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1
Irving Center for Clinical Research, and Departments of
2
Medicine and
3
Pathology, Columbia University College of Physicians and Surgeons, New York, NY 10032.
4
New York Hospital-Cornell Medical Center, White Plains,
NY 10605.
a Address correspondence to this author at: Irving Center for Clinical Research, Columbia University College of Physicians and Surgeons, 630 West 168th St., PH10-305, New York, NY 10032. Fax 212-305-3213; e-mail gk49{at}columbia.edu.
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResults and DiscussionConclusionsReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionConclusionsReferences |
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Early reports of cleavages (nicks) in the hCG beta subunit at positions 44–45 and/or 47–48 (5)(6)(7)(8) have been followed by other reports relating to the extent of nicking in various hCG preparations (9) and to the biochemical and immunological characterization of nicked hCG (hCGn) (10). Cleavages in the hCG beta sequence have been shown to have profound effects on the biological activity of hCG, reducing the biological activity to <20% of the activity of the parent molecule (11). Nicking also substantially reduces the binding of hCG by many specific monoclonal antibodies (mAbs), some of which are in widespread use as assay reagents (11)(12). Hoermann et al. (13) established that nicking eliminates two distinct epitopes present on the intact hCG molecule. These investigations also determined in a study of hCGn in testicular cancer that hCGn was more prevalent in urine than in serum but that its determination did not increase the efficacy over measuring just the intact, non-nicked molecule as a tumor marker.
One consequence of this altered immunological recognition contributes to very extensive discrepancies in hCG values obtained from a variety of commercial hCG kits (14)(15). Although the presence of nicking has been demonstrated clearly by isolation and gel electrophoresis, analysis in biological specimens has generally been indirect, performed either by measuring total intact hCG (i.e., nicked + non-nicked) and subtracting intact non-nicked hCG from the total, the remainder being ascribed to hCGn. Another approach attempted to neutralize ("scavenge") the interfering molecules in assays that did not efficiently discriminate between the nicked and non-nicked form of hCG. However, there are problems, at least in our hands, associated with either of these approaches to assay specificity. We here detail our development of mAbs that distinguish nicked from non-nicked intact hCG, the development of an IRMA for its measurement, and our initial findings related to the assay of hCGn in various forms of clinical pregnancy by direct measurement without prior isolation or pretreatment of the clinical specimens. We also report our findings regarding the use of scavenger antibodies to neutralize the influence of the cross-reacting non-nicked hCG in the clinical specimens under investigation.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionConclusionsReferences |
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hCGn (preparation 813) was purified from the parent CR 127 by hydrophobic chromatography as described for non-nicked hCG above, which is a gradient modification of the earlier batchwise separation (17).
hCGn free beta subunit (hCGßn; preparation 834) was separated from CR 129 hCG free beta subunit (hCGß) by reversed-phase chromatography on a Vydac C4 column with 1 mL/L trifuoroacetic acid (TFA) in distilled water as buffer A and 1 mL/L TFA in acetonitrile as buffer B (10). The semipreparative column size was 22 cm x 10 mm i.d. The flow rate was 2 mL/min, and the gradient was 0–40% buffer B in 60 min.
The hCG beta core fragment (hCGßcf; preparation 455) was prepared from Diosynth crude commercial hCG by modification of the method of Blithe et al. (18), which included gel filtration, Concanavalin A chromatography, and anion-exchange chromatography, followed by reversed-phase chromatography in 1 mL/L TFA in acetonitrile. We added the reversed-phase step, which improves the amino acid analysis of the hCGßcf.
The hLH (AFP-8270B) and the hLH free beta subunit (AFP-3282) were kindly provided by the National Hormone and Pituitary Program, National Institute of Diabetes and Digestive and Kidney Diseases. The hLH beta core fragment was prepared as described by Birken et al. (19).
purification of mAbs
Immunoglobulins were purified from ascites by the Protein A
Monoclonal Antibody Purification System (Bio-Rad). The protein
concentration of pure antibodies was determined by amino acid analysis.
