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a Research and Development, N.V. Organon, 5340 BH Oss, The Netherlands
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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are inactive with FSH. Thus specific antibodies need to be obtained. With the final TR-IFMAs, rFSH and rLH levels were assessed during the estrous cycle and compared with those obtained with the more classical RIAs and fluoroimmunoassays (FIAs).
Two IFMAs for rFSH were developed with mMAbs against the recombinant human (rec h)FSHß subunit (FSH56A) attached to the wall and two different rabbit polyclonal antibodies (PAbs) against the subunit of rec hFSH (R93–2705) or recombinant rat (rec r)LH (R95–2715) conjugated with biotin as signal antibody. With both IFMAs, rFSH holo-molecules can be measured. Rat FSH standards could be assessed between 0.02 and 10 ng/ml with a detection limit of 0.05 and 0.24 ng/ml in buffer and serum, respectively. These detection limits in four IFMAs were 8- to 16-fold lower than those in RIAs and FIAs. This detection level allowed the measurement of FSH levels in serum of hypophysectomized (HYPEX) rats at 0.18 ng/ml. In serum of cycling rats, the FSH levels of the IFMA were 2-fold lower than those of the FIA, while in ovariectomized (OVX) rats the IFMA levels were comparable. A peak level of FSH was found during proestrus of Day 2 and gestation with both RIA and FIA, but with IFMAs at gestation only.
An IFMA for rLH was set up with mMAb (hCG77A) reacting with rLHß as capture and rabbit PAb to rec rLH (R95–2712) as signal antibody. Rat LH standard could be assessed between 0.001 and 10 ng/ml with a detection limit of 0.012 and 0.1 ng/ml in buffer and serum, respectively, which was 8-fold lower than that in RIA/FIA. In serum of HYPEX rats, LH was undetectable (< 0.04 ng/ml), whereas a high background level of 2.5 ng/ml was measured in the FIA. In serum of cycling rats, only a very low LH level of 0.14 ng/ml was measured, which strongly deviated from the level of 3.46 ng/ml with an FIA. The load of LH in serum of OVX rats was 2.91 ng/ml, which was 12-fold lower than that for the FIA. The peak level of LH was detected on proestrus Day 2 with RIA, FIA, and IFMA.
In conclusion, two IFMAs for rFSH and one for rLH have been developed with high sensitivity and specificity for intact gonadotropins. The LH pattern during the estrous cycle was comparable between IFMA, RIA, and FIA, although the overall level in the IFMA was much lower, as were HYPEX levels. The FSH pattern differed only on proestrus Day 2 in the IFMA from that of RIA/FIA, showing a peak level with RIA/FIA and a basal level with the IFMA. This implies that in RIA/FIA measurements, proteins other than intact FSH and LH interfere with the analysis at proestrus Day 2 for FSH and in HYPEX, cycling, and OVX rats for LH.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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For these preclinical reproductive studies, sensitive, fast, and reliable assays for the measurement of at least rat (r)FSH and to a minor extent for rLH are lacking [7–9]. A change in methodology from RIAs or fluoroimmunoassays (FIAs) toward IFMAs may overcome these sensitivity problems [9–12]. The detection in these IFMAs is based on long-term fluorescence of the lanthanide europium [13, 14] using an indirect biotin-streptavidin-europium-coupled assay. The basic principle of this sandwich assay is the enclosure of intact FSH or LH molecules between antibodies against their common and selective ß subunits [15, 16]. Thereby the subunit antibody is biotinylated and able to bind europium-labeled streptavidin.
