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The Journal of Clinical Endocrinology & Metabolism Vol. 84, No. 8 2826-2833
Copyright © 1999 by The Endocrine Society

Original Studies

The High Molecular Weight Insulin-Like Growth Factor-Binding Protein Complex: Epitope Mapping, Immunoassay, and Preliminary Clinical Evaluation

Javad Khosravi, Anastasia Diamandi, Jehangir Mistry and Radha G. Krishna

Diagnostics Systems Laboratories, Inc. (J.K., A.D.), Toronto, Ontario, Canada M5G 1X5; the Department of Laboratory Medicine and Pathobiology, Faculty of Medicine, University of Toronto (J.K.), Toronto, Ontario M5G 1L5, Canada; and Diagnostics Systems Laboratories, Inc. (J.M., R.G.K.), Webster, Texas 77598

Address all correspondence and requests for reprints to: J. Khosravi, Ph.D., Diagnostics Systems Laboratories, Inc. (Canada), Mount Sinai Hospital, Room 653, 600 University Avenue, Toronto, Ontario, Canada M5G 1X5.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Measurements of insulin-like growth factor I (IGF-I), IGF-binding protein-3 (IGFBP-3), and the acid-labile subunit (ALS) are important in assessing the GH-IGF axis. As nearly all IGF-I, IGFBP-3, and ALS circulate in a GH-dependent ternary protein complex, direct determination of the complex may be of significant analytical and clinical importance. We evaluated a panel of monoclonal antibodies (mAb) to human IGFBP-3 and classified them into four groups (G-1 to G-4). G-1 antibodies recognized epitopes that mapped at or near IGFBP-3 ligand (IGF)-binding site. This region overlapped with the G-2 defined region, which, in turn, overlapped with G-3 epitopes defined by one antibody (mAb 3). Only G-1 and G-3 antibodies paired without interference. mAb 9 recognized a conformational epitope (G-4), and mAb 10 was nonreactive. In pairwise mixed antibody evaluation, mAbs in G-2 and G-3 showed simultaneous binding to serum IGFBP-3 complexes in combination with an anti-IGF-I or an anti-ALS antibody. On this basis, two novel enzyme-linked immunosorbent assays (ELISAs) involving IGFBP-3/IGF-I (ELISA-1) and IGFBP-3/ALS (ELISA-2) recognition partners were developed, both demonstrating acceptable analytical performance characteristics. IGFBP-3 complexes measured by ELISA-1 and -2 in samples from normal individuals and subjects with GH deficiency, acromegaly, and GH receptor deficiency more tightly correlated with IGF-I, IGFBP-3, and ALS than IGF-II. ELISA-1 determinations were comparatively more age dependent and, in comparison to ELISA-2, showed better discriminations among the various sample groups, particularly among GH receptor deficiency, normal, and GH deficiency subjects. The development of IGFBP-3 complex ELISAs may simplify diagnostic applications and facilitate investigations of the physiological relevance of the ternary complex formation.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE SUPERFAMILY of insulin-like growth factors (IGFs), including IGF-I and -II, at least six high affinity IGF-binding proteins (IGFBP-1 to IGFBP-6), an acid-labile subunit (ALS), various cell surface receptors, and proteases, is intimately involved in the regulation of cellular growth and metabolism and may significantly influence the proliferative behavior of malignant tissues (1, 2, 3, 4). IGFs circulate mostly complexed with IGFBP-3, which in association with ALS forms an approximately 150-kDa ternary protein complex (3, 4, 5, 6, 7). Although ALS is reportedly capable of IGF-independent interactions (4, 8), its binding to IGFBP-3 primarily depends on IGFBP-3 occupancy by IGF-I or -II (9, 10). Under normal conditions, nearly all of the circulating IGFs remain ternary complexed, and smaller proportions are associated with other IGFBPs or exist in the free form (3, 4, 5, 6, 7, 8, 9, 10). Although the precise biological relevance of the ternary complex formation has not been clearly defined, the circulating levels of its constituents, and thus the formation of the complex, are highly GH dependent (3, 4, 5, 6, 7, 11, 12).