Purification of mAbs was checked by electrophoresis in polyacrylamide
gel in the presence of sodium dodecyl sulfate according to the method
of Laemmli (20).
iodination of hormones and mAbs
Antibodies and hormones were labeled with
125I by the chloramine T method (21).
immunization of mice and cell fusion
Balb/c mice were immunized with hCGn (preparation 813) according
the following protocol: the first immunization was performed by the
subcutaneous injection of 15–20 µg of immunogen per animal in
complete Freund's adjuvant; the second immunization was performed 2
weeks later by injection of the same amount of hormone in incomplete
Freund's adjuvant. On days 21 and 28, the mice received
intraperitoneal immunizations, using 15 µg of antigen in
phosphate-buffered saline (PBS) for each animal. Immune sera were
tested in a liquid phase RIA using [125I]hCGn. Mice
with a high immune response were boosted with 15 µg of hormone and,
after 3 days, used for fusion.
Splenocytes from immunized mice were fused with cells of myeloma line X63-Ag8.653 (American Type Culture Collection) 3 days after the booster injection, according to the method of Kohler and Milstein (22) as described by Kovalevskaya et al. (23). The splenocyte:myeloma cell ratio was 4:1 or 6:1. Polyethylene glycol 4000 (Sigma Chemical Co.) was used as the fusing reagent. After fusion, cells were distributed in six microtitration plates that contained mouse peritoneal feeder cells and cultured for 1 week in hypoxanthine-aminopterin-thymidine-RPMI 1640 containing 200 mL/L fetal calf serum (HyClone). One-half of the medium was replaced every 3 days. On days 12–14 post fusion, culture supernatants (100 µL) from the wells with cell clones were screened for the presence of antibodies to hCGn, using a liquid phase RIA (vide infra). Positive selected cells were cloned at least two times by the limiting-dilutions method on mouse peritoneal feeder cells. Balb/c mice received intraperitoneal injections of the subclones (0.5 x 101 to x 101 cells/mouse), and mAbs were purified from the ascites as describe previously.
Isotypes of mAbs were determined using the ImmunoPure Monoclonal Antibody Isotyping Kit II (AP/PNPP; Pierce) according to the manufacturer's instructions for the antigen-dependent technique.
screening of primary clones and immune serum
Primary screening was carried out in a liquid phase RIA with
[125I]hCGn, using a procedure described previously
(24). Briefly, the binding buffer consisted of PBS
supplemented with 1 g/L bovine serum albumin (BSA) and 1 g/L sodium
azide. One hundred and fifty microliters of solution (containing
30 000–40 000 cpm [125I]hCGn) was added to 100 µL of
culture supernatant diluted 2.5:1 with PBS. Fifty microliters of 80
mL/L normal mouse serum was also added. This solution was incubated
first for 1 h at 37 °C and then overnight at 4 °C. The next
day, 500 µL of a 25 mL/L goat antimouse serum was added, and the
mixture was incubated for 1 h at 37 °C, followed by incubation
for 2 h at room temperature. The precipitate containing bound
radioactive hormone was separated by centrifugation and counted in a
gamma counter. Supernatants of positive clones were tested in the same
type of assay to check preliminary cross-reactivity with
[125I]hCG and [125I]hCGß. Immune serum
was used as a positive control.
competitive liquid phase ria
Our competitive liquid phase RIAs have been described previously
(23). Briefly, cell supernatants were used at those
dilutions at which ~40% of maximum antibody binding occurred in the
absence of unlabeled hormone. The following reagents were added to each
12 x 75 mm polystyrene tube: 100 µL of diluted supernatant,
30 000–40 000 cpm of [125I]hCGn in 300 µL of binding
buffer (PBS, pH 7.2, containing 1 g/L BSA, and 1 g/L sodium azide), 100
µL of competitor solution, and 100 µL of 80 mL/L normal mouse
serum. After incubation for 1 h at 37 °C and overnight at
4 °C, 1 mL of 25 mL/L goat antimouse serum was added as in the
primary screening. Affinity constants were calculated by homologous
competitive displacement assays using the personal computer version of
the program Ligand by Munson and Rodbard
(25).1
In our RIA format, cross-reactivity was calculated as a
percentage of the molar ratio of hCGn to competitor at 50% of maximum
binding.
irma for hCGn
Our methodology for the construction and validation of IRMAs has
been described fully (26). Briefly, the specificity of
the antibody pairs and their capacity for simultaneous binding to
antigen were determined as follows.