Such IFMAs are already commercially available for human (h)FSH in human serum samples (Delfia kit, EG&G Wallac, Breda, The Netherlands). One advantage of this particular IFMA is the very high degree of sensitivity (detection limit, 0.1 mIU/ml in serum), which stimulated us to mimic this approach for rat gonadotropins. In these commercial assays, the antibodies are directly labeled with europium. Although we succeeded in developing such an IFMA for hFSH, we decided to label the antibodies or antigens with biotin, subsequently followed by binding to europium-labeled streptavidin. This choice was made because coupling of biotin to a variety of compounds, proteins, and antibodies is a very simple procedure [17], whereas labeling of europium is troublesome. Since europium-labeled streptavidin is commercially available, this step can be avoided. A second advantage of IFMAs is that radiolabeled compounds, with all their radiation hazards and high instability, can be skipped. Although for rFSH these IFMAs are not yet available, for rLH, three IFMAs have already been described [9, 18, 19]. These LH IFMAs are based on the capture mouse monoclonal antibodies (mMAbs) against bovine LH and various signal antibodies, such as rabbit polyclonal antibodies (PAb) against rLH from NIDDK, mMAb against hLH, and rabbit PAb against ovine FSH. Since either it was difficult to obtain these antibodies in large amounts or the antibody does not cross-react with rFSH, we started the search for new antibodies in-house. Because the subunit of FSH and LH is identical in amino acid constitution, we searched for a common subunit antibody active against both gonadotropins and active in both the FSH and LH assays.
Our IFMA technique with FSH and LH is based on the recognition of two noncovalently linked proteins, the so-called and ß subunits. Both human and rat FSH and LH have been characterized as such, and a very high structure resemblance between the subunits of these species has been identified: e.g., for the subunit, the resemblance is 74.1%; for the FSHß subunit it is 82.9%; and for the LHß subunit it is 71.6% [15, 20–23]. Thus immunological cross-reactivity between human and rat FSH or LH antibodies is very likely. This similarity is very essential, since so far only one PAb against rFSH and another one for rLH are available from NIDDK for the quantification of FSH and LH in RIAs. Thus the development of an IFMA for rFSH and rLH depends on the identification of specific mMAbs or PAbs against rFSHß and rLHß and rLH subunits and/or the raising of new MAbs in mice and/or PAbs in rabbits. When recombinant rFSH and rLH became available [24, 25], PAbs were raised in rabbits against these gonadotropins, while the selection of mMAbs is still in progress. For the setup of the rFSH and rLH assays, three procedures were used (Table 1).
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Mouse MAbs against hFSH, hLH, and hCG and PAbs raised in rabbits against a) hFSH, b) recombinant (rec h)FSH, c) rec hFSH, d) hCG, e) recombinant rat (rec r)LH, f) FG6006, FG6007, and FG5020 (bulk productions of MAb-anti-rec hFSHß clone FSH56A), and g) FG6008 and FG5021 (bulk productions of MAb-anti-hCGß clone hCG77A) were obtained from N.V. Organon and Diosynth (Oss, The Netherlands). Delfia europium(Eu)-labeled streptavidin, Delfia enhancement solution, Delfia plate washer, and fluorometer 1234 were obtained from EG&G Wallac. Rabbit antiserum against a) rFSH (anti-rFSH-S-11), b) rFSH standard rFSH-RP-2, and c) rFSH highly purified for iodination rFSH-I-8, as well as rabbit antiserum against a) rLH (anti-rLH-S-11), b) rLH standard rLH-RP-3, and c) rLH highly purified for iodination rLH-I-9 were supplied by the National Hormone and Pituitary Program, NIH, NIDDK, Ogden BioServices Corporation (Rockville, MD). Rec hFSH (B 52), rec hFSH (B 529), rec hLH (WKLH13P), and rec hCG (B WKHCG148E) (N.V. Organon and Diosynth)—all of human origin—were used for biotinylation, as were the highly purified rFSH-I-8 and rLH-I-9 preparations for iodination from NIDDK. Human LH (code i004) and rFSH (code i037) were obtained from UCB-Bioproducts SA, Campro (Veenendaal, The Netherlands). Nunc maxisorp microtiter plates (cat. no. 4–73709A) of polystyrene, specially developed for fluorescence assays, were obtained from Nunc (Roskilde, Denmark).