Methods for determination of the IGF system components have evolved considerably, and based on their inherent advantages, immunoassays have emerged as the most widely used alternative (13). However, the high level of association with IGFBPs has been a major concern in routine immunoassays of IGFs (13, 14). Present strategies to minimize IGFBP interference involve serum acidification to disrupt the ternary complexes, followed by procedures to remove IGFBPs or prevent them from reassociating with the IGF peptides (15, 16). Size exclusion acid chromatography is considered the gold standard method (17), but it is not practical for efficient high volume sample processing. The commonly used alternatives, such as acid-ethanol extraction (18), may be problematic, as removal of IGFBPs, particularly the smaller species, is invariably incomplete (14, 17, 19, 20, 21, 22), and/or IGFs may coprecipitate with IGFBPs, resulting in variable underestimations of their levels (23). Various means, including addition of IGF-II to the assay mixture, acid-ethanol cryoprecipitation, and use of analog tracers with low affinity for IGFBPs or acid-Bio-Spin chromatography (19, 20, 22, 24), have been devised to further minimize the interference of residual IGFBPs. However, these alternatives further add to the complexity of the assays and may not completely resolve the IGFBP interference problem. Although IGFBP-3 determinations are comparatively less problematic and have several inherent advantages (4, 13), the reported variable proteolysis of IGFBP-3 could adversely affect their detectability as different assays may recognize different IGFBP-3 variants (4, 13, 25). Although the potential value of the third member of the IGFBP-3 complex, ALS, have been long recognized (12), commercial ALS methods have only recently became available (11, 26). This new development should encourage a more detailed evaluation of ALS physiology and its potential diagnostic applications.

The clinical assessment of the IGFBP-3 complex components, particularly IGF-I, is further complicated by the differential effects of age, pubertal stage of development, and nutritional factors (27, 28, 29). The impacts of these variables are such that current recommendations appear to favor the combined use of IGF-I, IGFBP-3, and ALS in a younger age group, and IGF-I and ALS in adult subjects (15, 27, 28, 29, 30, 31, 32). Because of the GH dependency of the ternary complex formation and its potential involvement in systemic regulation of IGF/IGFBP-3 bioavailability (4), direct determination of the ternary complexes may represent a less problematic and physiologically more relevant alternative. This assumption is favored by the fact that the ternary IGFBP-3 complex normally encompasses nearly all of the circulating IGF-I and IGFBP-3 (3, 4, 5, 6, 7, 8, 9, 10) and as much as 50% of ALS (11, 12, 33).

To explore the feasibility of direct IGFBP-3 complex determination, we evaluated a panel of monoclonal antibodies to human IGFBP-3 and initially mapped them for IGFBP-3 binding by pairwise analysis in both competitive and noncompetitive enzyme-linked immunosorbent assays (ELISAs) and in ligand binding inhibition assays. Based on evaluation of the candidate antibodies in mixed capture/detection combinations with anti-IGF-I, anti-IGF-II, and anti-ALS antibodies, two novel ELISAs (ELISA-1 and -2) were developed. In ELISA-1, IGFBP-3 complexes are captured by an anti-IGFBP-3 mAb, and the complexes are detected with an anti-IGF-I mAb (IGFBP-3/IGF-I combination). Similarly, in ELISA-2, IGFBP-3 complexes captured by a different anti-IGFBP-3 mAb are detected by an anti-ALS polyclonal antibody (pAb; IGFBP-3/ALS combination). We here present development and performance characteristics of these novel ELISAs and report the preliminary comparative evaluation of ELISA-1 and ELISA-2 in the assessment of GH status.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Samples and materials

Ethylenediamine tetraacetate (EDTA)-plasma samples from subjects with untreated GH receptor deficiency [GHRD; five males (aged 1–32 yr; mean age ± SD, 17 ± 14.5) and and 6 females (aged 2.3–65 yr; mean age, 29.9 ± 25)] and age-matched normal controls [eight males (aged 1–27 yr; mean age, 13.3 ± 10.4) and eight females (aged 2–67 yr; mean age, 36.6 ± 23.7)] were obtained from Dr. Jaime Guevara-Aguirre, Institute of Endocrinology, Metabolism, and Reproduction (Quito, Ecuador), as previously described (34). Serum samples from consenting adults with acromegaly (n = 8) and GH deficiency (n = 5) were provided by Dr. John Miell, Department of Medicine, Kings College School of Medicine (London, UK). These samples were stored at -70 C for at least 1 month until use. Fresh random serum samples from adults (n = 42), obtained from the Clinical Biochemistry Laboratory at Mount Sinai Hospital (Toronto, Canada), were stored at 4 C and used for these studies within 48 h.

