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Clinical Chemistry 52: 286-297, 2006. First published November 23, 2005; 10.1373/clinchem.2005.058172
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(Clinical Chemistry. 2006;52:286-297.)
© 2006 American Association for Clinical Chemistry, Inc.

Other Areas of Clinical Chemistry

Feasibility Study of New Calibrators for Thyroid-Stimulating Hormone (TSH) Immunoprocedures Based on Remodeling of Recombinant TSH to Mimic Glycoforms Circulating in Patients with Thyroid Disorders

Sandrine Donadio1, Aurélie Pascual1, Jos H.H. Thijssen2 and Catherine Ronin1,a

1 Laboratory of Neuroglycobiology, Université de Provence, UMR 6149 et GDR 2590 CNRS/Université de Provence, Marseille, France.
2 Universitair Medish Centrum Utrecht, Utrecht, The Netherlands.

aAddress correspondence to this author at: Laboratoire de Neuroglycobiologie, Pôle 3C Université de Provence, Batiment 9-Case B, UMR 6149 CNRS/Université de Provence et GDR 2590, 3 Place Victor Hugo, 13331 Marseille Cedex 03, France. Fax 33-488-576-804; e-mail catherine.ronin{at}up.univ-mrs.fr.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Background: Differences between the glycosylation patterns of a pituitary thyroid-stimulating hormone calibrator (pitTSH) and serum samples have been shown to be responsible for nonidentical epitope expression and for introducing discrepancies in TSH measurements. We studied the feasibility of developing new candidate reference materials by remodeling recombinant TSH (recTSH) to generate potential mimics of serum TSH.

Methods: Terminal sialylation and/or inner fucosylation of recTSH were remodeled by a combination of enzyme treatments followed (or not) by lentil lectin-Sepharose affinity chromatography. The resulting TSH preparations were screened for epitope similarity in 23 immunoassays mapping 3 antigenic clusters common to the pitTSH 2nd International Reference Preparation (IRP) and the recTSH 1st IRP and then challenged against a pool of 63 patients with increased serum TSH (>60 mIU/L).

Results: pitTSH was poorly correlated with serum TSH, with a mean (SD) slope of 2.124 (0.001), in contrast to recTSH [slope, 1.178 (0.056)]. Comparison of variably sialylated preparations with recTSH gave slopes of 0.860 (0.057) for desialylated TSH, 1.064 (0.057) for {alpha}2,3/6-oversialylated recTSH, and 0.953 (0.033) for {alpha}2,6-resialylated recTSH, indicating that TSH forms enriched in sialic acid closely resemble serum TSH. Further testing against serum TSH showed satisfactory agreement with both TSH preparations containing {alpha}2,6-sialic acid [slopes, 1.064 (0.057) and 0.953 (0.033)], particularly in the absence of nonfucosylated forms [0.985 (0.044)].

Conclusions: Glyco-engineered recTSH preparations enriched in sialic acid and inner fucose are promising candidates for future reference materials. These preparations may have advantages over existing preparations used for standardizing TSH measurements.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Thyroid-stimulating hormone (thyrotropin; TSH) 1 is a member of the glycoprotein hormone family that includes pituitary and placental gonadotropins. All members are noncovalently linked {alpha}ß heterodimers, consisting of a common {alpha}-subunit and a unique ß-subunit, which confers biological and immunologic specificity to the hormone (1)(2)(3). TSH contains 3 N-glycosylation sites with oligosaccharide structures whose composition varies according to the source of the hormone. Pituitary TSH (pitTSH) contains mainly biantennary glycans with a terminal N-acetylgalactosamine (GalNAc) sulfate signal and low sialic acid content (1), whereas recombinant TSH (recTSH) produced in Chinese hamster ovary (CHO) cells specifically terminates in {alpha}2,3-linked sialic acid (4)(5)(6)(7) and recTSH produced in yeast lacks sialic acid (8) or insect cells(9). Interestingly, TSH isolated from the sera of patients with primary hypothyroidism displayed increased sialylation (10)(11)(12)(13) and decreased inner fucosylation (14). These glycosylation patterns are clinically relevant because hormone clearance is governed by hepatic clearance of plasma glycoproteins (Scheme 1 ). Hepatic receptors specific for GalNAc sulfate (15), asialo-glycoproteins(16)(17)(18)(19)(20), mannose (21)(22)(23), or fucose(21)(24) rapidly capture TSH forms containing these glycan structures. This leads to mostly highly branched and sialylated TSH forms in the circulation, in contrast to the forms stored in the pituitary gland (11)(12). Hepatic uptake is not altered in liver insufficiency because physiologic concentrations of circulating TSH have been reported in some patients with chronic (25) or fatty (26) liver disease or liver cirrhosis(27).


