Assaying glycoprotein hormones — The influence of glycosylation on immunoreactivity

Storring, P. L.

doi:10.1016/0167-7799(92)90292-4

Cited by 56 publications

(22 citation statements)

References 46 publications

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“…In some ways these are similar to difficulties previously experienced in FSH assay development (Storring 1992). The presence of multiple molecular forms in biological fluids in a variety of species (Robertson et al 1987, Sugino et al 1993, Yokoyama et al 1995, together with varying degrees of glycosylation, combined with the possibility of proteolysis during sample handling all complicate follistatin assay development.…”

Section: Discussionmentioning

confidence: 84%

Development, validation and application of an ultra-sensitive two-site enzyme immunoassay for human follistatin

Evans

Muttukrishna²,

Groome³

1998

Journal of Endocrinology

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Recent studies have found follistatin to be an important regulator of activin bioactivity. Whilst a number of assay formats have been described, all are of limited sensitivity and require the use of isotopes. Many use polyclonal antibodies. Furthermore, a wide range of follistatin preparations have been used as standards, complicating inter-laboratory comparison.We now describe an ultra-sensitive two-site enzyme immunoassay using a pair of mouse monoclonal antibodies raised against follistatin 288. The presence of sodium deoxycholate and Tween 20 in the diluent gave results for total (free and activin-dissociated) follistatin. The assay had a detection limit of <19 pg/ml and recovery of spiked follistatin 288 from amniotic fluid, serum, seminal plasma, human follicular fluid and granulosa cell conditioned medium averaged 100·7 7·5%, 89·1 5·5%, 98 4·9%, 96 7·2% and 123·9 11% respectively. The intra-and interplate coefficients of variation were <5%. An excess of activin-A (50 ng/ml) prior to assay did not affect follistatin recovery. Inhibin-A, inhibin-B, activin-A, activin-B and activin-AB had minimal cross-reactivity (<0·3%). However, follistatin 315 had a significant cross-reaction (9·9%).Serially diluted human samples gave dose-response curves parallel to the standard. Pooled human follicular fluid contained high concentrations of follistatin (242 ng/ml). Follistatin was also found in maternal serum during pregnancy (first trimester 0·8 ng/ml, third trimester 2·8 ng/ml), normal male serum (0·45 ng/ml), amniotic fluid (sixteen week 3·63 ng/ml, term 0·89 ng/ml), seminal plasma (2·4-30 ng/ml) and human granulosa cell conditioned media (0·44 ng/ml). Serial serum samples taken throughout the menstrual cycle of ten women showed fluctuating follistatin concentrations (0·62 ng/ml) with no apparent relationship to the stage of the cycle. Interestingly, pooled serum from postmenopausal women appeared to have higher follistatin levels than any of the normal women (1·4 ng/ml).The possible presence in certain samples of mixtures of follistatin isoforms with different immunoreactivities poses major problems of interpretation in this and all other current follistatin immunoassays. Further work is needed to identify the major immunoreactive forms in different tissues and fluids. Nevertheless, the new assay has a number of advantages over previous assays and should prove a useful tool for various clinical and physiological studies.

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Section: Discussionmentioning

confidence: 84%

Development, validation and application of an ultra-sensitive two-site enzyme immunoassay for human follistatin

Evans

Muttukrishna²,

Groome³

1998

Journal of Endocrinology

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“…These carbohydrate structures can affect many of the protein properties including pharmacokinetics, bioactivity (Takeuchi et al, 1989), secretion, in vivo clearance, solubility (Narhi et al, 1991), recognition, and antigenicity (Storring, 1992;Wasley et al, 1991). The glycosylation profile of these glycoproteins can be affected by the production process in culture, including the host cell line (Goochee, 1992;Goto et al, 1988;Kagawa et al, 1988;Sheeley et al, 1997), mode of culture (Gawlitzek et al, 1995;Jenkins and Curling, 1994;Schweikart et al, 1999), extracellular environment, and the protein itself (Jenkins et al, 1996;Reuter and Gabius, 1999).…”

Section: Introductionmentioning

confidence: 98%

The effect of dissolved oxygen on the production and the glycosylation profile of recombinant human erythropoietin produced from CHO cells