Recombinant human IGF-I, IGF-II, and IGFBP-2–6 were obtained as described previously (11). IGFBP-1, ALS, and [125I]IGFBP-3 (5 x 106 cpm/mL; 0.1 mol/L sodium phosphate, pH 7.4) were obtained from Diagnostics Systems Laboratories, Inc. (Webster, TX). Other materials and chemicals were obtained as previously described (35). Polyclonal (pAb) and monoclonal antibodies (mAb 1–10) to human IGFBP-3 were raised by Diagnostics Systems Laboratories, Inc. against nonglycosylated recombinant IGFBP-3 (rIGFBP-3) and purified by affinity chromatography. The specificities of the mAbs for synthetic fragments of IGFBP-3 have been recently described (36). Anti-IGF-I (IGF-I mAb 1 and 2), anti-IGF-II (IGF-II pAb), against recombinant human IGF-I and IGF-II, respectively, and anti-ALS antibodies (ALS pAb 1 and 2), against synthetic N-terminal (ALS1–34) and C-terminal (ALS551–578) regions of human ALS (11), were also developed by Diagnostics Systems Laboratories, Inc. The ELISA assay buffers, standard matrix buffer, stopping solution, and coating and blocking buffers were described previously (35).

Procedures

Procedures for antibody or rIGFBP-3 coating to microwells and for antibody conjugation to horseradish peroxidase (HRP) or biotin have been previously described (14, 35, 37). A fresh pool of human sera was assigned 100 arbitrary units/L IGFBP-3 complexes and used for ELISA-1 and ELISA-2 standardization. Standards were prepared by appropriately diluting the serum pool in the standard matrix buffer to give reference standard values of 0.78–50 arbitrary units/L. The standards were stable for up to 3 days at 4 C and for more than 2 months at -20 C or lower.

IGFBP-3 epitope mapping

Pairwise sandwich ELISA. Anti-IGFBP-3 mAb 1–10 were evaluated for simultaneous binding to IGFBP-3. Each antibody (500 ng) was immobilized onto microwells and reacted with 0.0–100 ng/mL rIGFBP-3. After 1 h of room temperature incubation and washing, the wells were treated with 10 ng/well of each of the remaining HRP-labeled anti-IGFBP-3 mAbs, and the reaction was developed colorimetrically (35). Increases in optical density (OD) of 3 times the background signal or more, between 3 times the background to less than 1 OD, between 1 OD to 2 OD or more, and more than 2 OD were taken as indication of no simultaneous pairing, weak paring, moderate pairing, and strong pairing, respectively.

Pairwise competitive ELISA. Anti-IGFBP-3 mAb 1–10 were evaluated for competitive binding to IGFBP-3. Each biotinylated mAb at a predetermined dilution was mixed with increasing amounts of each of the remaining unlabeled mAbs, and 50 µL of the mixture were added in triplicate to rIGFBP-3-coated microwells. After 2-h incubation and washing, wells were incubated with HRP-labeled streptavidin and developed colorimetrically (35). Decreases in OD of less than 20%, 20–60%, and more than 60% were taken as an indication of noninterfering, moderately interfering, and strongly interfering binding of the mAb pair to IGFBP-3.

IGF blocking of anti-IGFBP-3 antibody binding activity. The ability of IGFs to block anti-IGFBP-3 mAb binding activity was evaluated. In brief, [125I]IGFBP-3 (1 x 106 cpm/mL) was mixed with 0.0–0.75 µg/mL IGF-I or IGF-II and incubated for 2 h, and 100 µL were added in triplicate to anti-IGFBP-3 mAb-coated microwells. After 2-h incubation and washing, the wells were counted for bound radioactivity (Packard RIASTAR, Mississauga, Canada). A mAb binds at or near IGF-binding site when its binding is decreased by 30% or more in response to IGFBP-3 preincubation with the IGFs.