Buffers
All buffers contained 50 mM Tris-HCl (pH 7.8), 0.15 M NaCl, 1 g/L BSA, and 0.5 g/L sodium azide (elution buffer). The blocking buffer was enriched with 100 g/L sucrose and 5 g/L skim milk; assay buffer 1 with 5 g/L skim milk, 20 µM diethylenetriamine pentaacetic acid (DTPA), and 0.2 ml/L Tween 20; assay buffer 2 with 5 instead of 1 g/L BSA, 0.5 g/L bovine gamma globulin, 20 µM DTPA, and 0.2 ml/L Tween 20; and wash buffer with 0.2 ml/L Tween 20. Moreover, the wash buffer contained only 5 instead of 50 mM Tris-HCl (pH 7.8).
Blood Samples
Blood samples were collected from ovariectomized (OVX), hypophysectomized (HYPEX), cycling, or randomly taken female Wistar rats. Serum samples were prepared and stored at -20°C until assayed.
Immunization Procedure for rec hFSH, rec hFSH, and rec rLH
Bastard Chinchilla rabbits were immunized with 100 µg hormone (dissolved in 0.2 ml saline and emulsified in 0.2 ml Freund's complete adjuvant) by two i.m. injections in the back of each animal. At Weeks 6, 10, and 14, these animals were boosted again with 100 µg hormone (dissolved again in 0.2 ml saline but emulsified in 0.2 ml incomplete Freund's adjuvant) by two s.c. injections in the back of each animal. At Weeks 4, 6, 8, and 12, blood samples were taken, and at Week 16 the animals were exsanguinated. If the antibody titers of one of the animals at Week 12 appeared to be low, an extra booster was given to that animal at Week 18, followed by exsanguination at Week 20.
IgG Isolation 1
Sodium sulfate was slowly titrated to culture medium of mMAbs until a concentration of 180 g/L was reached, followed by shaking for at least 15 min at room temperature (RT). The immunoglobulin was centrifuged at 1000 x g for 15 min at 20°C. The pellet was washed twice with 3 ml 180 g/L sodium sulfate in water and dissolved in 10 mM sodium phosphate (pH 7.4) with 0.15 M NaCl without sodium azide, which disturbs biotinylation.
IgG Isolation 2
Avid Al (product 3100–25; Bioprobe International, Tustin, CA), an affinity gel designed to bind immunoglobulins from mammalian and avian species and prepared with a low molecular weight synthetic compound as ligand, was used. Nine milliliters of gel in a column was used per 3 ml PAb serum; the column material was regenerated with 20 ml 20% methanol and equilibrated with 20 ml 10 mM sodium phosphate and 0.15 M NaCl buffer (pH 7.4). Serum diluted 4-fold in this buffer was loaded on the column and washed with 25 ml of buffer. The IgG was eluted with 20 ml of 50 mM acetic acid adjusted with HCl to pH 2.8. The IgG was immediately neutralized.
Biotinylation of Proteins
For the biotinylation of proteins, 1 volume of 15 nmol protein per milliliter was diluted with 0.15 M NaCl and incubated for 30 min at RT with 1 volume 0.5 M carbonate buffer, pH 9.0, and 500-fold molar excess of sulfo-NHS-LC-biotin as crystals (Pierce, Rockford, IL). The conjugate was desalted on PD10 columns (Amersham Pharmacia, Uppsala, Sweden) in elution buffer without BSA and stored at -20°C in concentrated form.
Coating of Antibodies
Coating of IgG to Nunc maxisorp plates was carried out by diluting IgG in 0.15 M NaCl to a final volume of 100 µl/well with simultaneous addition of 100 µl of 50 mM carbonate buffer (pH 9.5). The plates were mixed, sealed, and incubated for 3 days at 4°C. In order to prevent nonspecific binding, the wells were blocked with 360 µl blocking buffer for 1 h at RT. The blocking buffer was removed from the plates; this was followed by storage in a humidified box in a refrigerator for a maximum of 4 wk.