Figure 1
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  		Scheme 1. Metabolic clearance of TSH glycoforms.

The pituitary gland produces TSH containing (fucosylated) biantennary glycans with a GalNAc sulfate signal and low sialic acid content. The pituitary extract, however, contains a mixture of synthetic mannose-rich intermediates and mature complex forms. TSH forms with terminal mannose ({circ}), GalNAc sulfate ({blacktriangleup}-SO4–]), N-acetylglucosamine ({blacksquare}), and galactose (•) and/or core fucose ({diamondsuit}) are rapidly captured by specific liver receptors, and as consequence, blood TSH is enriched in highly branched and sialylated glycoforms.

It has been widely assumed that antibodies recognize peptidic determinants of TSH with virtually no effect from changes in glycosylation; however, earlier studies indicated that the presence or absence of specific glycans modified the immunoreactivity of TSH, particularly when these changes occurred in the ß-subunit (28)(29)(30). Furthermore, pituitary and recombinant forms of TSH origin usually share high cross-reactivity but clearly do not behave identically (5)(31). More recently, our laboratory has shown that monoclonal anti-TSH antibodies can be divided into 2 groups depending on their capacity to bind TSH forms with altered glycosylation (32). Indeed, some antibodies were unable to bind sialylated forms of TSH, whereas others failed to detect nonfucosylated TSH (32). Because both alterations are known to occur in patients developing common thyroid disorders, particularly hypothyroidism (10)(11)(12)(13)(14), it may be that some antibodies fail to detect disease-related TSH glycoforms, particularly during the onset of hypothyroidism.

Measurement of TSH as an indicator of thyroid function has become one of the most popular tests in laboratory medicine (33), but inter- and intralaboratory variations in TSH measurements have been observed over the past 20 years (34)(35)(36)(37)(38)(39)(40)(41)(42)(43). Assays routinely measure a nondetermined, highly heterogeneous mixture of glycoforms and possibly degradation products of TSH against a Reference Material consisting of a quite distinct combination of glycoforms composed of immature and mature biosynthetic intermediates (44). Both preparations may eventually share common glycoforms, but they should not be assumed to behave identically (45). Ideally, the best candidate for a Reference Material should display the same epitope distribution as TSH circulating in healthy individuals and in patients with thyroid disorders. However, there are indications (10)(11)(12)(13)(14) that the circulating forms of TSH differ between euthyroid and hypothyroid persons. In this study, we aimed to identify the sources of variation in TSH measurements and designed a feasibility study to develop TSH preparations that resemble as closely as possible the wide array of serum forms of TSH.

TSH circulates in such low amounts that measurement of the protein concentration and/or structural characterization of the glycan structure are not possible. We have attempted immunopurification of TSH, but although the antibodies bound biantennary TSH forms, they failed to isolate the whole array of glycoforms suspected to circulate in patients. To overcome these technical limitations, we used a recombinant preparation of TSH with well-established polypeptide and glycan structures [ (32) and Morelle et al., "Site-Specific Glycosylation of Recombinant Human Thyrotropin," manuscript in preparation] that contained many of the glycoforms considered to be present in serum. We produced a panel of recTSH preparations differing in glycosylation patterns (Scheme 2 ). To properly assess the similarities in epitope distribution, we used pairs of antibodies mapping 3 remote antigenic clusters of the TSH molecule as present in each of the candidate preparations. To our knowledge, this is the first report of a method that enables estimation of epitope distribution on a molar basis and can be used to produce new candidate reference materials for further standardization of TSH measurements as recommended by European Directive DIV 98/79/CE.


Figure 2
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  		Scheme 2. Production of recTSH preparations varying in glycosylation pattern.