Simultaneous mixed antibody binding to native IGFBP-3 complexes. Anti-IGFBP-3 mAb 1–10 were evaluated for binding to native IGFBP-3 complexes in pairwise combinations with anti-IGF-I, anti-IGF-II, or anti-ALS antibodies. In brief, 50 µL of a fresh serum pool or the zero standard were incubated for 2 h in quadruplicate microwells precoated with each IGFBP-3 mAb. After washing, each quadruplicate set of serum/zero standard-treated wells was incubated with HRP-labeled anti-IGF-I, anti-IGF-II, or anti-ALS antibodies, and the reaction was developed colorimetrically (35). Increases in OD of 3 times the background or less, between 3 times the background to less than 1 OD, and between 1 and 2 OD or more indicates no binding, moderate, or strong simultaneous binding of the two antibodies to IGFBP-3 complexes, respectively.

ELISA of IGFBP-3 complexes

Complexed IGFBP-3/IGF-I (ELISA-1). The assay involves combination of IGFBP-3 mAb 3 and IGF-I mAb 1 as capture and detection antibodies, respectively. In the assay, standards or serum samples (25 µL of 10- to 40-fold diluted) added to antibody-coated microwells are incubated for 1 h at room temperature. After washing, the wells were incubated as described above with 100 µL of the appropriately diluted anti-IGF-I-HRP conjugate, and the reaction was developed colorimetrically (35).

Complexed IGFBP-3/ALS (ELISA-2). The assay is based on a protocol similar to that described above, involving combination of IGFBP-3 mAb 2 and ALS pAb 1 as capture and detection antibodies, respectively.

Complexed IGFBP-3 ELISA validation procedure

The analytical performance characteristics of the assays were determined as previously described (11, 14). Cross-reactivity was analyzed by assaying IGF-I (up to 300 µg/L); IGF-II (up to 3000 µg/L); IGFBP-1, -2, and -4–6 (up to 500 µg/L); and IGFBP-3 (up to 4.3 mg/L) added to the standard matrix buffer. Stability was assessed by storing aliquot of fresh serum samples (n = 3) at room temperature, 4 C, and -20 C and then analyzing them on days 0, 2, and 3 by ELISA-1 and ELISA-2. Aliquots of a set of standards were also stored at the above temperatures and similarly analyzed.

Other assays and data analysis

IGF-I, IGF-II, IGFBP-3, and ALS were analyzed by immunoassay kits manufactured by Diagnostics Systems Laboratories, Inc. The performance characteristics of these assays have been recently described (11, 14, 38, 39). The data were analyzed using data reduction packages included in the Labsystems Multiskan microplate ELISA reader (Labsystems, Helsinki, Finland) with cubic spline (smoothed) curve fit. Statistical analysis was performed using the Microsoft Corp. Excel 97 Statistical Package (Microsoft Corp., Redmond, WA). Descriptive data are presented as the mean and SD unless otherwise specified. Linear regression analysis performed by the least squares method, and correlation coefficients were determined by the Pearson method.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Epitope map of IGFBP-3 complex

The antigenic profile of IGFBP-3 and its binding relation to IGF-I and ALS were grossly defined in four different binding experiments involving a panel of 10 mAbs to IGFBP-3, 2 mAbs to IGF-I, 1 pAb to IGF-II, and 2 pAbs to the N- and C-terminals of ALS as described in Materials and Methods.

Assessment of IGFBP-3 mAbs in two-site sandwich ELISAs identified 31 of the possible 100 combinations and classified them into 4 groups. Group I included mAbs 5, 6, and 8; group II included mAbs 1, 2, 4, and 7; group III included mAb 3; and group IV included mAb 9. Antibodies in the same group did not bind simultaneously to IGFBP-3, but demonstrated weak to strong pairing with antibodies in other groups. Pairing of antibodies in groups I and II appear to depend, to some extent, on whether a given antibody was used for coating or detection, presumably due to epitope conformational changes induced by binding of the first antibody to the solid phase and/or IGFBP-3. The strongest pairing signals were generated between antibodies in groups I and III (i.e. mAb 3). mAb 9 and mAb 10 were unable to form a sandwich with each other or with the remaining mAbs. mAb 9 demonstrated strong binding to IGFBP-3 only in combination with mAb 3.

