A convenient homogeneous enzyme immunoassay for estradiol detection

Abstract

A convenient homogeneous enzyme immunoassay for estradiol is described. Unlike heterogeneous immunoassays, which require time-consuming separation steps or expensive automated systems, homogeneous immunoassays, wherein all reagents are freely suspended in bulk solution, can be simple and fast without costly instrumentation. The key component of this assay system, an estradiol-reporter enzyme conjugate, was prepared by covalently binding β-estradiol-6-(O-carboxymethyl)oxime to glucose-6-phosphate dehydrogenase (G6PDH) by an N-hydroxysuccinimide-enhanced, carbodiimide-mediated coupling reaction. The estradiol-G6PDH activity can be repressed up to 46% upon anti-estradiol antibody binding. The lower detection limit of the assay is 1 nM estradiol in aqueous solution, and the standard curve is linear on logit-log scale-up to 6.7 µM estradiol. A detection limit of 11.5 nM in estradiol-spiked human serum samples suggests the feasibility of applying this assay to monitor estradiol levels for the prediction and prevention of ovarian hyperstimulation syndrome.

Dexamethasone-(C₂₁-phosphoramide)-[anti-EGFR]: molecular design, synthetic organic chemistry reactions, and antineoplastic cytotoxic potency against pulmonary adenocarcinoma (A549)

“… 96 – 99 A relatively modest array of organic chemistry reaction schemes have been described for covalently bonding dexamethasone and other steroid core pharmaceuticals to biologically relevant molecular platforms. Besides covalent compounding with other low molecular weight pharmaceuticals, 100 corticosteroids have most commonly been covalently bound to a spectrum of high molecular weight platforms of natural origin, such as albumin, 23 – 25 , 101 – 106 cardiolipin, 107 , 108 chondroitin sulfate (sulfated glycosaminoglycan), 29 glucose-6-phosphate dehydrogenase, 109 horseradish peroxidase, 110 , 111 spermine, 112 and cloned fusion proteins. 113 Pharmaceuticals with a steroid motif have additionally been covalently bound to artificial or semiartificial molecular platforms, such as amino-PEG (eg, α-methoxy-ω-amino-PEG), 27 chitosan, 26 dextran, 114 N -(2-hydroxypropyl) methacrylamide, 115 amine-modified polysaccharides, 116 poly- l -glutamic acid (polypeptide configuration), 117 1-dodecylthio-2-decyloxypropyl-3-phophatidic acid, 118 , 119 lipid nucleosides, 120 N -(2-hydroxypropyl)methacrylamide polymer, 75 benzodiazepine receptor ligands, 121 , 122 4-( N )-valeroyl, 4-( N )-lauroyl, and 4-( N )-stearoyl, 123 4-fluoro[ 18 F]-benzaldehyde derivatives (diagnostic positron-emitting radionucleotide), 124 polyamidoamine dendrimer, 28 and peptide hormone antagonists.…”

“… 28 Some corticosteroid/steroid hemisuccinate analogs (eg, C 21 position) are commercially available or they can be synthesized utilizing succinic anhydride reagent in combination with sodium sulfate, pyridine, chloroform, acetone, and benzene/hexane. 24 , 105 , 106 , 109 , 125 Similarly, covalent corticosteroid/steroid –( O -carboxymethyl)oxime analogs can be synthesized “in-house” utilizing a wide spectrum of reagents including –( O -carboxymethyl)hydroxyamine in combination with diazomethane, N -nitroso- N -methylurea, aluminum isopropoxide, ethyl acetate, ether, acetone, benzene, KOH, and HCl reagents. 105 , 106 Carboxylation of corticosteroids/steroids through the introduction of glutarate, 28 hemisuccinate, 24 , 109 , 125 or –( O -carboxymethyl)oxime 24 , 109 can potentially occur at hydroxyl groups located at the C 3 , C 6 , C 11 , C 17 , or C 21 positions within the chemical structure of steroid core pharmaceuticals.…”

“… 24 , 105 , 106 , 109 , 125 Similarly, covalent corticosteroid/steroid –( O -carboxymethyl)oxime analogs can be synthesized “in-house” utilizing a wide spectrum of reagents including –( O -carboxymethyl)hydroxyamine in combination with diazomethane, N -nitroso- N -methylurea, aluminum isopropoxide, ethyl acetate, ether, acetone, benzene, KOH, and HCl reagents. 105 , 106 Carboxylation of corticosteroids/steroids through the introduction of glutarate, 28 hemisuccinate, 24 , 109 , 125 or –( O -carboxymethyl)oxime 24 , 109 can potentially occur at hydroxyl groups located at the C 3 , C 6 , C 11 , C 17 , or C 21 positions within the chemical structure of steroid core pharmaceuticals. Alternatively, some but not all corticosteroids/steroids can be obtained commercially as hemisuccinate 24 , 26 , 109 or –( O -carboxymethyl)oxime analogs.…”

“…105 , 106 Carboxylation of corticosteroids/steroids through the introduction of glutarate, 28 hemisuccinate, 24 , 109 , 125 or –( O -carboxymethyl)oxime 24 , 109 can potentially occur at hydroxyl groups located at the C 3 , C 6 , C 11 , C 17 , or C 21 positions within the chemical structure of steroid core pharmaceuticals. Alternatively, some but not all corticosteroids/steroids can be obtained commercially as hemisuccinate 24 , 26 , 109 or –( O -carboxymethyl)oxime analogs.…”

Glucose‐6‐phosphate dehydrogenase mutant D306C has a higher inhibition rate for enzyme multiplied immunoassay of cholyglycine

“…Since Rubenstein et al. pioneered the enzyme multiplied immunoassay technique (EMIT) approach for homogeneous enzyme competitive immunoassays, EMIT assays are now routinely used in clinical analysis. Glucose‐6‐phosphate dehydrogenase (G6PDH) from Leuconostoc mesenteroides is the most commonly used enzyme in EMIT assays in which analytes in the sample compete with analytes labeled with G6PDH (G6PDH–analyte conjugates) for antibody‐binding sites .…”