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Andrology Laboratory, 3 ANZAC Research Institute & Concord Hospital, University of Sydney, Sydney, New South Wales 2139, Australia
NV Organon,4 5342 Oss, The Netherlands
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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subunit as a detection probe, with signaling amplified by europium-labeled streptavidin. The mFSH IFMA lowered the detection limit 34-fold (5 vs. 170 pg/sample) compared with standard mFSH RIA. The mFSH IFMA demonstrated parallelism of response to dilutions of castrated mouse serum and rat FSH but no cross-reactivity with hFSH and mLH or hLH, whereas the RIA demonstrated nonparallel cross-reactivity with hFSH. The IFMA has a wide analytical range, with a good precision profile for within- and between-assay reproducibility. Because the IFMA is a sandwich-type assay with strict dimer-specificity by design, the lower readings and recovery obtained were compared with the RIA when both assays used a pituitary-purified mFSH assay standard that contained isolated or fragmented subunits as well as intact dimeric FSH. When used with mouse serum sample, the mFSH IFMA demonstrated the expected increases following orchidectomy as well as markedly enhanced sensitivity to very low levels of endogenous mFSH in gonadotropin-deficient mice. Furthermore, the IFMA measured mFSH with fidelity in both intact and orchidectomized male mice without any interference from transgenic hFSH. The greatly enhanced sensitivity, specificity, and technical convenience of this mFSH IFMA will allow wider application of FSH measurements to very small blood samples in immature and mature mice as well as transgenic models.
follicle-stimulating hormone, pituitary hormones, Sertoli cells, spermatogenesis
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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subunit identical to that of LH, hCG, and thyrotropin combined with a ß subunit homologous with those of the other three members of the glycoprotein hormone family. The ß subunit confers receptor specificity and biological activity, and the subunit ensures dimer stability and enhances receptor binding and potency. Follicle-stimulating hormone is crucial for gonadal development, for follicle recruitment and ovulation in females, and for Sertoli cell proliferation and spermatogenesis in males. It shares with LH a common cell of origin (gonadotrope) and hypothalamic-releasing hormone (GnRH) as well as highly homologous heptahelical membrane receptors. These closely intertwined evolutionary and biological relationships make it difficult to discern the individual roles of FSH and LH. Major advances have recently been made using the modern tools of recombinant (and, therefore, pure) gonadotropic hormones and genetic mouse models deleting individual hormones, receptors, and their subunits [2]. For the present study, the mouse is currently the optimal experimental species, because its reproductive system and genome are well characterized. Therefore, the mouse is the most readily manipulated and cost-effective option for experimentation and colony breeding.
A critical requirement for such research is the ability to measure mouse (m) FSH with high sensitivity and specificity adaptable to very low blood sample volumes. For decades, RIAs were widely used for measuring human (h) [3] and rodent (r) [4] FSH. More recently, these have been superseded by more sensitive, two-site immunoassays using highly specific monoclonal antibodies together with amplified, nonradioactive readouts (fluorescence, enzymatic) [5]. Among these, time-resolved immunofluorimetric (IFMA) assays, based on long-lasting fluorescence of the lanthanide europium [6], has greatly enhanced sensitivity and range of detection compared with previous FSH assay technologies [7]. Furthermore, commercial reagents are available for either specific hFSH or in-house Delfia (Perkin-Elmer, Turku, Finland) immunoradiometric assays [6, 8]. Two dual-site IFMA assays with high sensitivity and specificity for rat (r) FSH were recently reported and validated for