IFMA for rFSH (I)
Plates coated with mMAb-anti-rec hFSHß (FG5020, FG6006, or FG6007; 0.5 µg IgG per well) were pretreated with 200 µl wash buffer, shaken for 10 min, and washed 4 times. Then 200 µl of rFSH standard (rFSH-RP-2) or sample solution was added into a well in a concentration range of 0.02 to 10 ng/ml in assay buffer 1, followed by the addition of 100 µl rabbit PAb-anti-rec hFSH-biotin label (29.8 ng biotinylated IgG per well from antiserum R93–2705) also in assay buffer 1. The plates were sealed, shaken for one night at RT, and washed 4 times with wash buffer; then 100 µl of assay buffer 2 was added, followed by 100 µl of streptavidin-Eu (> 1 000 000 cps). The plate was shaken for 1 h at RT. This was followed by 6 washes with wash buffer, addition of 100 µl enhancement solution, and shaking for 10 min at RT, after which Europium fluorescence was measured with a time-resolved fluorometer (model 1234; Wallac, Turku, Finland).
IFMA for rFSH (II)
The IFMA for rFSH II was the same as that for rFSH I, with the exception of the second antibody PAb-anti-rec rLH-biotin label (43.4 ng biotinylated IgG per well from antiserum R95–2715).
IFMA for rLH (III)
The IFMA for rLH III was the same as that for rFSH I with the exceptions of mMAb-anti-hCGß (FG5021 or FG6008; 1.0 µg IgG per well), the rLH standard rLH-RP-3 in a concentration range of 0.001 to 10 ng/ml, and PAb-anti-rec rLH-biotin label (77 ng biotinylated IgG per well from antiserum R95–2712).
RIA for rFSH and rLH
Heterogenous assays were carried out with 125I-labeled rFSH and rLH molecules and antibodies from NIDDK. Two hundred microliters of rFSH or rLH was added in test tubes followed by 100 µl of rabbit anti-rFSH or rabbit anti-rLH (NIDDK) serum (100 µl 30 000 times diluted serum for FSH or 100 µl 10 000 times for LH) and preincubation for 30 min. Thereafter 100 µl of 125I-labeled rFSH or rLH was added and incubated for 18 h at RT. This was followed by the addition of 0.5 ml of a cellulose suspension conjugated with anti-rabbit immunoglobulin. After an incubation of 30 min under shaking, 2 ml of double-distilled water was added, followed by centrifugation at 3000 x g and decantation. The sediment was counted in a gamma counter.
FIA for rFSH and rLH
Plates were coated with goat anti-rabbit antibody (0.5 µg/well), blocked with BSA, and washed 4 times with wash buffer. Rabbit anti-rFSH or rabbit anti-rLH (NIDDK) serum (100 µl 30 000 times diluted serum of FSH or 10 000 times of LH) was incubated and shaken for 1 day at RT or stored for 3 days in a refrigerator. The wells were then washed 4 times with wash buffer. The plates can be stored for at least 1 mo in a humidified plastic box in the refrigerator. Before use the plates were washed again 4 times with wash buffer. Then 200 µl of rFSH/rLH or sample dilution in a concentration range of 0.1–31.6 ng rFSH/ml assay buffer 2 or 0.0316–10.0 ng rLH/ml assay buffer 2 was put into a well, sealed, and preincubated for 1 day at RT. This procedure was followed by the addition of 50 µl rFSH-biotin (99.5 ng rFSH/316 fmol per 50 µl per well) or 50 µl rLH-biotin (4.9 ng rFSH/163 fmol per 50 µl per well) and incubation for 2 h at RT. Subsequently the wells were washed 4 times with wash buffer, after which 100 µl streptavidin-diethylenetriamine tetra acetic acid-Eu solution (1 000 000 cps, 4.4 mol/Eu per mole streptavidin) in elution buffer and 100 µl assay buffer 1 was added and shaken for 1 h at RT. This was followed by 6 washes with wash buffer, addition of 100 µl enhancement solution, and shaking for 10 min at RT, after which Eu fluorescence was measured with the time-resolved fluorometer.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Since there is a high similarity in the amino acid sequences of human and rat gonadotropins, specific antibodies that cross-react with both human and rat hormones were selected. To this end, an in-house collection of mMAbs and rabbit PAbs against hFSH (22 MAb/4 PAb), hLH (10 MAb), and hCG (16 MAb/2 PAb) were screened for cross-reactivity in an FIA with rec hFSH-, rec hFSH-, rec hLH-, rec hCG-, rFSH-, and rLH-biotin.
