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Articles |
1
Diagnostic Systems Laboratories (Canada) Inc., Toronto, ON, Canada.
2
Department of Clinical Biochemistry, University of
Toronto, Toronto, ON, Canada.
3
Diagnostic Systems Laboratories, Webster, TX.
a Address correspondence to this author at: Diagnostic Systems Laboratories (Canada) Inc., Mount Sinai Hospital, Room 653, 600 University Ave., Toronto, ON, Canada M5G 1X5. Fax 416-586-8479.
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Key Words: indexing terms: insulin-like growth factors • insulin-like growth factor binding proteins • phosphorylation • ELISA
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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IGFBP-1, synonymous with placental protein-12 (10) and the
pregnancy-associated endometrial 
1-globulin
(11), is a 25-kDa protein expressed and secreted by
hepatocytes, ovarian granulosa cells, and decidualized endometrium
(12)(13)(14)(15)(16). IGFBP-1 is present in serum, is the predominant
binding protein in amniotic fluid, and is the major IGFBP in fetal and
maternal circulation (12)(17)(18)(19). Because of
the reported inverse relation between birth weight and both fetal and
maternal IGFBP-1 concentrations, a critical role for IGFBP-1 in fetal
growth and development has been suggested
(12)(19). In normal individuals, circulating
IGFBP-1 concentrations fluctuate rapidly by 10-fold or more in response
to the acute changes in insulin concentrations that have been reported
to inhibit IGFBP-1 gene transcription and, thus, its production by the
liver (12)(13). Because of the apparent
regulation of IGFBP-1 production by insulin and other glucoregulatory
hormones, a significant role for IGFBP-1 in glucose counterregulation
has been also proposed (12)(20).
A number of reports have shown that IGFBP-1 is capable of both inhibition as well as augmentation of the IGFs' actions (4)(21)(22)(23)(24). These observations may be explained by the findings that differential phosphorylation of IGFBP-1 could significantly alter its affinity for the IGFs (25) and, therefore, its capacity to influence their cellular responsiveness. Analysis of IGFBP-1 by anion-exchange chromatography and nondenaturing polyacrylamide gel electrophoresis has identified up to five IGFBP-1 variants, differing only in their degree of phosphorylation. Various cell types such as Hep G2, decidual, and liver cells were found to secrete predominantly phosphorylated forms, whereas amniotic fluid and fetal serum contained substantial amounts of nonphosphorylated and lesser-phosphorylated variants (4)(16)(25)(26). In contrast, in the sera of normal adults only a single highly phosphorylated variant of IGFBP-1 has been detected (27). The IGFBP-1 profile of normal adult serum may, however, change under certain conditions such as during pregnancy or in subjects with Laron syndrome (LS), when nonphosphorylated and lesser-phosphorylated variants are also expressed (20)(27). Dephosphorylation of IGFBP-1 has been proposed as a possible mechanism for increasing IGF bioavailability (4)(27). The nonphosphorylated IGFBP-1 isoforms have reportedly four- to sixfold lower binding affinity for IGF-I as compared with the phosphorylated variants (25). The stimulation and inhibition of the IGF actions have been found in association with the nonphosphorylated and phosphorylated IGFBP-1 variants, respectively (4).
Changes in the phosphorylation state of IGFBP-1 have also been reported to result in significant change in its immunoreactivity (27). As a result of differential recognition of the various IGFBP-1 phosphoforms by different antibodies, up to 11-fold differences in the circulating concentrations of IGFBP-1 in normal adults were observed. However, in serum samples from pregnant subjects the antibodies demonstrated only twofold variations in the mean IGFBP-1 concentrations (27). Because variable antibody recognition of the IGFBP-1 isoforms may obviously result in false estimates of actual concentrations or inappropriate interpretations of the IGFBP-1 determinations, development of immunoassays unaffected by the state of IGFBP-1 phosphorylation is urgently needed.