female rat serum [9]. These assays, using a monoclonal antibody to hFSHß as a capture antibody and a biotinylated human or rat subunit for detection, were noted to detect mFSH, but no details were published [9]. An IFMA using the human subunit detection tracer reportedly was able to measure high FSH levels, but no details of the mFSH assay validation were reported [10]. For studies of a transgenic mouse line developed to express dimeric hFSH [11], it was necessary to distinguish with reliability in vivo between biologically active hFSH and mFSH in both castrated and intact mice. We now report validation of an IFMA for mFSH based on the previously reported rFSH IFMA [9] reagents and show that it is highly sensitive, specific, and capable of measuring mFSH without interference from hFSH in vivo.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Mouse FSH was measured by double-antibody RIA using reagents from National Institute of Diabetes and Digestive and Kidney Diseases as described originally [12] and updated by the National Hormone and Peptide Program [13]. Briefly, 100 µl of serum or mouse standard solution (1–125 ng/ml, AFP5308D) were incubated with 100 µl of guinea pig anti-mFSH (AFP 1760191; final dilution, 1:200 000) and 10 000 cpm of iodinated rFSH (National Institutes of Health [NIH] I-9) overnight at room temperature. Rat FSH is iodinated by chloramine testosterone and separated from free iodine and fragments on a concanavalin A-lectin affinity column. Bound, iodinated FSH was separated from the unbound fraction by a second antibody precipitation using goat anti-guinea pig immunoglobulin G (Peninsula Labs) in a 10% polyethylene glycol (Merck) buffer by precipitation at 3000 x g and counted using a multigamma detector (LKB-Wallac 1261, Perkin-Elmer). Sample results were interpolated from the standard curve constructed from a sigmoidal curve of the response (bound counts) for each standard plotted against dose (standard). The bound counts were normalized by expressing them as a proportion of those for the zero standard (B/B0).
Serum levels of mLH were measured by liquid-phase, double-antibody RIA using NIH reagents as described previously [14]. Briefly, incubations of 100 µl of serum or mouse standard (AFP 5306A) with 100 µl of antiserum (rLH S-11; final concentration, 1:750 000) for 24 h at 22°C were further incubated with 100 µl of iodinated rLH (I-9) for an additional 24 h and separated by second antibody/6% polyethylene glycol precipitation. Samples were assayed in duplicate, and the detection limit for the mLH RIA was 70 pg/ml.
mFSH IFMA
Coating of microtiter plates with anti-FSHß antibody The MAC anti-hFSHß 56A monoclonal antibody [9] was diluted in 150 mM NaCl to a concentration of 5 µg/ml, and then 100 µl were added to each microtiter well (Maxisorb; Nunc, Roskilde, Denmark). To the same well, 100 µl of 50 mM carbonate buffer (pH 9.6) were then added. The microtiter plate was mixed on an orbital shaker (LKB 1296-001 plateshaker, Perkin-Elmer, slow setting, 15 min) and then sealed and incubated for 1–3 days (but for at least 18 h) at 4°C. The microtiter plate was washed once (LKB 1296-024 platewasher, Perkin-Elmer) with 350 µl of wash buffer (50 mM Tris, 150 mM NaCl, 7.5 mM NaN3, 0.2 [v/v] Tween 20, pH 7.8), then blocked with 350 µl of blocking buffer (50 mM Tris, 150 mM NaCl, 7.5 mM NaN3, 0.2 [v/v] Tween 20 1% skim milk [Difco Becton Dickinson, Sparks], 10% [w/v] sucrose, pH 7.5) for 1 h at room temperature. The blocking buffer was then removed, and the microtiters plates were sealed and stored at 4°C until used (always within 24 h).
Biotinylation of anti-FSH antibody
The polyclonal anti-rFSH R95-2715 antibody was biotinylated in one volume of 15 nmol/ml of antibody diluted with 150 mM NaCl and incubated at room temperature for 30 min with one volume of 500 mM carbonate buffer (pH 9.00) and 500-fold molar excess of sulfo-NHS-lethal concentration-biotin as crystals (Pierce, Rockford, IL). The conjugate was desalted on PD10 columns (Amersham Pharmacia, Uppsala, Sweden) in Tris buffer (50 mM Tris, 150 mM NaCl, 7.5 mM NaN3, pH 7.8) and stored at –20°C in concentrated form.