A clearer picture emerged when the spatial distribution of the epitopes recognized was assessed in pairwise competitive ELISAs. This provided information on whether an epitope recognized by one antibody was distinct enough to allow noninterfering (independent) binding of a second antibody, or whether the epitopes recognized were completely or partially overlapping. In these experiments, nonpairing, moderately pairing, and strongly pairing antibodies, identified above, appeared to compete with one another strongly, moderately, or not at all, respectively. Only combinations of group I with group III (mAb 3) antibodies could simultaneously bind to IGFBP-3 without any interference. In IGF blocking experiments, preincubation of IGFBP-3 with IGF-I or IGF-II inhibited binding of group I mAbs to IGFBP-3 by more than 50%, thus positioning group I recognition epitopes at or near the IGF-binding site. Preincubation with the IGFs had no effect on the binding activity of group II and group III antibodies (Table 1Go).


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  		Table 1. Ligand (IGF) blocking of anti-IGFBP-3 binding activity

 
Finally, pairwise evaluation of the antibodies in mixed antibody ELISAs demonstrated the ability of mAbs in groups II and III to bind simultaneously to the naturally occurring (native) serum IGFBP-3 complexes in combination with an anti-IGF-I monoclonal antibody (mAb 1) or an anti-ALS polyclonal antibody (pAb 1). The strongest pairing signal was between IGFBP-3 mAb 2/ALS pAb 1 and IGFBP-3 mAb 3/IGF-I mAb 1 combinations. No simultaneous binding to IGFBP-3 complexes was observed when the anti-IGF-I and anti-ALS antibodies were evaluated or when an unrelated antibody (anti-PSA) was used as one of the binding partners (data not shown). The lack of sandwich formation between the anti-IGF-I mAb 1 and anti-ALS pAb 1 is noteworthy, as the finding may reflect structural proximity of the N-terminal of ALS to the IGFBP-3 ligand-binding site and, as such, direct involvement of ALS in modulating IGFBP-3 conformation and thus its affinity for the IGF peptides.

Complexed IGFBP-3 ELISA-1 and ELISA-2

The mixed antibody combinations identified were instrumental in the development of two novel ELISAs capable of direct quantification of IGFBP-3/IGF-I (ELISA-1)- and IGFBP-3/ALS (ELISA-2)-containing complexes. The optimized protocols were established by evaluating the effects of various technical manipulations on the analytical performance of the assays as previously described (14, 35). There was no cross-reactivity with IGF-I (up to 300 µg/L), IGF-II (up to 3000 µg/L), IGFBP-2 and -4–6 (up to 500 µg/L), and IGFBP-3 (up to 4.2 mg/L). Performance characteristics of the complex IGFBP-3 ELISA-1 and ELISA-2 are summarized in Table 2Go.


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  		Table 2. Complex IGFBP-3 ELISA-1-2 validation data

 
IGFBP-3 complexes analyzed in replicate aliquot of fresh serum samples stored at room temperature, 4 C, and -20 C demonstrated up to at least 3 days of stability. For both IGFBP-3/IGF-I and IGFBP-3/ALS complexes, recoveries at 4 and -20 C storage were 85% or more of the day 0 values. Serum-based standards stored and analyzed as described above demonstrated similar stability, and serum IGFBP-3 complexes stored at -20 C demonstrated more than 2 months of stability (data not shown).

Comparison of complexed IGFBP-3 with IGF-I, IGF-II, IGFBP-3, and ALS

Plasma samples from subjects with untreated GHRD (n = 11) and their age-matched normal subjects (n = 16) and serum samples from adults with acromegaly (n = 8) or GH deficiency (GHD; n = 5) were simultaneously analyzed by complexed IGFBP-3 ELISA-1 and ELISA-2 and by IGF-I, IGF-II, IGFBP-3, and total ALS ELISAs.