The recombinant compound (recTSH = 3Sial-recTSH) was enzymatically remodeled as described in the Materials and Methods to modify its sialic acid content. Neuraminidase treatment produced desialylated recTSH with no sialic acid (0Sial-recTSH), reaction of 3Sial-recTSH with an {alpha}2,6-sialyltransferase produced oversialylated recTSH (3/6Sial-recTSH), whereas sialylation of 0Sial-recTSH produced resialylated recTSH (6Sial-recTSH) containing only {alpha}2,6-linked sialic acid residues. 3/6Sial-recTSH and 6Sial-recTSH were further purified by lentil lectin affinity chromatography to isolate nonfucosylated 3/6Sial(-Fuc)-recTSH or 6Sial(-Fuc)-recTSH and fucosylated 3/6Sial(+Fuc)-recTSH or 6Sial(+Fuc)-recTSH, respectively. Symbols for the carbohydrate moieties are as given in the legend for Scheme 1Up .


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
materials
The monoclonal antibodies were a generous gift from Ortho-Clinical Diagnostics (Issy-Les-Moulineaux, France), Roche Diagnostics (Basel, Switzerland), and Bayer Diagnostics (Puteaux, France) or were purchased from Beckman-Coulter and Seradyn. The 2nd International Reference Preparation (IRP) for pitTSH (80/558) and the 1st IRP for recTSH (94/674) were from the National Institute for Biological Standards and Control (NIBSC, United Kingdom). Highly purified human pitTSH was from Biogenesis, and recombinant human TSH was from Seradyn.

Neuraminidase, bovine serum albumin (BSA), {alpha}-methylmannopyranoside, cacodylic acid, Triton X-100, CMP-N-acetylneuraminic acid, p-nitrophenyl phosphate, and Tween 20 were from Sigma. All culture reagents were from Invitrogen-Life Technologies. The streptavidin–alkaline phosphatase conjugate was from Jackson ImmunoResearch Laboratories, the Micro BCATM Protein Assay was from Pierce-PERBIO, the biotin labeling reagents were from Roche, lentil lectin-Sepharose was from Amersham-Pharmacia Biotech, and the chromatography columns and microtiter 96-well plates were from VWR International.

serum collection
Serum samples (0.2–1.5 mL) were collected from patients with overt primary hypothyroidism [TSH >50 mIU/L and free thyroxine <8 pmol/L] and from patients without thyroid glands, which had been removed years previously because of thyroid cancer. In these patients, the TSH-suppressive thyroxine therapy was stopped for ~2 months. Only sera with TSH concentrations >50 mIU/L and low free thyroxine (<5 pmol/L) were included in the collection. All samples were collected anonymously and stored at –20 °C. The measurements were performed on the ADVIA Centaur (Bayer). For this study, sera from 63 patients with TSH concentrations ranging from 60 to 150 mIU/L were used.

To enable conversions of serum TSH values provided to us as mIU/L into molar protein content (ng/L protein), we first established an in-house conversion factor by comparing a highly purified commercially available pitTSH to the 2nd human pitTSH (IRP 80/558; NIBSC). We checked the homogeneity of the highly purified pitTSH by sodium dodecyl sulfate–gel electrophoresis (data not shown) and by mass spectrometry (32). An epitope map of both preparations was made under saturating conditions at equilibrium to control for epitope identity (data not shown). Dose–response curves were constructed with used of aliquots of the highly purified preparation quantified by an ultrasensitive protein assay as described below. In this way, we confirmed that the appropriate conversion factor was 4.93 mIU/µg, very similar to that mentioned in the datasheet for the NIBSC 80/558 standard. Under these conditions, a serum TSH titer of 98.6 mIU/L was assigned the value 2 ng/100 µL.

protein assay
We determined the protein concentrations of all TSH preparations after modifying the experimental procedure for the protein assay by selecting recTSH as the in-house calibrator for protein determination. Protein content was determined by absorbance and amino acid composition to establish the molar content, irrespective of the carbohydrate composition. This value was then used throughout all subsequent determinations, which were carried out as recommended by the manufacturer. Briefly, 1 mL of recTSH internal standard or unknown sample was mixed with 1 mL of the working reagent provided with the assay and incubated in a 60 °C water bath for 1 h. After all of the tubes had cooled to room temperature, the absorbance at 562 nm was measured.

production of an engineered {alpha}2,6-sialyltransferase
To produce a soluble recombinant {alpha}2,6-sialyltransferase, we established a stable CHO cell line that produced ß-galactoside {alpha}2,6-sialyltransferase (CHO-K1/hST6Gal I) (46). The cells were grown at 37 °C in a 5% CO2 incubator, in DMEM–Glutamax-I medium supplemented with 100 mL/L inactivated fetal calf serum, fungizone (2.5 mg/L), gentamicin (50 mg/L), and geneticin (200 mg/L). The cell culture medium was collected after a 72-h period and concentrated ~15-fold by centrifugation with Amicon Centriprep units (Millipore). Batches were pooled, and the soluble enzymatic activity was determined as described previously (46). Under these conditions, the enzyme activity was estimated to be 60–100 U/L, and aliquots were stored at –20 °C.

remodeling of RECTSH sialic acid content
The enzymatic treatments of recTSH preparations are summarized in the 4 larger panels in Scheme 2Up .