Restelli

Wang

Huzel

et al. 2006

Biotech & Bioengineering

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Human recombinant erythropoietin (rHuEPO) was produced from Chinese hamster ovary (CHO) cells transfected with the human EPO gene. The cells were grown in batch cultures in controlled bioreactors in which the set-points for dissolved oxygen varied between 3% and 200%. The cell-specific growth rate and final cell yield was significantly lower under hyperoxic conditions (200% DO). However, there was no significant difference in growth rates at other oxygen levels compared to control cultures run under a normoxic condition (50% DO). The specific productivity of EPO was significantly lower at a DO set-point of 3% and 200% but maintained a consistently high value between 10% to 100% DO. The EPO produced under all conditions as analyzed by two-dimensional electrophoresis showed a molecular weight range of 33 to 37 kDa and a low isoelectric point range of 3.5 to 5.0. This corresponds to a highly glycosylated and sialylated protein with a profile showing at least seven distinct isoforms. The glycan pattern of isolated samples of EPO was analyzed by weak anion exchange (WAX) HPLC and by normal-phase HPLC incorporating sequential digestion with exoglycosidase arrays. Assigned structures were confirmed by mass spectrometry (MALDI-MS). The most prominent glycan structures were core fucosylated tetranntenary with variable sialylation. However, significant biantennary, triantennary, and non-fucosylated glycans were also identified. Detailed analysis of these glycan structures produced under variable dissolved oxygen levels did not show consistently significant variations except for the ratio of fucosylated to non-fucosylated isoforms. Maximum core fucosylation (80%) was observed at 50% and 100% DO, whereas higher or lower DO levels resulted in reduced fucosylation. This observation of lower fucosylation at high or low DO levels is consistent with previous data reported for glycoprotein production in insect cells.

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“…Like other glycoprotein hormones, EPO exists as mixtures of a multitude of isoforms that differ mainly in their glycosylation (Storring, 1992). Thus, each of the three N-glycosylation sites may be occupied by a range of different glycans, with some heterogeneity also among the glycans at the single O-glycosylation site (Sasaki et al, 1987;Takeuchi et al, 1988;Tsuda et al, 1988;Nimtz et al, 1993;Watson et al, 1994;Hokke et al, 1995).…”

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confidence: 99%

Relationships between the N‐glycan structures and biological activities of recombinant human erythropoietins produced using different culture conditions and purification procedures

et al. 2003

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Summary. Eight preparations of recombinant human erythropoietin (EPO) with differing isoform compositions were produced by using different culture conditions and purification procedures. The N-glycan structures of these EPOs were analysed using a recently developed profiling procedure and identified using matrix-assisted laser desorption ionization mass spectrometry. The specific activities of each of the EPOs were estimated by in vivo and in vitro mouse bioassays. The eight EPOs were found to differ in their isoform compositions (as judged by isoelectric focusing), their N-glycan profiles, and in their in vivo and in vitro bioactivities. N-glycan analyses identified at least 23 different structures among these EPOs, including bi-, tri-and tetraantennary N-glycans, with or without fucosylation or N-acetyllactosamine extensions, and sialylated to varying degrees. Mass spectrometry also indicated the presence of N-glycans with incomplete outer chains, terminating in N-acetylglucosamine residues, and of molecular masses consistent with phosphorylated or sulphated oligomannoside structures. The tetrasialylated tetra-antennary N-glycan contents of the eight rEPOs were found to be significantly and positively correlated with their specific activities as estimated by mouse in vivo bioassay, and significantly and negatively correlated with their specific activities as estimated by mouse in vitro bioassay. It was concluded that the tetrasialylated tetra-antennary N-glycan content of EPO is a major determinant for its in vivo biological activity in the mouse.

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Assaying glycoprotein hormones — The influence of glycosylation on immunoreactivity

Cited by 56 publications

References 46 publications

Development, validation and application of an ultra-sensitive two-site enzyme immunoassay for human follistatin

Development, validation and application of an ultra-sensitive two-site enzyme immunoassay for human follistatin

The effect of dissolved oxygen on the production and the glycosylation profile of recombinant human erythropoietin produced from CHO cells

Relationships between the N‐glycan structures and biological activities of recombinant human erythropoietins produced using different culture conditions and purification procedures

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