Bioluminescence Immunoassay for Thyroxine Employing Genetically Engineered Mutant Aequorins Containing Unique Cysteine Residues

Abstract

Genetically engineered one-to-one conjugates between an analyte and a protein label have been demonstrated to yield assays with better detection limits and performance characteristics than those prepared by conventional chemical conjugation methods. To date, the preparation of these conjugates has been limited to fusion techniques where a peptide analyte is fused in frame to the protein label. To further expand the range of analytes that can be detected by using genetic engineering techniques coupled with bioanalytical methods, we have employed site-directed mutagenesis to prepare one-to-one analyte-label conjugates that include nonpeptidic analytes such as drugs, vitamins, and hormones. Specifically, we have prepared mutants of the photoprotein aequorin containing single cysteine residues suitable for site-specific conjugation. Aequorin is a photoprotein that emits light at 469 nm and has been employed as a highly sensitive bioluminescent label in the development of binding assays for important biomolecules. We have performed polymerase chain reaction-based site-directed mutagenesis on apoaequorin to yield four mutant aequorins containing unique cysteine residues at positions 5, 53, 71, and 84 in the polypeptide chain for the purpose of site-specific conjugation to a model analyte. A maleimide-activated thyroxine was selected as the model analyte and site-specifically conjugated to the mutants through their unique cysteine residues. A heterogeneous assay for thyroxine was then developed by employing the genetically engineered aequorin mutants.

Antibody-labeled Gold Nanoparticles Based Immunosensor for the Detection of Thyroxine Hormone

“…For this reason, the measurement of the free T4 hormone concentration is prominent to analyze the thyroid status of patients in clinical diagnostics. 5 Various analytical techniques, such as high performance liquid chromatography coupled mass spectrometry (HPLC-MS), 6,7 time-resolved immunoassay, 8 dialysis, 9,10 ultra-filtrations, 11 polyacrylamide gel filtration, 12,13 radioimmunoassay (RIA), 14,15 enzyme-linked immunosorbent assay (ELISA), [16][17][18] mass spectrometry, 19,20 bioluminescent immunoassay, 21 chemiluminescent immunoassay (CLIA), 2 electrochemical immunoassay, 12,22 and surface plasmon resonance (SPR) 3 have been employed for the determination of T4. However, these methods, being the direct method are complicated and time-consuming and require sophisticated non-portable instruments and expertise.…”

“…Light sources (such as aequorin) used in bioluminescence assays have a relatively very low light output, resulting in less efficiency. 21 ELISA is time-consuming since it involves multiple incubation and washing steps. RIA is associated with the handling of harmful radioactive labels, expensive waste disposal and limited half-life.…”

Bioorthogonal Protein Conjugation: Application to the Development of a Highly Sensitive Bioluminescent Immunoassay for the Detection of Interferon-γ

“…In general, specific conjugation of bioluminescent proteins to antibodies is also a significant challenge due to the unavailability of unique amino acids with appropriate chemical functionality necessary for conjugation, which necessitates protein engineering to introduce unique cysteines if not detrimental to protein activity. 10,17,18 Further, a few important criteria to be considered while designing protein reporter–antibody conjugation include maintaining the structural and functional integrity of the biomolecules involved, reproducibility of the conjugation, control of biomolecule conjugation ratio, stability of the conjugates, ability to perform reaction in hydrophilic environments, and so forth. Many different orthogonal bioconjugation methods with superior performance over the traditional conjugation method have been developed that involve the use of less common amino acid residues.…”

“…Thus, far, bioluminescent proteins are conjugated to antibodies using specific and nonspecific chemical methods and genetic fusion, if the gene sequence for the antibody is available. − Nonspecific chemical conjugation can result in heterogeneous conjugates that can lead to loss of the functional property of the antibody as well as the bioluminescent reporter. In general, specific conjugation of bioluminescent proteins to antibodies is also a significant challenge due to the unavailability of unique amino acids with appropriate chemical functionality necessary for conjugation, which necessitates protein engineering to introduce unique cysteines if not detrimental to protein activity. ,, Further, a few important criteria to be considered while designing protein reporter–antibody conjugation include maintaining the structural and functional integrity of the biomolecules involved, reproducibility of the conjugation, control of biomolecule conjugation ratio, stability of the conjugates, ability to perform reaction in hydrophilic environments, and so forth. Many different orthogonal bioconjugation methods with superior performance over the traditional conjugation method have been developed that involve the use of less common amino acid residues. , However, these methods have not been used for conjugating bioluminescent proteins to antibodies.…”

Application of a Liposomal Bioluminescent Label in the Development of a Flow Injection Immunoanalytical System

“…The apoprotein (21 700 MW) of aequorin binds to an imidazopyrazine compound (coelenterazine) and molecular oxy-gen to form a stable photoprotein complex. [1][2][3][4][5][6][7][8][9][10][11] Upon addition of calcium ions, the photoprotein undergoes a conformational change leading to the oxidation of chromophore with the release of CO 2 and blue light (λ max ∼469 nm). 12 Flow injection analysis (FIA) is a widely used technique for the simple and rapid examination of environmental, agricultural, and clinical as well as pharmaceutical compounds.…”