Desialylated recTSH (0Sial-recTSH) was obtained by incubating recTSH with neuraminidase (enzyme activity, 20 mU/µg of protein) from Clostridium perfringens for 2 h at 37 °C, under gentle stirring in 100 mmol/L sodium acetate (pH 6.5) containing 2 mmol/L CaCl2 and 0.3 g/L BSA.

Oversialylatedf recTSH (3/6Sial-recTSH) was prepared by incubating recTSH for 2 h at 37 °C with in-house hST6Gal I enzyme (enzyme activity, 20 mU/µg of TSH) in the presence of 15 mmol/L CMP-N-acetylneuraminic acid, 2 mmol/L MnCl2, 1 g/L BSA, and 10 mL/L Triton X-100 in 50 mmol/L cacodylate buffer (pH 6.5).

Resialylated recTSH (6Sial-recTSH) was obtained by neuraminidase-agarose treatment (enzyme activity, 20 mU/µg of protein) in 50 mmol/L acetate (pH 6.5). The insoluble enzyme was removed by centrifugation at 750g for 1 min. Asialo-TSH was incubated with hST6Gal I as mentioned for 3/6Sial-recTSH.

The protein concentrations of all of the preparations were determined by protein assay as described above.

affinity chromatography on lentil lectin-sepharose
The fractionation of sialylated recTSH preparations is summarized in the 4 smaller panels at the bottom of Scheme 2Up .

Lentil lectin-Sepharose (1 mL) was equilibrated with 10 mL of buffer containing 10 mmol/L Tris-HCl (pH 8.0), 150 mmol/L NaCl, 1 mmol/L MgCl2, 1 mmol/L MnCl2, and 1 mmol/L CaCl2. We chose to use fractionation of remodeled recTSH (20 µg) on the lectin column as representative of ~1 L of serum with highly increased TSH. After incubation for 2.5 h at room temperature to facilitate interaction, the sample was packed into a 3-mL column. Unbound TSH was collected by centrifugation 10 times with 1 mL of buffer (buffer removed after every centrifugation and new buffer added). The bound fraction was collected by the same procedure and the same buffer supplemented with 500 mmol/L {alpha}-methylmannopyranoside (32). Finally, we determined the TSH content by testing the various fractions in an in-house ELISA, using the initial appropriate sialylated preparation with known molar content as the calibrator.

solid-phase immunoassays
A panel of 10 monoclonal antibodies was characterized recently (32) and was used throughout the study. All experiments were carried out at equilibrium. Microtiter plates were coated with monoclonal anti-TSH antibodies (1 µg) in 100 µL of 50 mmol/L PBS (pH 7.5) for 2 h at 37 °C. After each step, the wells were washed with PBS containing 0.5 mL/L Tween 20. The wells were then incubated with 20 g/L BSA in PBS to saturate the nonspecific binding sites. Portions (containing 2 ng of TSH, the half-maximal concentration of all assays) of the various preparations, lectin fractions, or serum samples were added to the wells and incubated overnight at 4 °C in PBS containing 1 g/L BSA. This buffer was used because the same binding was observed when recTSH was incubated in the presence of rabbit nonimmune serum or human hyperthyroid serum (data not shown). Bound TSH forms were detected by incubation for 2 h at 37 °C with 100 ng of 4 different biotinylated anti-TSH monoclonal antibodies. Visualization was with the streptavidin–alkaline phosphatase conjugate (100 ng) and p-nitrophenyl phosphate substrate (100 µg). All assays were performed in duplicate, and the data are reported as a mean of these values.

