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Sex hormone-binding globulin concentration: differences among commercially available methods.

Sex hormone-binding globulin (SHBG) is a protein that binds certain androgens and estrogens with high affinity (1, 2) and restricts the access of such steroids to tissues as long as the steroids remain bound to SHBG (3). Measurements of SHBG are often done on a clinical basis and can be used to determine the amounts of free and bioavailable estradiol and testosterone (3-5). Initially, the measurements were indirect (6-9), but radioirnmunoassays were developed (10, 11). Now several commercial methods are available to measure SHBG directly using the interaction of SHBG with an antibody. We have found marked differences in the values obtained by these methods.

Blood samples were obtained from 20 men (ages 21-30 years) and 20 women (ages 21-42 years) between 0800 and 1000. The men and women were healthy by history and a brief physical exam, and were taking no medications. The women all had regular menstrual cycles. The samples were allowed to clot, and the serum was collected, divided into aliquots, and stored at -80 [degrees]C until analyzed. Analyses were carried out within 3 months of blood collection. The protocol was approved by the Institutional Review Board of the University of Massachusetts Medical School, and informed consent was obtained from all subjects.

Methods A and B were from Diagnostic Systems Laboratories, method C was from Radim (Angleur, Belgium through Wein Laboratories, Succasunna, NJ, and now available through KMI Diagnostics, Minneapolis, MN), and method D was from Diagnostic Products Corporation. Methods A, B, and C were radioimmunoassays using an antibody to SHBG and an antibody to [gamma]-globulin. Method D was an immunometric assay, and method E was an indirect measurement using [[sup.3]H]dihydrotestosterone and a filter-paper disc to collect the SHBG as described (7,12).

Because the data did not follow a gaussian distribution, the analysis was done using log-transformed data. An analysis of variance indicated a significant difference between the means, and a Bonferroni correction was used to determine the significant differences between the means. A two-tailed t-test was used to analyze the differences between the means for men and women.

The characteristics of the methods tested are shown in Table 1. Methods A-D all used an antibody to SHBG, but the antibodies were raised in different species. The intraand interassay CVs were in the same range. A major difference was in the expected ranges for men and women, with wide variation for the maximum normal values for both men and women.

The results for the men are shown in Fig. 1. The highest mean value was for assay B, which was approximately fourfold higher than the mean values for assays A and C and twice as high as those of D and E. The mean values for assays A and C were nearly the same (P >0.05), but both means were significantly different (P <0.05) from those for assays D and E. The values for assay B were very different from those of the other assays, whereas D and E, although differing in methodology, were not different in their results (P >0.05).

The results for the women are also shown in Fig. 1. Similar to the results for men, the mean for method B was higher than all the others. Assays A and C were not significantly different, nor were methods D and E (P >0.05), but both assays A and C were significantly different from assays D and E (P <0.05).

When the means for men and women were compared within assays, the mean values for women were significantly (P <0.01) higher than the means for the assays in men.

The measurement of SHBG often is used clinically in investigations of hyperandrogenicity in women and hypogonadism in men. The SHBG concentration has been used, along with the testosterone concentration, to calculate the percentage and amount of non-protein, or free, testosterone circulating in the blood as well as the amount of albumin-bound testosterone (6,13, 14). The ratio of the concentrations of testosterone to SHBG has also been used as an index of androgenicity (4,15). Thus, the measurement of SHBG can be critical to the evaluation of certain patients. It is evident from our data that the result of these calculations can be very different depending on the method used to measure SHBG. The mean percentage of free testosterone calculated by the method of Rosner (14) varied from 1.6% for method B to 3.7% for method C, and the mean indices of androgenicity (testosterone/SHBG) were 0.29 and 0.57, respectively.

There are several reasons why the methods might give such disparate results. SHBG exists in several forms in the blood (16-18), in part because of differences in glycosylation (19, 20). Antibodies raised to SHBG may recognize these forms differently, and thus different antibodies could give disparate results in an immunoassay. Although human SHBG was always the antigen, the antibodies were raised in different species, and this could produce antibodies that recognize SHBG differently. The preparation of the antigens could differ and produce antibodies that recognize SHBG in dissimilar manners.

SHBG concentrations do differ between men and women, as our results indicate when the means for men and women were analyzed for each method separately. However, the differences between methods were the same in groups with high and low values for SHBG concentrations.

Because of the great disparity between the methods tested, it would be important to know exactly which method was used when trying to compare results reported by investigators. It would also be important to know the exact method identified by more than the source of the reagents because two of them have the same source.


Investigators must be careful in reviewing data on SHBG concentrations that the method used is clearly indicated to avoid misinterpretation.


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(2.) Dunn JF, Nisula BC, Rodbard D. Transport of steroid hormones: binding of 21 endogenous steroids to both testosterone-binding globulin and corticosteroid-binding globulin in human plasma. J Clin Endocrinol Metab 1981;53: 58-68.