We report for the first time development and performance characteristics of a novel noncompetitive ELISA for total IGFBP-1 that is unaffected by changes in its states of phosphorylation. The distinguishing feature of the proposed assay has been established by showing that the measured IGFBP-1 concentrations in various biological fluids remain virtually unchanged after sample pretreatment with alkaline phosphatase (ALP), which is known to readily dephosphorylate IGFBP-1 (16)(25)(27). Comparison of the present total IGFBP-1 ELISA (ELISA-1) with other ELISAs (ELISA-2 and ELISA-3) that appear to measure a subset of nonphosphorylated or lesser-phosphorylated IGFBP-1 isoforms and a commercially available IGFBP-1 IRMA is also presented.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Horseradish peroxidase (HRP) was obtained from Scripps Labs., San Diego, CA. Tetramethylbenzidine (TMB) microwell peroxidase substrate system was from Kirkegaard and Perry Labs., Gaithersburg, MD. Sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxyl-ate (sulfo-SMCC) and 2-iminothiolane were purchased from Pierce, Rockford, IL. Enzyme immunoassay-grade ALP was obtained from Boehringer Mannheim, Indianapolis, IN. All other chemical reagents were of highest quality and were obtained from Sigma Chemical, St. Louis, MO or Amresco, Solon, OH. Microtitration strips and frames were products of Costar, Cambridge, MA.
Recombinant human IGF-I and IGF-II were obtained from GroPep, Adelaide, Australia (distributed by DSL, Webster, TX) and recombinant nonglycosylated IGFBP-3 from Celtrix Pharmaceuticals, Santa Clara, CA. Recombinant human IGFBP-2 and IGFBP-4–6 were purchased from Austral Biologicals, San Roman, CA. IGFBP-I, purified from human amniotic fluid according to previously described methods (13)(28), was obtained from DSL. The preparation was calibrated against pure recombinant human IGFBP-1.
Five different IGFBP-1 mouse monoclonal antibodies and an affinity-purified goat polyclonal anti-IGFBP-1 antibody were obtained from DSL.
igfbp-1 elisa
The IGFBP-1 assay buffer was 0.05 mol/L Tris-maleate, pH 7.0, 9
g/L NaCl, 20 g/L bovine serum albumin (BSA), and 0.1 g/L thimerosal.
The antibody-HRP conjugate buffer was 0.02 mol/L sodium phosphate, pH
7.0, 9 g/L NaCl, 1 g/L CaCl2, 5 g/L BSA, and 0.1 g/L
thimerosal. The stopping solution was 2 mol/L sulfuric acid in
deionized water. The composition of the coating and blocking buffers,
as well as the wash solution, were as described previously
(29).
IGFBP-1 antibody coating to microtiter wells was performed at a concentration of 0.25–20 mg/L by using previously published methods (29). In brief, 0.1 mL of the capture antibody solution was added to each microwell and allowed to incubate overnight at room temperature. The wells were then washed with the wash solution and 0.2 mL/well of the blocking solution was added and allowed to incubate for 1 h as above. The wells were washed again before use.
The IGFBP-1 detection antibodies were coupled to HRP as previously described (30). The coupling reaction involved activation of the enzyme with sulfo-SMCC and its subsequent conjugation to the anti-IGFBP-1 detection antibody, which had been activated by 2-iminothiolane. The stock HRP-conjugated antibody solution was diluted at least 1000-fold before use.
IGFBP-1 calibrators were prepared by dissolving precalibrated IGFBP-1
from amniotic fluid in a protein-based buffer matrix (0.05 mol/L sodium
phosphate, pH 7.4, 9 g/L NaCl, 1 g/L BSA, 0.1 g/L thimerosal) or in
newborn calf serum (NBCS) containing 0.1 g/L thimerosal. The
preparation, diluted in the standard matrix to give IGFBP-1 values of
~0.5, 1.0, 2.0, 10, 40, 80, and 160 µg/L, was stable for up to 4
weeks at 4 °C and 
6 months at -20 °C or lower. The NBCS was
chosen as standard matrix because calf serum IGFBP-1 did not
demonstrate any cross-reactivity or interferences with IGFBP-1
measurement by the present methods; human IGFBP-1 serially diluted in
the protein-based buffer matrix or NBCS generated identical assay
response (data not shown). The quality-control samples were prepared by
supplementing NBCS at four different IGFBP-1 concentrations (~3–50
µg/L). The nominal concentrations of the control samples were
established by analyzing them in the IGFBP-1 ELISAs.
igfbp-1 elisa protocol
The IGFBP-1 ELISA was performed according to previously described
procedures after determination of optimal assay conditions
(31)(32). Calibrators or samples (0.025 mL)
were added in duplicate to the precoated wells, followed by addition of
the assay buffer (0.05 mL) and 1 h of incubation at room
temperature with continuous shaking. The wells were washed four times
and incubated with 0.1 mL/well of the appropriate anti-IGFBP-1
antibody–HRP conjugate (diluted in assay buffer to ~0.1–0.25 mg/L)
for 30 min at room temperature. The wells were washed five times with
the wash solution and 0.1 mL of the TMB/H2O2
substrate solution was added for an additional 10 min of incubation at
room temperature. Stopping solution (0.1 mL) was then added and
absorbance measured by dual wavelength measurement at 450 nm with
background wavelength correction set at 620 nm. Absorbance measurements
for all ELISAs were performed with the Labsystems Multiskan Multisoft
microplate reader (Labsystems, Helsinki, Finland).