Under these conditions, the panel of our preparations could be similarly calibrated with the 2nd IRP for pitTSH (80/558) and the 1st IRP for recTSH (94/674) despite a maximal binding capacity that varied by as much as 100% (47) (see Figs. S1 and S2 in the Data Supplement that accompanies the online version of this article athttp://www.clinchem.org/content/vol52/issue2/ ).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Very recent work from our laboratory has shown that changes in TSH glycosylation alter the expression of several epitopes (32) in the TSH molecule. Accordingly, we proposed that discordance among routine measurements of serum TSH originated from both the determinants targeted by the assays and the pitTSH calibrator, which does not display the same epitope pattern as serum TSH. In this study, we aimed at defining the feasibility of harmonizing TSH assays by use of new calibrators independent of the current IRPs. For this purpose, we estimated the epitope patterns of novel TSH preparations on a molar basis.

We used a panel of 23 assays to map 3 remote areas in the TSH molecule that were common to pituitary and recombinant TSH (32) (see Fig. S3 in the online Data Supplement). To identify the best mimic of serum TSH, we used samples from patients with highly increased TSH because they represent conditions in which the circulating hormone is present in nanograms per 100 µL of serum and can be quantitatively compared with the calibrators. Because the glycosylation pattern of TSH was found to significantly alter the expression of all 3 antigenic clusters, we designed a series of recTSH preparations with modified terminal and/or internal glycosylation and compared their respective epitope distributions before screening them against serum samples. Unfortunately, the TSH concentrations of euthyroid patients as well as patients with subclinical hypothyroidism are far too low to be measured in the current analysis.

comparative analysis of PITTSH, RECTSH, and serum TSH
As shown in Fig. 1A , the highly purified recombinant preparation (y) was poorly correlated with pitTSH (x) when tested at half-maximal concentrations and at equilibrium. We obtained a mean (SD) slope for the linear regression of 1.769 (0.055), which indicated that the monoclonal antibodies preferentially bound the recombinant preparation, as described recently (32). When pitTSH (x) was compared with serum TSH (y) from patients with TSH concentrations >60 mIU/L (Fig. 1B ), the slope was 2.124 (0.001), indicating that the pituitary preparation also poorly resembled the circulating hormone. On the other hand, recTSH (x) and serum TSH (y) immunoreacted very similarly, although not identically (Fig. 1C ), with a slope of 1.178 (0.056). These observations support the notion that the recombinant compound better mimicked circulating long-lived TSH glycoforms because it displays a more closely related glycosylation pattern (see Scheme 1Up ).


Figure 1
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  		Figure 1. Comparative evaluation of pitTSH, recTSH, and serum TSH.

Differential epitope mapping between pitTSH and recTSH (A), pitTSH and serum TSH from a pool of 63 patients (B), or recTSH (3Sial-recTSH) and serum TSH from the same pool (C) was based on the use of 23 defined immunoassays mapping 6 epitopes in 3 remote clusters (32). Typical experiments were carried out as described in the Materials and Methods. Concentrations of the various TSH forms (x and y axes) reflect absorbance at 405 nm.

To identify which TSH preparation(s) best fit(s) the circulating hormone, we engineered various recTSH forms with variable terminal and/or internal glycosylation and compared their epitope expression with that of the recTSH.

comparison between RECTSH and enzymatically remodeledRECTSH preparations
Although circulating TSH has been shown to have higher sialic acid content than does pitTSH (12)(13), the nature of the glycosidic linkage(s) has not been determined in the native or serum hormone. We therefore enzymatically remodeled the terminal sialylation of the recombinant hormone according to {alpha}2,3- and/or {alpha}2,6-linkages. Because CHO cells have been found to specifically sialylate glycans in the {alpha}2,3 position (4)(5)(6)(7), we used an {alpha}2,6-sialyltransferase produced in the laboratory (46) to introduce this alternative {alpha}2,6-sialylation. As summarized in Scheme 2Up , recTSH was treated with neuraminidase to remove sialic acid and produce 0Sial-recTSH or was reacted with {alpha}2,6-linked sialic acid to produce 3/6Sial-recTSH. In addition, desialylated TSH was enzymatically resialylated to produce a preparation designated 6Sial-recTSH, which contained {alpha}2,6-sialic acid exclusively. Accordingly, the slopes for the linear regression between the remodeled recTSH forms (y) and recTSH (x) were 0.860 (0.057) for 0Sial-recTSH (Fig. 2A ), 1.090 (0.084) for 3/6Sial-recTSH (Fig. 2B ), and 1.227 (0.029) for 6Sial-recTSH (Fig. 2C ). These results indicated that oversialylated and resialylated recTSH forms (Fig. 2, B and C ) are good candidates to mimic hormone sialylation as may occur in serum, at least after the onset of hypothyroidism (10)(11)(12)(13). We then concentrated our efforts on further refining these 2 preparations and adding a second level of heterogeneity, namely core fucosylation.