(3.) Siiteri PK, Murai JT, Hammond GL, Nisker JA, Raymoure SJ, Kuhn RW. The serum transport of steroid hormones. Recent Prog Horm Res 1982;38:457-510.

(4.) Carlstrom K, Gershagen S, Rannevik G. Free testosterone and testosterone/ SHBG index in hirsute women: a comparison of diagnostic accuracy. Gynecol Obstet Invest 1987;24:256-61.

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(8.) Corvol PL, Chrambach A, Rodbard D, Bardin CW. Physical properties and binding capacity of testosterone-estradiol-binding globulin in human plasma, determined by polyacrylamide gel electrophoresis. J Biol Chem 1971;246: 3435-43.

(9.) Rosner W. A simplified method for the quantitative determination of testosterone-estradiol-binding globulin activity. J Clin Endocrinol Metab 1972;34: 983-8.

(10.) Khan MS, Ewen E, Rosner W. Radioimmunoassay for human testosteroneestradiol-binding globulin. J Clin Endocrinol Metab 1982;54:705-10.

(11.) Cheng CY, Bardin CW, Musto NA, Gunsalus GL, Cheng SL, Ganguly M. Radioimmunoassay of testosterone-estradiol-binding globulin in humans: a reassessment of normal values. J Clin Endocrinol Metab 1983;56:68-75.

(12.) Longcope C, Hui SL, Johnston CC Jr. Free estradiol, free testosterone and sex hormone binding globulin in peri-menopausal women. J Clin Endocrinol Metab 1987;64:513-8.

(13.) Sodergard R, Backstrom T, Shanbhag V, Carstensen H. Calculation of free and bound fractions of testosterone and estradiol-17 [beta] to human plasma proteins at body temperature. J Steroid Biochem 1982;16:801-10.

(14.) Rosner W. Errors in the measurement of plasma free testosterone. J Clin Endocrinol Metab 1997;82:2014-5.

(15.) Brody S, Carlstrom K, Lagrelius A, Lunell N-O, Mollerstrom G, Poussette A. Serum sex hormone binding globulin (SHBG), testosterone/SHBG index, endometrial pathology and bone mineral density in postmenopausal women. Acta Obstet Gynecol Scand 1987;66:357-60.

(16.) Khan MS, Ehrlich P, Birken S, Rosner W. Size isomers of testosteroneestradiol-binding globulin exist in the plasma of individual men and women. Steroids 1985;45:463-72.

(17.) Strel'chyonok OA, Awakumov GV. Specific steroid-binding glycoproteins of human blood plasma: novel data on their structure and function. J Steroid Biochem 1990;35:519-34.

(18.) Cornelisse MM, Bennett PE, Christiansen M, Blaakaer J, Gluud C, Andersen JR, et al. Sex hormone binding globulin phenotypes: their detection and distribution in healthy adults and in different clinical conditions. Clin Chim Acta 1994;225:115-21.

(19.) Hammond GL, Bocchinfuso WP. Sex hormone-binding globulin/androgenbinding protein: steroid-binding and dimerization domains. J Steroid Biochem Mol Biol 1995;53:543-52.

(20.) Danzo BJ, Bell BW, Black JH. Human testosterone-binding globulin is a dimer composed of two identical protomers that are differently glycosylated. Endocrinology 1989;124:2809-17.

Charlotte Bukowski, [1] Mary Ann Grigg, [1] and Christopher Longcope [1,2] *

(Departments of [1] Obstetrics and Gynecology and [2] Medicine, University of Massachusetts Medical School, 55 Lake Ave. North, Worcester, MA 01655; * address correspondence to this author at: Department of OB/ GYN, University of Massachusetts Medical School, 55 Lake Ave. North, Worcester, MA 01655; fax 508-856-5933, e-mail
Table 1. Characteristics of the methods tested.

 A B

Assay type Double antibody Coated tube

Antibody Goat anti-SHBG - (a)

 Intraassay, (b) % 2.7 3.7
 Interassay, (b) % 4.0 8.7
Range of normal, (b) nmol/L
 Men 10-55 9-111
 Women 30-95 24-220


 C D

Assay type Double antibody Immunometric

Antibody Rabbit anti-SHBG Monoclonal murine
 Intraassay, (b) % 3.1 6.1
 Interassay, (b) % 8.5 8.0
Range of normal, (b) nmol/L
 Men 13-55 13-71
 Women 30-95 18-114


Assay type Indirect: filter
 paper absorbent

 Intraassay, (b) % 11
 Interassay, (b) % 8.0
Range of normal, (b) nmol/L
 Women 11-190

(a) Manufacturer could not disclose source of antibody.

(b) As stated by manufacturer.
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Title Annotation:Technical Briefs
Author:Bukowski, Charlotte; Grigg, Mary Ann; Longcope, Christopher
Publication:Clinical Chemistry
Date:Sep 1, 2000
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