Assay A precoated microtiter plate was washed four times with 350 µl of wash buffer. Standards (NIH-AFP5308D), samples, or quality controls in a volume of 25 µl were added to wells of the precoated microtiter plate followed by the addition of 175 µl of assay buffer (50 mM Tris, 150 mM NaCl, 7.5 mM NaN3, 20 µM diethylenetriamine pentaacetic acid (DTPA; Sigma, St Louis, MO), 0.2 (v/v) Tween 20, and 0.5% (w/v) skim milk to all wells. The microtiter plate was sealed and incubated on the plateshaker on slow setting at room temperature for 18 h. The plate was then washed four times with wash buffer. Five-hundred microliters of the concentrated stored biotinylated antibody was diluted to 0.1 µg/ml with assay buffer, and 200 µl were added to each well. The plate was sealed and incubated on the plateshaker for 2 h at 37°C. The plate was then washed four times with wash buffer. Five-hundred microliters of a 0.5 µg/L europium-labeled streptavidin solution (Perkin-Elmer) was diluted with 20 ml of strep buffer (50 mM Tris, 150 mM NaCl, 7.5 mM NaN3, 10 µM DTPA, 0.1 [v/v] Tween 20, 3 g/L of BSA, pH 7.8), and 200 µl were added to all wells. The plate was sealed and incubated on the plateshaker for 1 h at room temperature. The plate was washed six times with 350 µl of wash buffer, and 200 µl of enhancement solution (Perkin-Elmer) were added. After a 10-min incubation at room temperature on the plateshaker, fluorescence was measured with a 1234 Delfia fluorometer (Perkin-Elmer). FSH in the sample is directly proportional to the amount of biotinylated antibody bound so that the FSH concentration in samples was interpolated from standard dose-response curves that plot the fluorescence intensity (FI) against the standard concentration with the FI normalized by expressing it as a proportion of the maximal FI (FI/FImax) in that assay.
hFSH IFMA
Human FSH was measured using a commercial two-site IFMA (Delfia) hFSH assay kit (Perkin-Elmer) as previously described [11].
Assay Cross-Reactivity, Recovery, and Precision
Serial dilutions of hFSH (World Health Organization [WHO] 78/549), hLH (WHO 80/552), mLH (NIH-AFP5306A), rFSH (NIH-rFSH-RP2), and castrated mouse serum pool (n = 8) were used to establish cross-reactivity in the IFMA (dilutions in normal horse serum) and RIA (dilutions in hpg mouse serum).
Precision profiles were obtained for both RIA and IFMA from multiple assays (n = 7–8) using mFSH standard dilutions for within (n = 10 tubes/ dose) and reported as between- and within-assay coefficients of variation expressed as a percentage.
Recovery of mFSH was determined using pooled serum (quality-control samples) from hypogonadal (hpg) mice as blank mouse serum matrix that was spiked with mFSH standard (AFP5308D). For both RIA and IFMA, the recovery was expressed as the percentage of mFSH added.
Matrix effects were evaluated using mFSH standards (AFP5308D) made up in normal horse serum according to the standard assay protocol in sufficient quantities for multiple samplings. Four standard curves were run using 10, 20, 40, and 60 µl of the standards and complementary amounts of appropriate assay buffer to maintain constant the total incubation volume, salt concentration, and buffering capacities as in the standard assay protocol. The 60-µl standard curve was used to evaluate FSH concentrations in all samples, and these values were compared to expected nominal results by linear least-squares and nonparametric Passing-Bablok procedures for comparison of assays [15–17].
Mice, Surgery, and Hormone Treatments
Hypogonadal (hpg), transgenic hFSH (hFSH+, ß6 line) and FSH receptor-activating mutation (FSHR+, RR3 line) mice as well as transgenic lines crossed onto the hpg background were bred at the ANZAC Research Institute under standard housing conditions for mice. Genotypes were determined by polymerase chain reaction using DNA from proteinase K digests of tail snips as described previously for hpg [18], hFSH+ [11], and hFSHR+ [14] mice. The Animal Welfare Committee of the Central Sydney Area Health Service approved all animal handling procedures. Serum from FSHß subunit knock-out mice [19] was provided by Dr. N. Wreford (Monash University, Melb, Vic, Australia) [20].