From the FSH antibodies, mMAb FSH49A and FSH56A bound human and rFSH, but not hFSH, hLH, hCG, or rLH (Table 2). Only FSH56A could be produced in larger quantities and was used further. PAb Ch32826 showed a remarkable binding to rFSH, but was available only in very small amounts.
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From the hCG and hLH antibodies, mMAb hCG147B and hCG77A and rabbit PAb As349 showed cross-reactivity with hCG, hLH, and rLH, but not with rec hFSH, rec hFSH, and rFSH (Table 2). As349 was available only in small amounts. From the two selected mMAbs, hCG77A was chosen for rLH development because of its low blank value in the IFMA.
None of these antibodies showed overall binding to hFSH, hFSH, hLH, hCG, rFSH, and rLH. Therefore, none of these antibodies were directed to the subunit of rat hormones.
Preparation and Selection of PAbs Against Rec hFSH and Rec rLH
In order to obtain antibodies for the subunit of rFSH and rLH, PAbs were invoked in four rabbits with rec hFSH. Antibody titers were measured with biotinylated rec hFSH-, rec hFSH-, and rFSH-biotin in an FIA. Only rabbit PAb R93–2705 showed an obvious binding to rec hFSH, rec hFSH, and rFSH, implying -subunit selectivity to human as well as rat FSH. In an IFMA based on the capture mMAb-anti-hCGß (hCG77A) and PAb R93–2705 as label, only hLH showed binding, but not rLH from NIDDK (not shown). So PAb R93–2705 was directed to the human subunit and rFSH subunit and unexpectedly not to the subunit of rLH.
Since no rat antibodies against LH were found, 5 rabbits were immunized with rec rLH. The serum of 2 rabbits (R95–2712 and R95–2715) showed cross-reactivity with rLH- and rFSH-biotin labels in the FIA. These antibodies were directed against the rat subunit. From these 2 rat subunit antibodies, used as signal antibodies, R95–2715 showed the highest binding in an IFMA for rFSH and R95–2712 for rLH.
IFMA Development for rFSH (I)
IFMA I was set up with mMAb-anti-rec hFSHß (FSH56A) and PAb-anti-rec hFSH (R93–2705) as biotin label. This IFMA showed binding with rec hFSH, rFSH (standard RP-2) from NIDDK, rFSH for iodination from NIDDK, rFSH from UCB, rat pituitary extract, and rat serum (Fig. 1). In this IFMA the cross-reactivity of rec hLH was less than 0.03% with respect to rec hFSH (Fig. 1). The cross-reactivity of rFSH was 5.6% compared with that of rec hFSH, while the cross-reactivity of rLH was less than 0.25% compared with that of rFSH (Fig. 1). Only the holo-molecules of rec hFSH were bound in this assay, while the interaction of the individual subunits was very weak. Moreover, only the holo-rFSH (RP-2) and rat serum showed binding, whereas rLH did not interact with the antibodies (Fig. 1). Nearly linear binding curves for FSH were observed with dilutions of serum pools from OVX and cycling rats (Fig. 1). However, the binding of FSH from HYPEX serum was very low. A precision profile was made from 6 standard curves showing a high reproducibility and low coefficient of variation (CV) at concentrations above 0.05 ng rFSH/ml. The detection limit was assessed at 0.05 ng rFSH (RP-2)/ml based on a maximal intraassay CV of 15%. If a fixed amount of rFSH was added to serum samples of cycling rats, recovery measurements showed that a dilution of at least 4–8 times was needed to obtain recovery values of 67–82% of rFSH in these spiked rat sera (Table 3).