Figure 2
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  		Figure 2. Effect of sialylation on epitope distribution in remodeled recTSH glycoforms.

Typical experiments comparing 0Sial-recTSH recTSH (A), 3/6Sial-recTSH (B), and 6Sial-recTSH (C) with recTSH (3Sial-recTSH) were performed as in Fig. 1CUp . Concentrations of the various TSH forms (x and y axes) are in mIU/L.

comparison between oversialylated and resialylated RECTSH and RECTSH as a function of core fucosylation
Because core fucosylation has been reported to decrease in circulating TSH as hypothyroidism develops (14), we were interested in comparing the epitope distribution of TSH preparations varying in both terminal and inner glycosylation. Fractionation of remodeled preparations by lentil lectin chromatography yielded an unbound fraction that contained exclusively nonfucosylated TSH glycoforms and a bound fraction that contained TSH glycoforms with at least 1 fucosylated glycan at either 1 of the 3 N-glycosylation sites.

The nonfucosylated, oversialylated recTSH, designated 3/6Sial(-Fuc)-recTSH (Scheme 2Up ), displayed slightly decreased binding compared with 3/6Sial-recTSH (Fig. 3A ), giving in a slope of 1.046 (0.090), whereas the fucosylated fraction [3/6Sial(+Fuc)-recTSH; Fig. 3B ] was not significantly affected, as shown by the slope of 1.095 (0.075). These findings revealed that alteration in fucose content did not affect the estimation of 3/6Sial-recTSH. The results were similar when we used the 6Sial-recTSH preparation; the slopes were 0.840 (0.017) and 0.976 (0.024) for the nonfucosylated [6Sial(-Fuc)-recTSH] and fucosylated [6Sial(+Fuc)-recTSH] fractions, respectively (Fig. 3, C and D ). However, we noted that measurements became highly dispersed, indicating that antibody recognition was significantly affected when TSH lacked fucose (see also below).


Figure 3
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  		Figure 3. Effect of fucosylation on epitope distribution in remodeled 3/6Sial-recTSH and 6Sial-recTSH.

Shown are typical results of experiments comparing nonfucosylated 3/6Sial-recTSH (A), fucosylated 3/6Sial-recTSH (B), nonfucosylated 6Sial-recTSH (C), and fucosylated 6Sial-recTSH (D) with recTSH (3Sial-recTSH). All experiments were performed as in Fig. 2Up . Concentrations of the various TSH forms (x and y axes) are in mIU/L.

comparison between remodeled preparations and serum tsh
We then compared all preparations with a pool of sera from patients with increased TSH (Fig. 4 ). Basically, the slope varied widely, indicating that the TSH glycosylation pattern rather strongly alters the quantitative epitope expression and, consequently, the performances of the various assays.


Figure 4
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  		Figure 4. Quantitative correlation between serum TSH and the various remodeled forms of TSH.

Shown are typical results from experiments comparing serum TSH (y) from a pool of 63 patients with the various remodeled TSH forms (x): (A), 3/6Sial-recTSH; (B), 6Sial-recTSH; (C), 3/6Sial(-Fuc)-recTSH; (D), 3/6Sial(+Fuc)-recTSH; (E), 6Sial(-Fuc)-recTSH; (F), 6Sial(+Fuc)-recTSH. All experiments were performed as in Fig. 1CUp . Concentrations of the various TSH forms (x and y axes) are in mIU/L.

Slopes varied between 1.064 (0.057) for 3/6Sial-recTSH (Fig. 4AUp ) and 0.953 (0.033) for 6Sial- recTSH (Fig. 4BUp ) compared with 1.178 (0.056) for the initial 3Sial-recTSH preparation (Fig. 1CUp ). This indicates that the {alpha}2,6-sialic acid content is important for mimicking circulating TSH.

Regarding the fucose content, we observed rather poor agreement between 3/6Sial(-Fuc)-recTSH and serum TSH [slope, 0.703 (0.031); Fig. 4CUp ]. In contrast, the 3/6Sial(+Fuc)-recTSH preparation (Fig. 4DUp ) had a slope of 0.985 (0.044), indicating more similarity between this preparation and serum TSH.