Immature male hpg or wild-type mice were orchidectomized via scrotal incision at 3 wk of age and underwent subdermal implantation of a single, 1-cm Silastic implant filled with crystalline testosterone or dihydrotestosterone [18] before being killed by cardiac puncture under anesthetic 6 wk later. Age-matched, untreated hpg and orchidectomized wild-type mice served as controls. Immature hFSHR+ and nontransgenic strain controls on either an hpg or wild-type background were studied untreated at 9 wk of age or 6 wk after administration of a single, 1-cm subdermal Silastic implant filled with crystalline testosterone at 3 wk of age.
Goserelin acetate implants (3.6 mg; Zoladex; AstraZeneca, UK) were administered subdermally under the dorsal skin into intact, mature (age, 18–22 wk), wild-type mice using the prefilled, single-dose syringe applicator under anesthesia. These sterile, biodegradable cylindrical implants release the GnRH agonist (goserelin acetate) from a lactide/glycolide copolymer rod for at least 28 days.
Experiment 1 Mature hFSH+ and nontransgenic-strain control mice were bled before or 10 days after bilateral orchidectomy.
Experiment 2 Mature wild-type mice were injected i.p. with 150 IU of recombinant hFSH (Org 32489; Puregon; NV Organon, Oss, The Netherlands) in 50 µl of saline vehicle. Two weeks before and 24 h after injection, mice underwent blood sampling (200 µl) via retro-orbital sinus puncture before terminal cardiac puncture at 48 h. Controls were injected with 50 µl of saline and underwent orbital vein blood sampling 2 wk before and 48 h after injection.
Experiment 3 Wild-type male mice underwent treatment for 28 (one implant) or 56 days (two consecutive implants at 28-day intervals) by subdermal administration of 3.6 mg of goserelin acetate implants or sham implantations and were killed at 6 mo of age. The goserelin acetate implants release GnRH analogue for 28 days, with the 56-day treatment group having a second implant administered after 28 days.
Experiment 4
Castrated wild-type weanling (age, 8–12 wk) male mice were implanted subdermally with 1-cm Silastic implants filled with a range of cholesterol-diluted testosterone that remained in place for 3 wk before mice were killed, serum stored for assay, and seminal vesicles weighed after emptying of secretions. Cholesterol-diluted testosterone was produced by mixing crystalline testosterone with crystalline cholesterol (Sigma) in various weight dilutions ranging from 1:3 to 1:400, and the mixed powder (total weight, 
1 g) was dissolved in 20 ml of 100% ethanol by gentle heating on a shaking roller for at least 48 h. The mixed steroid was recrystalized by evaporation at 37°C in a fume cabinet. When completely dried, the granules were powdered in a mortar and packed into the Silastic implants.
Blood was collected from all mice by retro-orbital sinus puncture (for nonterminal blood samples) or cardiac puncture (for termination of experiments) under ketamine/xylazine anesthesia. Serum was stored at –20°C until assayed for mFSH or hFSH by RIA and/or IFMA.
Statistical Analysis
Data were analyzed by ANOVA and expressed as the mean ± SEM using NCSS software (http:www.ncss.com) or CB Stat software (http://www.cbstat.com/) for method comparison regression [17]. Differences at P < 0.05 were considered to be statistically significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The IFMA for mFSH was 34-fold more sensitive than RIA. The IFMA could detect 5 pg/well (10% of dynamic range) using a 25-µl serum sample per well as compared to conventional RIA using a 100-µl serum sample per tube with a sensitivity of 170 ± 11 pg/tube at B/B0 = 0.9 (Fig. 1).
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The precision profile shows acceptable within-assay (RIA, 1.6–11.9%; IFMA, 0.3–12%) and between-assay (RIA, 1.1–11.1%; IFMA, 2.5–13.5%) coefficients of variation (Fig. 2).
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Recovery of mFSH from spiked sera (quality-control samples) was lower in IFMA than in RIA over the detectable range of the assay (Table 1).