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IFMA Development for rFSH (II)
IFMA II was developed with a different PAb-anti-rec rLH (R95–2715) as biotin label, which showed cross-reactivity with only rFSH and rLH, not with hLH or monkey FSH (not shown). Rat FSH RP-2 and rec rFSH did bind with parallel curves. The precision profile was better, while the detection limit and intraassay CV were identical to those of IFMA I.
FSH levels in rat OVX, cycling, and HYPEX serum sample pools were found proportionally to serum sample dilution, while recovery measurements were similar to those of IFMA I (Table 3). OVX, cycling, and HYPEX serum sample dilution curves showed parallel lines with the RP-2 standard (Fig. 2).
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IFMA Development for rLH (III)
IFMA III was developed with mMAb-anti-hCGß (hCG77A) and PAb-anti-rec rLH (R95–2712) as biotin label. In this IFMA, mMAb-anti-hCGß (batch hCG77A) reacted with hCG, hLH, and rLH, but not with hFSH and rFSH, while PAb R95–2712 showed cross-reactivity with both rFSH and rLH. In this IFMA, native pituitary and purified rLH was bound, while hLH (UCB), rec hLH, rFSH (NIDDK RP-2), and monkey LH showed in this assay 2%, 0.8%, 0.3%, and no cross-reactivity, respectively (Fig. 3). A precision profile was made from 6 standard curves, leading to a detection limit of 0.012 ng rLH (RP-3)/ml based on a maximal intraassay CV of 15%.
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If the serum pool of cycling rats was spiked with a fixed amount of rLH, a dilution of at least 8-fold was needed to obtain recoveries of 60% or higher (Table 3). LH levels in rat OVX, cycling, and HYPEX serum sample pools were also found proportional to serum sample dilution (Table 3). OVX serum pools showed parallel binding curves with respect to the standard RP-3. However, cycling and HYPEX serum pools contained very low LH levels in this IFMA, so no parallel binding curves could be obtained (Fig. 3).
Measurement of Pituitary and Recombinant Hormones
The cross-reactivity of highly purified rFSH for iodination rFSH-I-8 and highly purified rLH for iodination rLH-I-9 from NIDDK were compared in the newly developed IFMAs with the recombinant preparation of rFSH and rLH and were expressed in the corresponding FSH standard rFSH-RP-2 and rLH standard rLH-RP-3 from NIDDK. Rat FSH I-8 contained 1.25 versus 1.32 mg RP-2/mg and rec rFSH contained 2.64 versus 2.06 mg RP-2/mg in the IFMA and FIA, respectively. In the IFMA, rLH I-9 and rec rLH contained 1.64 and 1.54 mg RP-3/mg, respectively. Rec rFSH showed almost 2-fold higher binding values with respect to the pituitary hormone in both IFMA and FIA, while rec rLH was comparable to the pituitary hormone in the IFMA.
Sensitivity and Precision
The detection limit of the developed IFMAs was read from the corresponding precision profiles at a maximum CV of 15% interpolated from the mean standard curve. The sensitivity of these IFMAs for rFSH and rLH was much better than that from RIAs and FIAs (Table 4) and varied in the range from 2- to 60-fold lower levels. The RIAs and FIAs were less influenced by higher serum concentrations in comparison with the IFMAs. The advantage in sensitivity of the rFSH IFMA is very obvious, whereas the sensitivity of rLH IFMAs, if a sample dilution of 8-fold is taken into account, is still 8-fold. The mean intraassay CV (%) were for the rFSH serum samples (IFMA II) 4.3% (n = 60) and for rLH serum samples 4.7% (n = 60).