Regression analysis of data showed a high degree of correlation between IGFBP-3 complexes vs. IGFs, IGFBP-3, and total ALS ( Figs. 1–4GoGoGoGo). The strongest correlations were observed in comparisons with IGF-I levels, whereas correlations against IGF-II were relatively poor. Although poor correlations between IGF-II and IGF-I/IGFBP-3 complexes may not be surprising because of their differential responses to age and GH, a better correlation between IGF-II and ALS/IGFBP-3 complexes measured by ELISA-2 may be expected, as the later includes measurements of both IGF-I- as well as IGF-II-bound ternary complexes. However, our observation of equally poor correlation between IGF-II and measurement of ALS-based complexes by ELISA-2 may be reflective of the wide difference in age and GH dependency of IGF-II vs. IGF-I, IGFBP-3, and ALS as well as the compounding effects of abnormal physiologies, as the samples analyzed were from subjects with various GH-related disorders. The flattened low end appearance of correlation graphs is primarily due to the significant differences in the relative levels of the various analytes as a function of age (Fig. 5Go) and not necessarily to poor low end concordances. In fact, correlations in the GHRD range were the same as those involving all samples (data not shown). Although the number of normal samples was small, changes in the levels of IGFBP-3 complexes vs. age paralleled those in IGF-I, ALS, and IGFBP-3, with levels showing a significant rise during puberty (Fig. 5Go). In comparison to those by ELISA-2, determinations by ELISA-1 appeared more discriminating among the various sample groups, particularly among GHRD, normal, and GHD subjects, and the overall immunoreactivity patterns detected more closely resembled those obtained for IGF-I (Fig. 6Go).



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  		Figure 1. Comparison of complexed IGFBP-3 with IGF-I. EDTA plasma samples from subjects with untreated GHRD (n = 11) and age-matched normal subjects (n = 16), and serum samples from adults with acromegaly (n = 8) or GHD (n = 5) were assayed. The correlations of IGF-I values (x) vs. IGFBP-3/IGF-I (y = 1.03x + 25.2; r = 0.96; P < 0.005) and IGFBP-3/ALS (y = 0.39x + 11.3; r = 0.98; P < 0.005) measured by ELISA-1 and -2, respectively, are shown. Values are the mean of duplicate measurements.

 


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  		Figure 2. Comparison of complexed IGFBP-3 with IGF-II. EDTA plasma samples from subjects with untreated GHRD (n = 11) and age-matched normal subjects (n = 16) and serum samples from adults with acromegaly (n = 8) or GHD (n = 5) were assayed. The correlations of IGF-II values (x) vs. IGFBP-3/IGF-I (y = 1.63x + 245; r = 0.56; P = 0.124) and IGFBP-3/ALS (y = 0.66x + 104; r = 0.61; P < 0.066) measured by ELISA-1 and -2, respectively, are shown. Values are the mean of duplicate measurements.

 


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  		Figure 3. Comparison of complexed IGFBP-3 with IGFBP-3. EDTA plasma samples from subjects with untreated GHRD (n = 11) and age-matched normal subjects (n = 16) and serum samples from adults with acromegaly (n = 8) or GHD (n = 5) were assayed. The correlations of IGFBP-3 values (x) vs. IGFBP-3/IGF-I (y = 194x + 173; r = 0.91; P = < 0.005) and IGFBP-3/ALS (y = 68.1x + 65; r = 0.91; P < 0.005) measured by ELISA-1 and -2, respectively, are shown. Values are the mean of duplicate measurements.

 


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  		Figure 4. Comparison of complexed IGFBP-3 with ALS. EDTA plasma samples from subjects with untreated GHRD (n = 11) and age-matched normal subjects (n = 16) and serum samples from adults with acromegaly (n = 8) or GHD (n = 5) were assayed. The correlations of total ALS values (x) vs. IGFBP-3/IGF-I (y = 152x + 10.4; r = 0.93; P = 0.< 0.005) and IGFBP-3/ALS (y = 7.1x + 50; r = 0.91; P < 0.005) measured by ELISA-1 and -2, respectively, are shown. Values are the mean of duplicate measurements.