igfbp-1 elisa validation procedures
The lower limit of detection (sensitivity) was determined by
interpolating the mean plus 2SD of 12 replicate measurements of the
zero calibrator (NBCS). The intraassay CVs were determined by replicate
analysis (n = 12) of four samples at IGFBP-1 concentrations of
~3–50 µg/L in one run and interassay CVs by duplicate measurement
of the samples in 9–12 separate assays. Recovery was assessed by
adding 25 µL of exogenous IGFBP-1 diluted in the standard matrix
(NBCS) to 225 µL of three serum samples and analyzing the
supplemented and unsupplemented samples. Percent recovery was
determined by comparing the amount of added IGFBP-1 with the amount
measured after subtracting the endogenous IGFBP-1 concentrations.
Linearity was tested by analyzing three serum samples serially diluted
(2- to 32-fold) in the zero calibrator of the assay.
igfbp-1 irma
IGFBP-1 concentrations were also determined by using an IGFBP-1
IRMA (DSL). Calibrators or samples (0.1 mL) and
125I-labeled anti-IGFBP-1 antibody were added to
anti-IGFBP-1-coated tubes. The tubes were incubated at room temperature
for 16–20 h, washed, and counted (LKB 1275 Minigamma counter;
Pharmacia LKB Biotechnology, Uppsala, Sweden). Calibrators ranged from
0.8 to 200 µg/L. Intra- and interassay CVs are ~3.4–6% within the
assay range. The assay has no cross-reactivity with other human IGFBPs.
molecular sieve chromatography
A fresh serum (0.2 mL) was subjected to size-exclusion
chromatography on a precalibrated 600 x 7.5 mm Bio-Sil SEC-250
HPLC column (Bio-Rad Labs., Richmond, CA) as described
(30)(32). The column was preequilibrated and
eluted with 0.05 mol/L Tris-HCl, pH 7.2, 9.0 g/L NaCl at 0.5 mL/min
with collection of 1.0-mL fractions. Distribution of IGFBP-1
immunoreactivity was determined by IGFBP-1 ELISAs.
dephosphorylation of igfbp-1
Dephosphorylation of IGFBP-1 was achieved by sample pretreatment
with ALP by using a similar procedure described previously
(16)(25)(27). Briefly, ALP
dissolved in 10 µL of 1 mol/L diethanolamine (DEA), pH 9.5,
containing 0.5 mmol/L MgCl2 was added to a 200-µL aliquot
of the sample, mixed, and incubated at room temperature for 2 h.
The untreated control aliquot received 10 µL of the DEA buffer and
was similarly incubated. ALP-treated and untreated samples were then
analyzed.
data analysis
IRMA and ELISA data were analyzed with data reduction
packages included with the respective instrumentation, both based on
cubic spline (smoothed) curve fit. Other statistical analyses were
performed with the Statworks statistical software package (Starlight
Network, Mountain View, CA) on an Apple Macintosh SE computer.
Descriptive data are presented as the mean, median, and SD unless
otherwise specified. Linear regression analysis was performed by the
least-squares method and correlation coefficients were determined by
the Pearson method.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Because of the possible antibody recognition of antigenic determinants
at or near the ligand (IGF-I or IGF-II) binding site of IGFBP-1,
ELISA-1–4 were tested for potential interferences by free IGFs. In
these experiments, IGF-I was added to a buffer-based IGFBP-1
preparation (~8 µg/L) to ensure efficient IGF-I–IGFBP-1 complex
formation rather than IGF-I binding to other IGFBPs that would be also
present if serum had been used. As shown in Table 1
, IGF-I at concentrations up to 200 µg/L did not interfere
with IGFBP-1 measured by ELISA-1 but showed some interferences in
ELISA-3 at relatively high concentrations (50 µg/L). In contrast,
ELISA-2 showed significant interferences by free IGF-I at
concentrations 5 µg/L, and ELISA-4 falsely overestimated the
expected IGFBP-1 concentrations and was therefore discontinued. In
ELISA-2, the interfering effects of the free IGF-I added to actual
serum samples occurred at significantly higher concentrations (10
µg/L). Because these concentrations of free IGFs may be above the
expected endogenous concentrations available for binding to IGFBP-1,
ELISA-2 was further evaluated. However, the accuracy of results
obtained by ELISA-2 may be questionable, as the assay is incapable of
detecting IGFBP-1 molecules that may be present in association with
IGF-I or IGF-II.