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FSH and LH Measurements in Rat Serum Samples
The FSH levels measured in the OVX serum pool were similar for IFMA I and FIA, while a 2-fold difference was observed between IFMA II and FIA. The IFMA values of FSH in the cycling serum pool were about half of the levels found with the FIA (Table 3). In the 15 HYPEX samples, arbitrary FSH levels between 1.28 to 4.57 ng/ml were measured by FIA, whereas with the IFMA, nearly all levels were smaller than 0.16 or equal to 0.26 ng RP-2/ml.
The LH levels in the OVX serum pool were more than 10-fold lower with use of the IFMA as compared with the FIA (Table 3). The IFMA responses of LH in the cycling serum pool were about 25 times lower than the FIA results. The HYPEX samples did show the same phenomenon for LH as for FSH, as again, with IFMA all the levels were below 0.06 ng LH/ml and with FIA between 1.62 and 5.03 ng/ml. This implies high basal levels in the FIA.
FSH and LH Measurements During the Rat Estrous Cycle
The FSH levels in the rat estrous cycle assessed by RIA, FIA, and IFMA I are compared in Figure 4A. The duration of the estrous cycle was 5 days. The estrous cycle can be divided into diestrus Days 1 and 2 (A1 and A2), proestrus Days 1 and 2 (E1 and E2), and estrus (G). FIA levels were equal to or slightly lower than RIA values, whereas IFMA values decreased to at least 4-fold lower levels on A1, A2, E1, and E2 and to 2-fold lower levels at G in comparison with FIA and RIA values. The FSH peak reached an optimum at G in the IFMA and was high on both E2 and G in the RIA and FIA. The rLH levels were also measured by RIA and FIA in these estrous cycle samples and are presented in Figure 4B. Both RIA and FIA showed an LH peak at E2, while more basal levels were assessed at A1, A2, E1, and G.
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In another rat estrous cycle, a new sample set was analyzed for FSH and LH in the FIA and compared with the data from IFMA and an in vitro bioassay with Chinese hamster ovary cells based on cAMP production via the FSH receptor [24] (Fig. 5A). The FSH levels measured at A1 by IFMA were slightly lower than those of the FIA, and 4-fold lower with IFMA at A2 and E 900, 1700, and 1800 h. The FSH levels measured at A1 by bioassay were slightly lower than those of the IFMA, 2-fold higher with the bioassay at A2 and E 0900 h, and equal to the IFMA levels at E 1700 and 1800 h. The FIA, IFMA II, and bioassay values indicate a clear FSH peak at G, while at E 1900 h an increase was observed with all three assays, which was most prominently marked in the FIA and to a somewhat lesser extent in the bioassay. With FIA and IFMA, an LH peak was shown at the end of E at 1800 and 1900 h, while an increase was already observed with the IFMA at E 1700 h. The IFMA III values of the LH peak were nearly 6 times lower than those of the FIA (Fig. 5B), but the overall rise was more than 6- and 10-fold in the FIA and IFMA, respectively.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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subunits, and the amino acid identities of , FSHß, and LHß subunits correspond with those of the rat 74.1%, 82.9%, and 71.6%, respectively [15, 20–23]. Antibodies that cross-react with both FSH and LH can be expected to be antibodies directed against the subunit. The structural similarity of the ß subunit for human, ovine, bovine, porcine, and rat LH was suggested previously by Leiva [18]. The structural similarity between the and ß subunits of humans and rats allowed the design of homologous and heterologous assays. In the presently developed IFMAs for rFSH and rLH, a combination of mMAbs against human gonadotropins and PAbs raised against human or rat gonadotropins were used. Thus, it is not necessary to use homologous antibodies. The superior sensitivity of these IFMAs, relative to the RIA and FIA based on the reagents of NIDDK, allows the determination of the very low levels of intact FSH and LH hormones as observed during the ovulatory cycle. These IFMAs are sensitive and specific for the native and recombinant gonadotropins and show parallelism between pituitary and serum samples.
