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A discordance between sex hormone-binding globulin (SHBG) measurements by 2-site ELISAs was investigated using pairings of various "in house" SHBG antibodies together with a concordant control. The 2-site monoclonal ELISAs used the same base coat (11F11) and discordance was observed with one top coat monoclonal antibody (7H9) and also when a polyclonal SHBG antibody was paired with the basecoat antibody (11F11). Sialidase treatment of the discordant sample and purified SHBG revealed increased levels using 7H9 whereas there was no change in SHBG in the concordant sample. Conversely, following sialidase treatment, the discordant sample showed no change in SHBG measured using the other monoclonal antibody pairings whereas the SHBG levels in the concordant sample declined following sialidase using the same monoclonal antibody pairings. This implicated glycosylation as a factor in antibody recognition and synthetic peptides spanning the two N-linked and one O-linked glycosylation regions showed that SHBG recognition by monoclonal antibody 7H9 could be disrupted by a peptide spanning the O-linked glycosylation site. Hence rather than immunoassay discordance being attributed to heterophile antibodies or other circulating antibodies here it can be likely attributed to glycosylation affecting antibody recognition and hence the measurement of SHBG., (Copyright © 2020 Elsevier B.V. All rights reserved.)

The measurement of vitamin D-binding protein (VDBP) by immunoassay has been confounded by variable antibody recognition of the Gc1s, Gc1F and Gc2 phenotypes. This has led to spurious conclusions regarding vitamin D status in different ethnic groups. In order to overcome these problems there is a requirement for VDBP antibodies that are unaffected by phenotype status. Here we report the generation and testing of three monoclonal antibodies to VDBP which recognise linear epitopes and are unaffected by vast molar excesses of synthetic peptides spanning these phenotypic domains. These IgG1 kappa antibodies were purified and biotinylated to allow suitable pairings to develop a sandwich ELISA for circulating VDBP. The VDBP ELISA is unaffected by actin and confirms that VDBP levels are significantly reduced in sepsis patients and non-sepsis intensive care patients compared to normal healthy subjects. Levels of VDBP along with total 25OH vitamin D3 can be used to calculate free 25OH vitamin D3 levels and these compare well with consensus values determined independently. The VDBP ELISA meets acceptable performance criteria and as such can be used in conjunction with total 25OH vitamin D3 to determine the free 25OH vitamin D3 status in various cohorts., (Copyright © 2018 Elsevier B.V. All rights reserved.)

The predominant carrier of cortisol in circulation is corticosteroid-binding globulin (CBG) which is a non-functional member of the family of serine protease inhibitors. Corticosteroid-binding globulin possesses an exposed elastase sensitive loop and upon cleavage it adopts a "relaxed" conformation promoting the delivery of cortisol to sites of inflammation. Recently we have developed monoclonal antibodies which recognise only the intact exposed elastase loop, including an N-glycosylation site, which, in concert with another monoclonal antibody to CBG, offered the potential for the determination of intact and total CBG which may both be present in circulation. Here we validate these parallel ELISAs and show that like total CBG there is little diurnal variation of intact plasma CBG. Furthermore in a normal reference population the majority of CBG is in the intact or active form but a significant level of apparently cleaved CBG is evident. In some subjects there is gross discordance between total CBG and intact CBG implying a predominance of apparently cleaved CBG in circulation and this significantly affects calculated free cortisol levels. Gross differences in total and intact CBG levels may not be due to differences in N-glycosylation affecting antibody binding as CBG levels are unaffected by PNGase F treatment., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Objectives: Plasma retinol-binding protein (RBP) has been linked to insulin resistance and cardiovascular risk, yet little is know of its natural variation in plasma. We examined this in normal subjects and compared plasma levels and variability in lean subjects and subjects with the metabolic syndrome., Methods: We established an "in house" ELISA for plasma RBP and measured levels in 20 normal subjects over daylight hours and 2 subject groups, either lean or classified with the metabolic syndrome., Results: Plasma RBP in normal subjects did not vary over the day with no differences between males and females. There was also no difference in plasma RBP levels and between the age- and sex-matched lean subjects compared to the metabolic syndrome group., Conclusion: The lack of variation in plasma RBP in normal subjects and the lack of difference between plasma RBP in normal and metabolic syndrome subjects suggest the link between plasma RBP and insulin resistance is tenuous. Investigating a large cohort over the diabetic non-diabetic spectrum may clarify this issue.

A direct enyzme-linked immunosorbent assay (ELISA) for urinary immunoreactive methylphenidate (Ritalin), in which a standard 96-well microtiter plate is used, is described. For this ELISA, a methylphenidate-thyroglobulin conjugate is immobilized to the microtiter plate and competes with methylphenidate in the standard or urine sample for antibody-binding sites. After washing, the sheep methylphenidate antibody bound to immobilized methylphenidate is detected with peroxidase-labelled goat antisheep IgG. Following a further wash, tetramethylbenzidine is added, color is developed, and the plate is read at 450 nm on an ELISA plate reader. This method is unaffected by drugs of abuse and is suitable for routine use in the toxicology laboratory.