 


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  		Figure 5. IGF axis components levels vs. age. IGF-I, IGF-II, IGFBP-3, ALS, and complexed IGFBP-3 levels were measured by ELISA-1 and ELISA-2 in EDTA-plasma samples obtained from 16 normal subjects. For each analyte, the individual sample values as a percentage of the corresponding mean values are plotted vs. age.

 


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  		Figure 6. Distribution plot of IGF axis components and complexed IGFBP-3. EDTA-plasma samples from subjects with untreated GHRD (n = 11) and age-matched normal subjects (n = 16) and serum samples from adults with acromegaly (n = 8) or GHD (n = 5) were assayed for IGF-I, IGF-II, IGFBP-3, total ALS, and complexed IGFBP-3 by ELISA-1 and ELISA-2. Values represent the mean of duplicate measurements.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Clinical assessment of GH-related disorders and monitoring of GH therapeutic regimes increasingly involve biochemical determinations of IGF-I, IGFBP-3, and ALS (15, 27, 28, 29, 30, 31). As nearly all blood IGF-I and IGFBP-3 (3, 4, 5, 6, 7, 8, 9, 10) and as much as about 50% of ALS (11, 12, 33) are normally associated with the 150-kDa ternary protein complex, direct determination of the complex may represent a logical alternative. This novel approach would eliminate the need for complex sample pretreatment procedures, allowing combined determination of the IGF axis components and potentially providing better clinical information. Depending on the assay design, the ternary as well as binary complexes may be quantified, whereas contributions by fragments and less tightly bound or dissociable variants are eliminated.

To explore this possibility, a panel of 10 IGFBP-3 mAbs was evaluated in three different binding assays and classified into four groups (groups G-I to G-IV). The data provided detail information on binding characteristics of the antibodies and grossly defined the spatial arrangement of the antigenic regions. Group I antibodies (mAb 5, 6, and 8) recognized epitopes that mapped at or near the IGFBP-3 ligand (IGF)-binding site. Two of these antibodies (mAb 5 and 8) have been recently shown by Western immunoblot analysis to recognize epitopes at both the N-terminal (IGFBP-31–97) and the C-terminal (IGFBP-3, ~200–264) of IGFBP-3, whereas the third antibody (mAb 6) was found to react only with the N-terminal fragment (36). The observed differences may be explained by the possibility that the N-terminal sequences recognized by mAb 6 contribute to the formation of the IGF-binding site on IGFBP-3. As the ligand-binding site of IGFBP-3 is thought to involve both N- as well as C-terminal sequences, antibodies that bind to such conformational epitopes may compete strongly for binding to the native molecule, as defined in the present study, but bind distinctly to the denatured sequences detected by Western immunoblot analysis. However, our findings of ligand binding site specificity for antibodies that reportedly bind to both N- and C-terminals of IGFBP-3 are consistent with the idea that both N- and C-terminal sequences of IGFBP-3 contribute to the formation of the IGF-binding site (36, 40).

Although overlapping epitopes were recognized by group I and II antibodies, the binding of group II antibodies (mAb 1, 2, 4, and 7) was not affected by IGF binding to IGFBP-3. The antigenic region recognized by group II antibodies was therefore mapped to an area distant from the IGFBP-3 ligand-binding site. Again, group II mAbs could not be distinguished from each other, as they appeared to bind to overlapping epitopes. This is consistent with the reported Western immunoblot specificity of group II antibodies for the intermediate sequences of IGFBP-3 (IGFBP-398–159) (36). The third antigenic region was defined by only one antibody (mAb 3) that paired overlappingly with group II antibodies, but nonoverlappingly with group I antibodies. Interestingly, this antibody has been found to recognize epitope within the N-terminal IGFBP-31–97 region (36), which, according to our findings, must not overlap with those involved in the formation of the IGF-binding site. A fourth antigenic epitope recognized by mAb 9 appeared to be a distinct conformational epitope, accessible only after binding of mAb 3 to IGFBP-3.

