. Hybridoma 1989;8:551-560.[ISI][Medline]
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