Influence of Different Length Spacers Containing Enzyme Conjugate on Functional Parameters of Progesterone Elisa

Abstract

In steroid enzyme immunoassay (EIA), there is an increase or decrease of labeled steroid recognition by antibody due to homologous and heterologous combinations of enzyme conjugate with immunogen that affects sensitivity of the assay. We have introduced three to 18 atomic length linkers between enzyme and steroid moieties and studied their effects on functional parameters such as sensitivity, ED(50), and specificity of progesterone enzyme immunoassays. Progesterone-3-carboxymethyloxime-bovine serum albumin (P-3-CMO-BSA) was used as an immunogen to raise the antiserum in New Zealand white rabbits. Five enzyme conjugates were prepared using 17-α-hydroxy-progesterone-3-carboxymethyloxime (17-α-OH-P-3-CMO) as carboxylic derivative of 17-α-hydroxy-progesterone and horseradish peroxidase (HRP) as label. These were 17-α-OH-P-3-CMO-HRP, 17-α-OH-P-3-CMO-urea-HRP (17-α-OH-P-3-CMO-U-HRP), 17-α-OH-P-3-CMO-ehylenediamine-HRP (17-α-OH-P-3-CMO-EDA-HRP), 17-α-OH-P-3-CMO-carbohydrazide-HRP (17-α-OH-P-3-CMO-CH-HRP), and 17-α-OH-P-3-CMO-adipic acid dihydrazide-6-aminocaproic acid-HRP (17-α-OH-P-3-CMO-ADH-6ACA-HRP). The influence of different atomic length linkers on sensitivity, ED(50), and specificity were studied with reference to label without linker. The results of the present investigation revealed that the incorporation of ADH-6ACA spacer in 17-α-hydroxy-progesterone-enzyme conjugate improved the sensitivity in antigen plus bridge heterologous EIA system. The presence of spacer in enzyme conjugate improved the sensitivity and specificity (cross-reactivity) in some antigen plus bridge heterologous assay of progesterone.

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.…”

“… 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. 125 The intent and purpose of covalently bonding corticosteroids to biologically relevant molecular platforms has most frequently been for diagnostic-related applications 24 , 80 , 101 – 104 , 110 , 111 , 113 , 116 and much less often for the development of advanced therapeutics. 28 , 112 In rare instances where covalent corticosteroid biopharmaceutical therapeutics have been designed, synthesized, and evaluated for efficacy 28 , 112 it has most frequently been to determine their capacity to suppress cellular inflammatory responses utilizing molecular delivery platforms that include or are analogous to anti-E selectin and anti-CD183.…”

“…Precarboxylation of corticosteroids and steroid core pharmaceuticals is an initial requirement for some organic chemistry reactions which is implemented prior to synthetically bonding them covalently to biologically relevant molecular platforms. 101 Corticosteroid/steroid precarboxylation can be achieved utilizing several different organic chemistry reaction schemes. Hydrazine acetic acid ethyl ester and heating to 120°C produce a hydrazino acetic acid analog of a corticosteroid/steroid and introduces a functionally available carboxyl group and an acid-cleavable hydrazino at the C 21 position.…”

Reversible testicular toxicity of piperine on male albino rats

“…Hormones such as follicular-stimulating hormone (FSH),[ 34 ] luteinizing hormone (LH),[ 34 ] testosterone (T),[ 35 ] progesterone (P),[ 36 ] and sex hormone-binding globulin (SHBG)[ 37 ] were assayed using standard methods.…”

A Direct Competitive Enzyme Linked Immunosorbent Assay for Progesterone Using Monoclonal Antibody

“…Given the inherent problems therein, different non-radioactive methods have been developed for measuring progesterone, such as chemiluminescence immunoassays (CLIA), (13,14) time-resolved fluorescence immunoassay (TRFIA), (15,16) and fluorescence polarization immunoassays (FPIA), (17)(18)(19) along with enzyme-linked immunosorbent assays (ELISA). (20)(21)(22)(23) Generally, for the measurement of steroids directly from serum, different reagents or a combination of reagents were used as displacer in the enzyme conjugate or sample dilution buffer to displace the steroid from the specific steroid binding protein. 1-anilinonaphthalene-8-sulfonate (ANS), (14) trichloroacetic acid (TCA), (24) and cortisol (Cor) (25) were used as displacer in the sample dilution buffer for measuring progesterone directly from serum.…”