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Serial dilutions of pooled castrated mouse serum and of rFSH standard were parallel to the mFSH standard in both assays (Fig. 3). Mouse FSH standard gave parallel dilution curves when diluted in hpg or horse serum (data not shown). Human FSH was undetectable in the mFSH IFMA but showed cross-reactivity with marked nonparallelism in the RIA. Neither IFMA nor RIA showed cross reactivity with mLH or hLH.
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Matrix effects were not detectable up to a total serum volume of 60 µl as judged by the excellent correlation (r = 0.9937 [least squares] and 0.962 [nonparametric]) and unit slope (1.000: 95% confidence interval [CI], 0.960– 1.044 [least squares]; 0.988: 95% CI, 0.950–1.037 [Passing-Bablock nonparametric]) with no significant trend in regression residuals in the regression of observed on expected values for mFSH in the presence of between 10 and 60 µl of normal horse serum diluent.
Assay of Serum Samples
Nontransgenic mice Mouse FSH was detectable in all samples from wild-type normal male mice by IFMA (n = 97) and by RIA (n = 16). Dilution of a normal mouse serum gave virtually identical readings whether diluted in hpg or horse serum (Fig. 4). Using the same pituitary-purified mFSH as standard, the IFMA (13.7 ± 0.7 ng/ml, n = 97) gave consistently lower values than RIA (25.1 ± 2.3 ng/ml, n = 16) (Fig. 5). The mFSH IFMA values in these serum samples averaged 54.6% of the RIA mFSH values (Fig. 6)—that is, similar to the recovery in the spiked samples (average recovery, 57%) (see Table 1).
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In hypogonadal (hpg) mice, mFSH was undetectable in the RIA (n = 7) but detectable at very low levels by IFMA (0.36 ± 0.06 ng/ml, n = 15 [including seven undetectable]). Following testosterone or dihydrotestosterone treatment, mFSH levels remained undetectable in all mice by RIA but were detectable in all mice when measured by IFMA (testosterone treatment, 0.88 ± 0.15 ng/ml, n = 12; dihydrotestosterone treatment, 0.47 ± 0.04 ng/ml, n = 4).
Castration increased mFSH in both IFMA (59.2 ± 5.6 ng/ml, n = 9) and RIA (78.5 ± 2.2 ng/ml, n = 8) assays. Following administration of testosterone, mFSH concentrations were suppressed to levels comparable with intact mice by IFMA (7.8 ± 1.5 ng/ml, n = 4) and RIA (25.3 ± 4.1 ng/ml, n = 7).
Transgenic mice
Mice with an inactivated FSHß subunit (FSHß knock-out) had undetectable mFSH levels in the IFMA and in RIA, whereas transgenic mice expressing transgenic hFSH (ß6 line) had normal blood levels of mFSH by IFMA (10.6 ± 1.6 ng/ml, n = 12) and by RIA (32.3 ± 10.8 ng/ml, n = 5) (Fig. 5). By IFMA, mFSH was similar in FSHR+ (17.1 ± 4.5 ng/ml, n = 7) to wild-type normal mice (13.7 ± 0.7 ng/ml, n = 97). In hypogonadal (hpg) mice expressing human activating mutant FSH receptor (RR3 line), mFSH was detectable at low levels by IFMA (1.5 ± 0.8 ng/ml, n = 16).
Experiment 1 To characterize the IFMA in mice with both endogenous mFSH and transgenic hFSH secretion, both transgenic and wild-type nontransgenic mice were bled before and after orchidectomy. These mice had similar levels of endogenous mFSH (transgenic mice: 16.6 ± 3.7 ng/ml, n = 5; wild-type mice: 14.5 ± 1.4 ng/ml, n = 4), and both groups increased their mFSH by four- to fivefold following orchidectomy. In parallel, measurements of transgenic hFSH in mouse serum showed similar levels of hFSH in transgenic mice whether intact (6.9 ± 0.7 IU/L, n = 4) or after orchidectomy (6.4 ± 0.9 IU/L, n = 5). By IFMA, hFSH levels were undetectable in intact and castrate nontransgenic wild-type mice (Fig. 7).