The immunochemical behavior of 6Sial-recTSH without or with core fucosylation is presented in panels E and F, respectively, of Fig. 4Up . The 6Sial(-Fuc)-recTSH fraction was definitely the preparation most distant from serum TSH [slope, 0.496 (0.0630; Fig. 4EUp ], whereas the presence of fucose in 6Sial(+Fuc)-recTSH produced somewhat better agreement with the serum pool [slope, 0.724 (0.063); Fig. 4FUp ], but the agreement was not sufficient for that preparation to be a successful mimic of serum TSH. In addition, the variation in values was indicative of the influence of fucose in antibody recognition.

overall evaluation of the remodeled tsh preparations
In this study, we purposely avoided any use of Reference Materials, which could have biased the quantitative epitope mapping. Rather, all of the remodeled and chromatographic fractions were compared on a molar basis, as described in the Materials and Methods.

The data for the various TSH preparations tested are summarized in Table 1 . Mean values were calculated from at least 3 independent experiments for each comparison with recTSH, and with at least 2 independent experiments for comparison with serum TSH.


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  		Table 1. Summary of linear regression analysis for TSH preparations and serum samples from patients with thyroid disorders.1

Correlation coefficients (r2) were >0.900 in 62.5% of the paired mappings, indicating satisfactory correlation with serum TSH, whereas for others, including pitTSH and resialylated preparations, the results were more divergent, as indicated by coefficients ≤0.855. Slopes for the linear regression between each fraction (x) and the pooled sera (y) ranged from 0.496 for 6Sial(-Fuc)-recTSH to 2.124 for pitTSH, demonstrating up to a 4-fold difference in antibody recognition throughout the panel of assays. Interestingly, slope values ranged from 0.953 to 1.178 for 3 preparations varying in sialic acid content, suggesting that sialylated TSH glycoforms are definitely in close agreement with serum TSH. Among these preparations, fucosylated fractions displayed intercept values close to 0 compared with nonfucosylated preparations, indicating that internal glycosylation may play an additional role in the ability of assays to measure the widest spectrum of glycoforms. It thus appeared that remodeling a recTSH preparation may provide several good molecular mimics of circulating TSH for further harmonization of TSH tests.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
This study aimed at establishing the feasibility of selecting potential candidate(s) for a new Reference Material for TSH immunoprocedures. A first recombinant IRP was established recently (48), but there has not been clear use of this material because measurements calibrated against the pitTSH 2nd IRP (80/558) showed fairly broad dispersion. To overcome this limitation, we produced several new recTSH preparations and determined which may best satisfy epitope similarity with serum samples. Our antibody panel targeted 3 antigenic clusters present in both Reference Materials, the pitTSH 2nd IRP (80/558) and the recTSH 1st IRP (94/674) (32)(47). From there, we designed a procedure to measure epitope expression as it may vary in circulating forms of TSH, independently of the routinely used calibrators.

TSH, like many other circulating glycoproteins, is known to exhibit large variations attributable to glycosylation under pathophysiologic conditions (3). As a result, discordances in TSH measurements originate from the nonidentical epitope expression between serum samples and the calibrator (30)(31)(49)(50). In addition, epitope expression may be altered because TSH glycosylation changes during hypothyroidism. We thus wanted to define a procedure that can properly assess the changes in immunologic potency that occur during most common thyroid disorders, particularly hypothyroidism (11). We therefore produced several TSH preparations and compared them with TSH in sera from patients with thyroid disorders. We took advantage of an engineered TSH preparation produced in CHO cells because this cell line contains the enzymatic machinery to produce a complex serum-type glycosylation, except for {alpha}2,6-sialylation (4)(5)(6)(7). This glycosidic linkage is of particular importance because we recently showed that epitope expression of both pituitary and recombinant TSH is under the influence of terminal sialylation (32).

Using TSH produced in CHO cells as starting material, we designed enzymatic procedures to remodel the sialic acid content in recTSH and fractionated the resulting preparations according to their core fucosylation to produce 7 well-defined glycoform subsets. We mapped all of these preparations by use of a panel of 23 immunoassays targeting 6 distinct antigenic determinants and subsequently compared all them against serum. We finally identified 3 potential candidates that displayed epitope expression comparable to TSH circulating in hypothyroid patients. At present, the next step will be to measure the frequency of the appearance of disease-related forms when primary or hypothalamic hypothyroidism develops, as well as other thyroid diseases. To this end, efforts are currently being made to adapt the differential screening used in this study to automated methods.