Simultaneous binding of group II and III antibodies to serum IGFBP-3 complexes in pairwise combinations with an anti-IGF-I or an anti-N-terminal ALS antibody indicates exposure of IGF-I as well as ALS antigenic domains and accessibility for antibody binding. This finding is in agreement with the reported identification of an antigenic domain on IGF-I that remains exposed after IGF-I interaction with IGFBP-1, IGFBP-3, or type 1 IGF receptor (41). However, the lack of simultaneous binding of the present anti-IGF-I and anti-ALS antibodies to serum IGFBP-3 complexes may indicate steric hindrance, suggesting close binding proximity of the N-terminal of ALS to the IGF-binding site on IGFBP-3. This observation provides further support for the idea that ALS may be an important modulator of IGFBP-3 affinity for the IGF peptides (8). Analogous to the cooperative effect of mAb 3 on immunoreactivity of mAb 9 described above, proximal ALS binding to IGFBP-3 ligand-binding site could cause conformational changes, leading to enhanced IGF binding affinity of IGFBP-3.

As we demonstrated, native IGFBP-3 complexes could be accurately quantified by ELISAs involving mixed capture/detection antibodies. ELISA-1, involving IGFBP-3/IGF-I recognition partners, is specific for IGF-I/IGFBP-3/ALS ternary complex and any binary IGFBP-3/IGF-I complex that could be potentially present. The assay measured similar levels in untreated and acid-treated samples quantified 2 h after neutralization to allow IGF/IGFBP-3 reassociation (7-fold final sample pretreatment dilution). As ELISA-2 involves IGFBP-3/ALS antibody combination, it quantifies both IGF-I- as well as IGF-II-based ternary complexes and any high affinity binary IGFBP-3/ALS complexes. Because acidification is known to dissociate and functionally inactivate ALS, ELISA-2 did not detect any immunoreactivity in the acid-neutralized samples (data not shown).

The high stability of the IGFBP-3 complexes at various temperatures and in diluted form was unexpected, but was instrumental in the success of the developmental process. The demonstrated dilution linearity of the assays suggests measurement of the tightly bound complexes, as antibody binding and/or sample dilution may induce dissociation and thus removal of the loosely held components. In several experiments involving randomly selected samples, IGFBP-3 complexes showed significant correlation with IGF-I, IGFBP-3, and ALS levels (data not shown). This was subsequently confirmed by the preliminary clinical evaluations involving samples from subjects with GHRD, their age-matched normal controls, and specimens from acromegalic and GHD adults. Overall, both ELISA-1 and ELISA-2 demonstrated high correlation with IGF-I, IGFBP-3, and ALS, indicating the importance of the ternary complex formation in the regulation of IGF bioavailability (3, 4, 5, 6, 7, 8, 9, 10, 11, 12). Although the clinical samples analyzed were relatively old and subjected to several freeze/thawing cycles, measurements by ELISA-1 appeared as effective as IGF-I determinations in discriminating among the various sample groups, particularly among GHRD, normal, and GHD subjects. Further studies involving well defined patient populations and their age-matched normal controls are obviously needed to better define the potential advantages as well as applications of the ternary complex measurements.

In summary, we described identification of at least four antigenic regions on IGFBP-3, defined by systematic evaluation of a panel of 10 IGFBP-3 mAbs. We identified antibodies specific for the ligand-binding region of IGFBP-3 that may be useful as analytical reagents or in investigations of IGFBP-3/IGFs interactions. The results of mixed antibody evaluations demonstrated identification of an antigenic domain on IGF-I that remain exposed in its native ternary complex configuration. These findings as well as the demonstrated stability of the complexes were instrumental in the development of novel ELISAs for direct quantification of circulating IGFBP-3 complexes. In comparison to those by ELISA-2, determinations by ELISA-1 involving IGFBP-3/IGF-I recognition partners appeared more discriminating among subjects with GH-related disorders. Development of IGFBP-3 complex ELISAs should simplify diagnostic applications and facilitate further investigations of the regulation and physiological relevance of ternary complex formation.


   Acknowledgments
 
We thank Drs. Jaime Guevara-Aguirre and John Miell for providing the clinical samples.

Received December 4, 1998.

Revised April 21, 1998.

Accepted May 10, 1999.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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