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Experiment 2 To evaluate the IFMA for detection of endogenous mFSH in mice injected to achieve high levels of exogenous hFSH, wild-type mice (n = 10 per group) were injected with recombinant hFSH. As a result, suppression was observed of both mFSH to 33% ± 3% (range, 19–49%) of their own preinjection serum mFSH baseline at 24 h and further suppression to 28% ± 5% (range, 16– 63%) of their own baseline serum mFSH at 48 h after hFSH injection (Fig. 8). At the corresponding times, hFSH was undetectable in all mice before injection, rising to 722 ± 60 U/L at 24 h and 152 ± 8 U/L at 48 h. The saline-injected controls remained at 89% ± 16% (range, 33–200%) of their preinjection baseline at 48 h.
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Experiment 3 To evaluate the IFMA for detection of endogenous mFSH during gonadotropin suppression by a GnRH analogue, mice (n = 7–8 per group) were treated with a depot GnRH analogue (goserelin acetate; Zoladex) for 28 or 56 days and killed at 6 mo of age. The GnRH analogue-treated mice that exhibited complete suppression of testosterone (1.7 ± 0.6 nmol/L at 28 days and 0.7 ± 0.3 nmol/L at 56 days vs. 20.0 ± 5.8 nmol/L for sham-treated) and mLH (0.09 ± 0.01 ng/ml at 28 days and 0.04 ± 0.01 ng/ml at 56 days vs. 0.40 ± 0.11 ng/ml for sham-treated mice) still showed readily detectable mFSH remaining at approximately 40% of pretreatment baseline (15.7 ± 1.2 ng/ml) after 28 day (6.3 ± 0.9 ng/ml) and 33% of pretreatment baseline after 56 days (5.1 ± 0.6 ng/ml) of GnRH analogue treatment (Fig. 9).
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Experiment 4 To evaluate the IFMA in detection of endogenous mFSH suppressed by administration of physiological doses of testosterone, intact mature wild-type mice (n = 6 per dose) were castrated and treated for 3 wk with a wide range of testosterone doses produced by depot subdermal Silastic implants filled with testosterone diluted by increasing amounts of cholesterol. Mouse FSH levels were not significantly suppressed by testosterone despite dose-dependent positive androgenic effects on the seminal vesicles (Fig. 10).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Various assay formats have been developed to measure FSH, including bioassay and immunoassay using radiometric, enzymatic, and fluorometric readout methodologies [5]. Modern in vitro bioassays improve on the insensitivity of the original in vivo bioassays [21], but only immunoassays have the sensitivity needed for FSH measurement in the small blood samples available from mice. Among the immunoassays, the original double-antibody RIAs for rodent gonadotropins [4] were an important technical advance in reproductive biology research. However, the growing need for greater sensitivity and specificity as well as nonradioactive readout formats have led to improvements in assay performance. Comparative studies of FSH quantitation in human sera have shown IFMA methods to have superior performance in terms of assay sensitivity and range [5, 7]. Despite the availability of a commercial IFMA for hFSH for many years, IFMA assays for experimental animals has lagged behind, with researchers continuing to use RIA for mFSH [22] until IFMAs were developed for rLH [23] and rFSH [9, 24], but their application to the mFSH assay has not been fully developed.