Background: Adrenal function is commonly assessed by measuring plasma cortisol following synthetic ACTH (synacthen) challenge. Generally little regard is given to plasma levels of corticosteroid-binding globulin (CBG). We have developed and validated an enzyme-linked immunosorbent assay (ELISA) for CBG and together with plasma cortisol calculated the "free cortisol index" as an additional parameter for assessing adrenal function., Methods: A monoclonal antibody was raised against CBG. The antibody was characterized by Western blotting and used with a polyclonal antibody to develop a direct ELISA for CBG. Together with total plasma cortisol, the free cortisol index was derived and correlated with an "in-house" ligand binding method for assessing free cortisol. The free cortisol index was also used as an adduct to total plasma cortisol in assessing adrenal function., Results: The ELISA has acceptable performance and the free cortisol index correlates well with free cortisol determined by ligand binding. In addition, we show that CBG levels following synthetic ACTH (synacthen) show a modest but significant decline., Conclusion: We conclude that the measurement of both plasma CBG and total cortisol to derive the free cortisol index may provide an additional parameter in the interpretation of the short synacthen test and that there is a decline in plasma CBG over this test., (Copyright 2002 Elsevier Science B.V.)

Four monoclonal antibodies to human sex hormone-binding globulin were raised and characterized. Three of the four antibodies recognised different antigenic determinants on SHBG. Two of the distinct antibodies were useful for Western blotting and recognized a major 48 kDa band in human plasma as well as a 46 kDa minor component. Carbohydrate residues do not form part of the antigenic determinants of these two antibodies, although one of these showed increased signal following removal of N-linked oligosaccharides. Some of the antibodies were selected to form a basis of a same-day, non-competitive, enzyme-linked immunosorbent assay (ELISA) for SHBG in plasma. The assay employs a purified IgG2a SHBG monoclonal antibody adsorbed to the wells of a microtitre plate. After blocking any further adsorption to the plate, standards or diluted patient samples were added for a 5-h incubation at room temperature, after which the plate was washed and antibody-bound SHBG was detected with an anti-SHBG IgG1 monoclonal antibody followed by peroxidase-labeled antimouse-IgG1 and o-phenylenediamine substrate. The assay correlated well with an existing 2-day ELISA for SHBG in plasma using polyclonal antibodies and also correlated with a dihydrosterone (DHT) ligand-binding assay. The monoclonal antibody-based ELISA shows excellent performance characteristics and is unaffected by added testosterone or estradiol.

Mice were immunized with 5-androstene-3 beta-ol-7,17-dione-7-CMO:bovine serum albumin (DHEA-7-O-CMO-BSA) or 5-androstene-3 beta-ol-17-one hemisuccinate-bovine serum albumin (DHEA-3HS-BSA) conjugates and monoclonal antibodies were produced, characterized, and selected for maximum DHEAS binding. Of these hybridomas, four clones from DHEA-3HS-BSA-immunized mice had acceptable criteria for the development of a competitive enzyme-linked immunosorbent assay (ELISA) for DHEAS in plasma. One hybridoma supernatant from DHEA-7-O-CMO-BSA-immunized mice showed 360% cross-reactivity to both androsterone sulfate and epiandrosterone sulfate. This allows the possibility of the direct determination of androsterone sulfate and epiandrosterone sulfate in plasma after correction for the DHEAS contribution. Both ELISAs employ a DHEA-3HS-thyroglobulin conjugate adsorbed to the wells of a standard 96-well microtiter plate. DHEAS in the standards or diluted plasma sample competes with immobilized DHEA-3HS-thyroglobulin for antibody-binding sites. Antibody is detected with anti-mouse-lg peroxidase by further washing, adding o-phenylenediamine substrate, and reading the absorbance at 492 nm. The ELISAs are simple, reproducible, and reliable and, to our knowledge, they are the first tests employing monoclonal antibodies to DHEAS.

Cortisol mouse monoclonal antibodies were produced and characterized. Of the four clones studied, supernatant from one clone (A2), compared with other cortisol monoclonal antibodies, showed minimal cross-reactivity to other C21 steroids and was suitable for the direct determination of cortisol in plasma by enzyme-linked immunosorbent assay using a standard 96-well microtiter plate. The enzyme-linked immunosorbent assay uses the immobilized antigen approach, in which cortisol in plasma samples or standards competes with immobilized steroid for antibody-binding sites. After washing, the cortisol antibody bound to the wells of the microtiter plate is detected with antimouse immunoglobulin conjugated to horseradish peroxidase. Following further washing, o-phenylenediamine substrate is added. The enzyme-linked immunosorbent assay is robust and semiautomated. The mean +/- SD recovery from plasma was 97% +/- 6%. Precision studies on three different plasma pools showed mean coefficients of variation of 7.6% and 8.6% for within- and between-assay variation, respectively. The satisfactory performance criteria allow its use in the routine laboratory.

Monoclonal antibodies to pregnanediol-3-glucuronide were produced and characterized. One of three clones investigated provided antibody suitable for a direct urinary enzyme-linked immunosorbent assay (ELISA). The ELISA uses a pregnanediol-thyroglobulin conjugate adsorbed onto the wells of a standard 96-well microtiter plate. Pregnanediol-3-glucuronide in standards or diluted urine competes with the immobilized steroid for antibody-binding sites. After washing, mouse monoclonal antibody bound to the plate is probed with antimouse immunoglobulin peroxidase. After further washing, o-phenylenediamine substrate is added and, finally, the absorbance is read at 492 nm. The ELISA shows excellent performance and agreement with the previous gas chromatographic method. The ELISA is ideal for aiding the assessment of ovarian function in the routine laboratory.

ELISA technology using immobilized steroid conjugates has allowed the rapid screening and characterization of hybridoma supernatants. Although cortisol-3CMO-BSA was the immunogen, a monoclonal antibody with exceptionally high cross-reactivity (greater than 1000%) to prednisone was obtained. Characterization of the antigenic determinants shows a requirement for overall glucocorticoid 21-hydroxyl and 17 alpha-hydroxyl groups with additional high specificity for the 1,2-double bond and 11-position. There is potential use for this antibody in the assay of prednisone.