Most existing antibodies target 3 antigenic regions in the TSH molecule designated as I, II, and III, and our panel of assays divided them into 2 groups based on the different binding capacities. We previously showed that each cluster included at least 2 close epitopes (32). As a result, all determinants could be further mapped by use of 23 pairs of antibodies representing 6 distinct formats: I/II, I/III, II/I, II/III, III/I, and III/II. The III/II format gave the highest binding capacity because this combination of antibodies targeted all glycoforms independently of changes in TSH glycosylation, i.e., fucosylated as well as nonfucosylated (cluster III) and sialylated as well as nonsialylated (cluster II) glycoforms. In contrast, the formats I/II, I/III, and Ia/Ib gave lower binding capacities; these formats all target cluster I, the main immunogenic region, which has been characterized as dependent on changes in glycosylation (32). Some antibodies thus displayed preference for some glycoforms and thereby induced discordances among assays, particularly when these assays had been calibrated against the pitTSH standard. Our study identified the cause of differences in TSH values over 3 generations of assays. Very interestingly, it also indicated to us that there were no limitations in the use of some existing antibodies to improve the accuracy of TSH measurements.

Our findings demonstrate that the expression pattern of TSH circulating in patients with increased TSH concentrations did not match with all the tested preparations, particularly not with a highly purified pituitary preparation that was immunologically identical to the official reference preparation, pitTSH 2nd IRP. In contrast, recTSH preparations showed much better agreement with serum TSH. Remodeling of sialic acid content with {alpha}2,3-, {alpha}2,3/6-, or {alpha}2,6-linkages made it possible for us to explore the influence of 4 different terminal types of sialylation, despite the fact that the nature of TSH sialylation in serum cannot be determined. Indeed, we found that both the presence and the nature of sialic acid were of outmost importance for optimizing antibody recognition. The apparent role of fucosylation is to add a further level of complexity because the preference of some antibodies for nonfucosylated forms of TSH induced a broad divergence with serum samples, whereas fucosylated preparations showed closer similarities with serum TSH. This indicates that discordance in measurements can arise when an internal calibrator lacks fucose, such as TSH genetically engineered in yeast or insect cells. There is currently an ongoing active debate on defining the subnormal range of TSH by automated measurements; thus, new clinical investigations are needed. In this respect, our study indirectly supports a change toward new calibration of TSH measurements by providing new procedures and calibrators. To further explore this possibility, clinical studies will be shortly conducted within a working group of the IFCC Scientific Division.

We favor the view that a new calibrator is needed so that serum TSH can be measured with higher accuracy and possibly on a molar basis. Most of the formats that we tested were quite divergent when the calibrator was missing sialic acid, as in pitTSH, or a core fucose, as in nonfucosylated recTSH fractions. The current use of a pituitary IRP is likely to mask part, if not all, of the influence of sialic acid and may explain the disperse measurements encountered previously with the recombinant TSH in 1999 and still noted in most third-generation assays (43). In this respect, our strategy also suggests that recognition of disease-related forms of TSH may be further optimized by antibodies selection and construction of a Reference Material Procedure for TSH, which is not available at present. We hope that the best candidate(s) produced in this study will satisfy most clinical situations and shortly will be selected as new Reference Materials to meet the need of standardizing thyroid function testing.


   Acknowledgments
 
This work was jointly supported by the Centre National de la Recherche Scientifique, the University of Provence, and the European Community through a grant on "In Vitro Diagnostic Procedures in Diagnosis and Monitoring of Thyroid Disease" (G6RD-CRT-2001-00587; Rudolf Lequin, coordinator). The Community is not responsible for any use that might be made of the data appearing within this report. The authors are solely responsible for the content of this study, which does not represent the opinion of the Community.


   Footnotes
 
1 Nonstandard abbreviations: TSH, thyroid-stimulating hormone (thyrotropin); pitTSH, pituitary thyroid-stimulating hormone; GalNAc, N-acetylgalactosamine; recTSH, recombinant thyroid-stimulating hormone; CHO, Chinese hamster ovary; NIBSC, National Institute for Biological Standards and Control; BSA, bovine serum albumin; IRP, International Reference Preparation; hST6Gal I, ß-galactoside {alpha}2,6-sialyltransferase; 0Sial-recTSH, desialylated recombinant thyroid-stimulating hormone; 3/6Sial-recTSH, oversialylated recombinant thyroid-stimulating hormone; and 6Sial-recTSH, resialylated recombinant thyroid-stimulating hormone.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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