The present study demonstrates an IFMA with very high sensitivity and specificity for mFSH. In addition to the advantages of an IFMA in terms of its rapidity and nonradioactive readout with long shelf-life reagents, this assay has markedly increased (34-fold) sensitivity compared with RIA using standard NIH mFSH reagents, confirming analogous findings for IFMA of rLH and rFSH compared with standard rRIA [9, 23]. The enhanced sensitivity and specificity of the present mFSH IFMA now allows accurate measurement of mFSH, being just detectable in the blood of hypogonadal (hpg) mice while remaining undetectable with conventional RIA [18, 25]. This high sensitivity potentially allows application of this FSH IFMA to the very small blood samples available from prenatal and infant mice as well as to the serial blood samples of individual mice, in which, despite limited serum that can be obtained, this assay makes it feasible to use the mouse as its own control to reduce the impact of between-animal variability. Using this mFSH IFMA, it has been possible to demonstrate the suppression of endogenous mFSH by high doses of exogenous hFSH in serial sampling studies, whereas more physiological secretion of transgenic hFSH did not have such effects. This effect presumably results from increased endogenous inhibin B negative feedback on pituitary secretion of mFSH [26]. Similarly, the physiological role of gonadal inhibin B also may explain our observation that physiological steady-state delivery of low doses of testosterone did not suppress mFSH in castrated male mice, whereas more potent androgens could achieve full FSH suppression [27]. By contrast, sustained delivery of a depot GnRH analogue to intact male mice produced partial suppression of mFSH, whereas mLH and testosterone were completely suppressed.
The highly homologous nature of FSH subunits both within and between mammalian species has allowed the construction of very sensitive two-site assays with species specificity based on judicious selection from a range of poly- and monoclonal antibodies. Based on optimization as reported previously [9], this IFMA for mFSH features as a capture antibody a monoclonal antibody to hFSHß and as a detection antibody a polyclonal antibody to rat subunit. The location of epitopes on different subunits ensures that the IFMA will detect only intact dimeric mFSH, ensuring high specificity for intact dimers, which presumably correspond most accurately with FSH biological activity, because either subunit alone or fragments of FSH are unlikely to retain bioactivity. Monoclonal antibodies have the advantage of being readily produced in bulk: Larger amounts are required for plate-coating, whereas the highly sensitive and rodent-specific detection probe ensures species specificity. Elimination of the nonparallel cross-reactivity with hFSH in the standard mFSH RIA has particular advantages for studies involving expression of transgenic hFSH in the mouse. The high species specificity of this IFMA has allowed detection of mFSH in the presence of transgenic hFSH without any interference from each other, something not excluded in the standard mFSH RIA, which exhibits cross-reactivity and nonparallelism with hFSH. We have developed transgenic mouse models expressing transgenic hFSH [11] or an activating mutation of the hFSH receptor [14] on a gonadotropin-deficient mouse background to isolate FSH from LH effects in regulating spermatogenesis [28]. These studies created a need to measure both hFSH and mFSH concentration in the same serum samples without interference. Our present findings show not only that this can be achieved, but that it has advantages over the standard mFSH RIA used in previous studies. The residual cross-reactivity with rFSH might be further eliminated by the use of capture and detection antibodies raised to mFSH subunits. In addition, the high sensitivity of the mFSH IFMA assay has been crucial for rapid, nonlethal screening of homozygous FSHß knock-out mice (unpublished results).
The high dimer specificity of this IFMA has other consequences, such as lower readings and recovery of spiked mFSH standard in mouse serum as well as mouse samples. A key principle of biochemical assays is to compare like with like, so the validity of FSH assays depend not only on the integrity (precision, reproducibility) of the methodology but also on the nature and, especially, the purity of the assay standard. The lower mFSH concentrations in normal male mice by the IFMA compared with RIA therefore, in theory, may reflect either nonhomogeneity of the purified pituitary-derived mFSH standards and/or the epitope specificity of the FSH antibody used in the RIA. A conventional immunoassay like the RIA may detect either or both subunits or fragments thereof as well as intact dimeric FSH, all of which could lead to overestimation of blood FSH concentration in samples. The consistently lower serum mFSH levels and recovery of mFSH standard spiked into serum samples suggests that the suboptimal purity of the current mFSH standard, purified from pituitary extracts, may be primarily important in this discrepancy between assays and could be rectified by the availability of an agreed homogenous recombinant mFSH assay standard.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence: D.J. Handelsman, ANZAC Research Institute, Sydney NSW 2139, Australia. Fax: 61 29 767 9101; djh{at}anzac.edu.au
Received: 25 June 2004.
First decision: 12 July 2004.
Accepted: 18 August 2004.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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