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igfbp-1 elisa
The IGFBP-1 ELISA protocols were optimized for analytical
performance. This was easily achievable, as the ELISA-1–3 are based on
a common monoclonal capture antibody and a common two-step immunoassay
protocol that allows detection of the bound IGFBP-1 by each of the
three different detection antibodies in a separate step (i.e., the
second step).
The protocol optimization was based on the initial evaluation of a number of factors that could potentially affect detection limit, dynamic range, precision, and delayed sample addition. The best performances were obtained with a coating antibody concentration of 5 mg/L (500 ng/0.1 mL per well), a detection antibody concentration of ~0.1–0.25 mg/L (10–25 ng/0.1 mL per well), a sample size of 0.02 mL, a first- and second-step room temperature incubation of 60 and 30 min, respectively, and a 10-min substrate development step. With this protocol, the differences in assay results caused by 1–20 min delays between addition of the same samples into the coated wells was <10%.
The standard range and performance characteristics of ELISA-1–3 are
summarized in Table 2
. Analysis of IGFBP-2 and IGFBP-4–6 (up to 500 µg/L), IGFBP-3
(up to 4.2 mg/L), and insulin (up to 200 µg/L) added to the zero
calibrator or a calibrator preparation of ~8 µg of IGFBP-1/L did
not show any cross-reactivity or interferences in IGFBP-1 ELISA-1–3.
Addition of IGF-I (up to 200 µg/L) and IGF-II (up to 120 µg/L) to
the assay zero calibrator followed by IGFBP-1 analysis by ELISA-1–3
did not show any cross-reactivity (data not shown).
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To evaluate the distribution and molecular mass of immunoreactivity
detected, a fresh serum sample was fractionated by molecular sieve HPLC
and fractions were assayed for IGFBP-1. As expected, a major peak at
~30 kDa, corresponding to the reported molecular mass of IGFBP-1
(12), was detected by ELISA-1–3. However, there appear to
be significant differences in the peak IGFBP-1 intensity measured by
the three methods (Fig. 1
).
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It is now well established that production of IGFBP-1 by the liver is
inversely regulated by insulin so that in normal individuals, serum
IGFBP-1 should fall rapidly after a meal, whereas increases should
occur during fasting (12)(13). To evaluate the
assay response to meal-induced IGFBP-1 suppression, timed serum samples
taken from two apparently normal individuals during glucose tolerance
testing were assayed. As shown in Fig. 2
, the expected postmeal decline in IGFBP-1 was clearly
registered by ELISA-1–3, further confirming the specificity of the
assays for IGFBP-1. However, in another sample, only ELISA-1 was able
to demonstrate decreasing IGFBP-1 concentrations. The IGFBP-1
concentrations measured by ELISA-2 and ELISA-3, on the other hand, did
not show the expected fall in values. This observation might be related
to changes in the phosphorylation status of IGFBP-1, which could alter
its molecular structure and, thus, its binding to different IGFBP-1
antibodies. (see below).
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igfbp-1 concentrations in physiological fluids
The IGFBP-1 ELISA-1–3 measured considerably different
concentrations in randomly selected serum samples and SF (Table 3
). The median serum value measured by ELISA-1 was 3.3- and
13-fold higher than the concentrations measured by ELISA-2 and ELISA-3,
respectively. Furthermore, regression analysis of values by ELISA-2 and
-3 showed relatively poor correlations in comparison with ELISA-1, with
significant scattering of data points around the regression line (Fig. 3
). The median IGFBP-1 concentrations by ELISA-1–3 in SF were
19.5, 3.5, and 2.1 µg/L, respectively. In contrast, the
concentrations of IGFBP-1 measured by ELISA-1–3 in AF and CSF were
relatively similar (Table 3
).
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effect of igfbp-1 dephosphorylation
To investigate whether the apparent discrepancy in IGFBP-1
measurement was caused by differential antibody recognition of the
IGFBP-1 variants, the effect of IGFBP-1 dephosphorylation on the
response of ELISA-1–3 was examined.
Experiment 1.
