Development of molecular and immunological techniques for the genotyping and detection of haptoglobin, a biomarker of human health

Inflammation is the body’s immediate response mechanism to disturbances in tissue homeostasis caused by acute or chronic stimuli, such as infections, stress, autoimmune reactions, or mechanical injuries. A reliable way to identify when the body is undergoing an inflammatory process is by measuring specific molecules, such as acute-phase proteins (APPs). An APP that significantly increases during inflammation is haptoglobin (HP), a protein whose primary function is to bind free hemoglobin in the blood. HP concentration in plasma can vary with different pathologies and is commonly tested in individuals with autoimmune diseases and inflammatory bowel disease (IBD). Structurally, haptoglobin is a tetrameric molecule composed of two light α-chains (α1 or α2) and two heavy β-chains, linked together by disulfide bonds. The 83 amino acids (aa) α1 chain is smaller than the 145 aa α2 chain. The extra amino acids in the α2 chain contain an additional cysteine residue that causes the formation of complex tertiary and quaternary structures. The full HP protein is encoded by the HP gene and its two allelic variants, HP1 and HP2, are responsible for the variation in α1 chain and α2 chain, respectively. HP2 contains a 1700 bp DNA segment, which is missing in HP1, derived from an intragenic duplication of the HP1 allele. Additional allelic variation found in the HP1 allele can be further observed in the HP1F and HP1S haplotypes. Depending on the combination of these inherited alleles, three genotypes can be formed: HP1-1, HP2-1 and HP2-2 with estimated worldwide frequencies of 16%, 46%, and 38%, respectively. The individual’s HP genotype defines which haptoglobin isoform is present, which can directly influence the effectiveness of HP binding to free hemoglobin in the bloodstream. The HP1-1 isoform has strong antioxidant properties due to its simple dimer structure, presenting increased hemoglobin-binding efficiency and hemoglobin-clearing capacity. In contrast, the HP2-2 isoform has a more complex protein structure and therefore a reduced hemoglobin-binding efficiency and antioxidant properties. These differences have become of interest in research due to their potential association with disease susceptibility, particularly in diabetes, cardiovascular conditions, and inflammatory disorders. Given the importance of HP genotypes and HP protein level in biological fluids, the determination of both in an individual could be a useful tool for the identification of the predisposition and presence of several health conditions. In this study, HP genotyping was performed using qPCR in order to distinguish between the HP1 and HP2 alleles and simultaneously differentiate between the HP1 haplotypes. It was demonstrated that the proposed protocol has the advantage of being faster and requires less DNA than the conventional PCR protocol. However, some inconsistencies were observed in the obtained genotypes, explained by the presence of a single nucleotide polymorphism (SNP) in the annealing region of one of the primers used in qPCR. On the other hand, during the ELISA experiments, the monoclonal antibody (MAb) effectively recognized the HP recombinant protein at different concentrations, showing a positive correlation between the protein concentration and the signal obtained. However, the polyclonal antibody (PAb) is thought to lack specificity for haptoglobin and may be interacting with other molecules, as it demonstrated non-specific binding to HP. Additional studies are required to evaluate alternative PAbs for the development and validation of the sandwich ELISA for measuring haptoglobin levels in serum. Moreover, further studies are necessary to elucidate a possible association between HP levels and the HP genotype.