The conventional competitive assays for the measurement of rFSH and rLH have two major drawbacks: insufficient sensitivity [7–9] and a low specificity due to the interaction with both free ß subunits and intact gonadotropins. Our results confirm those of Haavisto et al. [9] and Illera et al. [19], who also found 10-fold lower LH levels in HYPEX rat male serum samples in immunometric assays in relation to the more classical RIAs and FIAs. Moreover, in our assays, different ratios between IFMA and FIA for FSH and LH levels were detected in the experimental groups. The ratio between FIA and IFMA increased from OVX to cycling to HYPEX rat serum sample groups. In OVX, cycling, and HYPEX serum samples, the FSH levels measured with the IFMA with respect to the FIA were decreased by 1-, 2-, and more than 8- to 25-fold, respectively. Also the LH levels measured with IFMA with respect to the FIA were decreased by 10-, 25-, and more than 25-fold, respectively. This effect is probably due to the high background activity of the FIAs and RIAs, causing an overestimation of the serum levels under cycling and HYPEX conditions. A similar discrepancy has been found between RIAs and IFMAs with human serum [26]. An alternative explanation might be that the and ß subunits are not secreted from the pituitary in the same amount. Since the IFMA assesses only the holo-hormones and the competitive immunoassay detects both the free ß subunit and the holo-hormone, differences between these assays might arise. However, very low FSH and LH levels are expected in HYPEX rats and very high levels in OVX rats. Both expected values are obtained with the IFMAs I and II for FSH and with the IFMA III for LH, but not with their FIAs. This suggests that the recently obtained IFMA values of cycling animals are likely to be more realistic, thus allowing us the ability to monitor small changes in FSH and LH during the estrous cycle. This has indeed been demonstrated for human serum samples in puberty with IFMAs versus competitive immunoassays [12].
With respect to the estrous cycle, the change in FSH during diestrus, proestrus, and estrus are more pronounced with the IFMA than with the FIA procedures. This difference is very pronounced between late E and G for FSH and during E at 1700, 1800, and 1900 h for LH. The obtained RIA and FIA values of FSH and LH during the estrous cycle in this study have also been observed in other studies [5, 6, 27–29]. However, the first FSH peak on proestrus, which normally coincides with the LH peak, was in this study present in FIA and RIA, but disappeared in the same serum samples with the IFMA. This may be attributable to the use of the Orga rats, which have an estrous cycle of 5 instead of 4 days, or to the measurement of intact gonadotropins in the IFMA. As the LH increase is clearly visualized at E 1700, 1800, and 1900 h and these serum samples did not contain enhanced FSH levels in two independent cycles, it is conceivable that free ß subunits of FSH are released together with LH, which might lead to the higher amounts of FSH measured with the FIA at E2 in the first cycle and at E 1800 and 1900 h in the second cycle. In the case of the FSH peak during gestation, measured with the FIA, IFMA, and bioassay, no LH was released, which is again in agreement with earlier reports [5, 6, 27–29]. Taken together these data suggest that a further evaluation of the FSH and LH patterns during the ovulation period is necessary with these new IFMA procedures. Moreover, very recent information showed that these antibodies are also useful for the detection of FSH and LH in mice.
In conclusion, two IFMAs for rFSH and one for rLH have been developed that have high sensitivity and specificity for intact gonadotropins. The LH pattern during the estrous cycle is comparable between IFMA, RIA, and FIA, although the overall level is much lower, as are HYPEX levels in the IFMA. The FSH pattern differs only on proestrus Day 2 in the IFMA from that of RIA/FIA, showing a peak level with RIA/FIA and a basal level with the IFMA. This implies that in RIA/FIA, measurements of proteins other than intact FSH and LH interfere with the analysis at proestrus Day 2 for FSH and in HYPEX, cycling, and OVX rats for LH.
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FOOTNOTES |
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1 Correspondence: W.G.E.J. Schoonen (RE 2218), Lead Discovery Unit, N.V. Organon, Molenstraat 110, P.O.Box 20, 5340 BH Oss, The Netherlands. FAX: 31 412 662542; w.schoonen{at}organon.oss.akzonobel.nl 
Accepted: November 11, 1999.
Received: August 23, 1999.
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