Seven serum samples (200 µL) were
incubated as described with increasing concentrations of ALP (0–100
U). The enzyme-treated and untreated samples were then analyzed by
ELISA-1–3. As represented in Fig. 4
, there were no significant differences in the concentrations of
IGFBP-1 measured by ELISA-1 before and after ALP treatment
(P = 0.337 by a two-way ANOVA), suggesting equivalent
recognition of all IGFBP-1 variants (nonphosphorylated and
phosphorylated). However, concentrations detected by ELISA-2 and
ELISA-3 increased progressively in response to increasing amounts of
ALP treatment and reached a plateau at concentrations close to the
amount measured by ELISA-1. The increases in IGFBP-1 concentrations
detected by ELISA-2 and ELISA-3 were highly significant (P
<0.001 by ANOVA). In this and subsequent experiments, ELISA-3
initially detected only 5–10% of IGFBP-1 measured by ELISA-1 but
almost 80% of the total concentrations after IGFBP-1
dephosphorylation. Analysis of 100–1000 U of ALP added to IGFBP-1
calibrators did not show any cross-reactivity or interferences in
ELISA-1–3.
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Experiment 2.
To further evaluate the differential
antibody recognition of IGFBP-1 by ELISA-1–3, the SF, AF, and CSF
samples were treated with ALP (at 0 and 100 U/200 µL) as above and
assayed. In SF, the assay responses to IGFBP-1 dephosphorylation were
similar to those described in experiment 1 for the serum samples.
ELISA-1 appeared to quantify total IGFBP-1 concentrations, as there was
no change in the mean ± SD value before (18.45 ± 12.5
µg/L) and after (18.70 ± 13 µg/L) ALP treatment. IGFBP-1
concentrations by ELISA-2 and ELISA-3 increased by ~2.2- and
6.4-fold, respectively, in response to ALP treatment, with
concentrations by ELISA-3 approaching those detected by ELISA-1 (data
not shown). In contrast, the IGFBP-1 concentrations (mean ± SD)
measured in AF by ELISA-1–3 were similar and did not show any
significant increases in response to ALP treatment. Similarly, the
IGFBP-1 concentrations in CSF demonstrated a relatively smaller
response to ALP treatment. Again, the IGFBP-1 values (mean ± SD)
measured by ELISA-1 before and after treatment with ALP were identical
(Table 4
).
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Experiment 3.
To confirm that phosphorylation of IGFBP-1
was responsible for the significantly lower IGFBP-1 concentrations
measured by ELISA-2 and -3, as well as the scattering of values around
the regression line in comparison with ELISA-1, fresh serum samples
(n = 21) were treated with ALP (100 U/200 µL) and then assayed.
Regression analysis showed excellent correlation between IGFBP-1
concentrations measured by ELISA-1 in untreated samples in comparison
with the corresponding values obtained by ELISA-2 and ELISA-3 after ALP
treatment (Fig. 5
). The IGFBP-1 concentrations by ELISA-2 and ELISA-3 in the
treated samples increased to within ~70–80% of those measured by
ELISA-1. As expected, ALP treatment had no significant effect on
IGFBP-1 concentrations measured by ELISA-1 in ALP-treated
(y) and untreated (x) samples (y
= -1.18 + 1.07x, r = 0.986,
Sy|x = 0.04, P =
<0.001).
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comparison with commercial igfbp-1 irma
ELISA-1 was compared with a commercially available IGFBP-1 IRMA.
Samples (n = 21) before and after ALP treatments were analyzed by
both methods. Regression analysis showed about two- to sevenfold higher
IGFBP-1 concentrations by ELISA-1, which resulted in significant
scattering of values in comparison with those measured by the IGFBP-1
IRMA. However, the correlation parameters improved significantly when
the IGFBP-1 concentrations in untreated samples by ELISA-1 were
compared with the concentrations in ALP-treated samples measured by the
IRMA (Fig. 6
). The median values by the IRMA in untreated and ALP-treated
samples were 6.8 and 20.5 µg/L, whereas the corresponding values by
ELISA-1 remained unchanged at 23.4 and 24 µg/L, respectively.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Although various competitive and noncompetitive immunoassays for IGFBP-1 have been available for almost two decades (12)(17)(33), a method unaffected by the state of IGFBP-1 phosphorylation, thus allowing determination of its total concentrations, has not been reported. The extent of the problem is such that the validity of the reported IGFBP-1 normal ranges, even in the nonpregnant adult population, which apparently expresses a single IGFBP-1 variant, has been recently questioned (27). The findings of variable expression of the IGFBP-1 isoform in biological fluids (4)(16)(25)(26) and changes in its circulating profile could further complicate analysis as well as interpretation of the IGFBP-1 measurements. In this context, IGFBP-1 antibodies have been reported to detect significantly different serum concentrations in nonpregnant subjects (up to 11-fold differences in the mean values), while measuring relatively similar concentrations during pregnancy (27). Similarly, in a recent evaluation of the effect of human IGF-I on IGFBP-1 concentrations in subjects with LS, an inverse relation between insulin and serum IGFBP-1 was detected with one antibody, whereas a direct relation was indicated when an antibody with a different IGFBP-1 specificity was used (20). This is in accordance with the present observation that in one subject, only ELISA-1 was able to clearly demonstrate the expected fall in IGFBP-1 concentrations in relation to a meal.
As we demonstrated in this report, ELISA-1 is capable of accurate determination of total IGFBP-1 concentrations. This was confirmed by demonstrating identical assay results in response to increment IGFBP-1 dephosphorylation under the condition that resulted in a progressive increase in IGFBP-1 concentrations measured by two other methods (i.e., ELISA-2 and ELISA-3). Although all three methods are based on a common capture antibody and detect a similar profile in HPLC fractions, the observed quantitative differences may not be surprising if differential antibody recognition of the various IGFBP-1 phosphoforms is considered. ELISA-1 consistently measured the highest concentrations in the various biological fluids examined; the medians in serum and SF were, respectively, ~3.3- and 5.5-fold higher than the corresponding concentrations by ELISA-2-, and ~13- and 9-fold higher than those by ELISA-3. However, the concentrations by ELISA-2 and ELISA-3 increased after ALP treatment to within ~66–98% of those measured by ELISA-1. Linear regression analysis of serum values measured by ELISA-2, ELISA-3, and a commercial IRMA showed a relatively poor correlation, and more importantly scattering of the data points, in comparison with those detected by ELISA-1. Again, excellent correlations were obtained only after ALP-treated samples were analyzed by the comparative methods. ELISA-1 was unaffected by IGFBP-1 phosphorylation and generated virtually identical results in response to ALP treatment.
The significant increase in the measured IGFBP-1 values by ELISA-2 and, more pronouncedly, by ELISA-3 after ALP treatment strongly suggests measurement of essentially nonphosphorylated and lesser-phosphorylated IGFBP-1 variants by these methods. This is in accordance with the findings that normal adult serum contains primarily a single highly phosphorylated IGFBP-1 variant that is readily dephosphorylated by ALP (27). As ELISA-3 is capable of a more dramatic response to dephosphorylation, it may be of greater value in monitoring changes in nonphosphorylated and lesser-phosphorylated IGFBP-1 subfractions.
The findings of similar IGFBP-1 concentrations in AF before and after ALP treatment by ELISA-1–3 may be expected, as AF reportedly lacks the highly phosphorylated IGFBP-1 variants (27), but contains predominant concentrations of nonphosphorylated and lesser-phosphorylated forms that are well-recognized by the three methods. The fact that all three ELISAs incorporate the same AF-derived calibrators should further minimize the expected differences. Antibodies that detect significant differences in serum IGFBP-1, but similar concentrations in AF, have been also reported by other investigators (27).
Our data suggest that the IGFBP-1 profile in CSF may be very similar to that present in AF, containing predominantly nonphosphorylated and lesser-phosphorylated variants. Relatively similar IGFBP-1 concentrations in untreated and ALP-treated samples were detected by ELISA-1–3. Again, only ELISA-1 generated identical measures before and after IGFBP-1 dephosphorylation. The higher concentrations of IGFBP-1 measured by ELISA-2 in response to ALP treatment might be due to overexpression of the predominant isoforms that are preferentially recognized by this method.
In summary, we describe the first report on development and validation of a highly specific and simple noncompetitive immunoassay (ELISA-1) for total IGFBP-1 in biological fluids. ELISA-1 is virtually unaffected by the state of IGFBP-1 phosphorylation, which significantly alters its immunoreactivity and thus accuracy of its measurements. Availability of this method should facilitate investigations of the physiological roles and potential diagnostic values of IGFBP-1.
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Footnotes |
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The following articles in journals at HighWire Press have cited this article:
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, ELISA-1; 
, ELISA-2; 
, ELISA-3.
, ELISA-3.