The capture antibody was adsorbed onto the wells of microtiter plates (Immulon IV; Dynatech) by incubating a 10 mg/L solution in coating buffer (0.2 mol/L bicarbonate, pH 9.5) overnight at 4 °C. The coating antibody solution was aspirated, and the plates were washed (wash solution: 9 g/L NaCl, 0.5 g/L Tween 20) and blocked with a 10 g/L solution of BSA in PBS containing 1 g/L sodium azide. After incubation with the BSA solution (minimum of 3 h at room temperature), the blocking solution was removed, the wells were washed again with wash solution, and 200 µL/well of the appropriate hCGn calibrators or potential cross-reacting molecules was added in phosphate buffer B (PBS containing 1 g/L bovine gamma globulin and 1 g/L sodium azide) or in hCG-free serum (for serum assay; Chemicon). After overnight incubation at 4 °C, the plates were again aspirated and washed, and 200 µL (50 000–100 000 cpm) of 125I-labeled antibody was added to the wells, which were again incubated for 24 h at 4 °C. The tracer was aspirated, the plates were washed with wash solution, the individual wells were placed in glass tubes, and the radioactivity was determined in a Packard Cobra gamma counter. Doses were determined by interpolation from a smoothed spline transformation of the data points.
The analytes tested for potential cross-reaction with the normal-pregnancy hCGn mAbs included hCG, hCGß, hCGßn, hCGßcf, hLH, the hLH free beta subunit, and the hLH beta core fragment. In the IRMA format, cross-reactivity was calculated as a percentage of the molar ratio of hCGn to tested analyte at 50% of maximum binding.
In addition to the hCGn assay, the B109-B108 IRMA for intact hCG was used (26).
recovery of hCGn
hCGn was added to hCG-free human serum and urine. hCGn was also
assayed as described above, using the B151-B604 assay in buffer B in
the presence of increasing concentrations of hCG (1.76–176 nmol/L)
with or without the addition of 10 mg/L B109 as a scavenger for
non-nicked hCG.
subjects
Down syndrome samples (n = 9) and control normal-pregnancy
urine samples (n = 99), in vitro fertilization (IVF)
normal-pregnancy samples (n = 42), IVF ectopic pregnancy samples
(n = 9), and spontaneous abortion samples (n = 12) were the
kind gift of Dr. L. Cole (Department of Obstetrics and Gynecology, Yale
University School of Medicine, New Haven, CT). Nine matched serum/urine
samples from the first trimester and five urine samples from the third
trimester were provided by Dr. A. Kelly at Columbia Presbyterian
Medical Center, New York, NY.
Trophoblast disease serum (17 samples) and urine (28 samples) were also obtained from Dr. Cole, but were collected by Dr. Edward Newlands (Charing Cross Hospital, London, UK).
All samples were stored frozen at -20 °C until they were assayed. Because extreme values of sample pH may interfere with antibody binding, the urine pH was adjusted with 1.0 mol/L Tris (pH 9.0, 50 mL/L of urine) before being assayed, so that the final pH was 7–7.4.
creatinine
Creatinine determinations were performed in a 96-well microtiter
plate format by a procedure adapted from Taussky (27).
statistical analysis of down syndrome vs normal-pregnancy
urine
Normal-pregnancy urine samples (n = 99) and Down syndrome
samples (n = 9) were tested for intact hCG in the B109-B108 assay
and for hCGn in the B151-B604 assay. Data were normalized to
creatinine. To minimize the effects of outliers on the analysis any
values greater or less than 2 SD from the mean were excluded from the
analysis. Even after removal of these extreme values, the data remained
highly skewed, and these remaining values were then log-transformed to
approximate gaussian distributions. The data were then analyzed by a
Generalized Additive Model approach (28).
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Results and Discussion |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionConclusionsReferences |
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. Amino acid sequence analysis detected no peptide bond nicks.
The most sensitive method for detecting peptide bond nicking involves
reduced sodium dodecyl sulfate gel electrophoresis followed by blotting
and staining of the blot with antibodies to the hCGß COOH-terminal
region (17). Such blots of hCGn or hCGßn indicate two beta
bands, one representing Beta48–145 and the other
representing Beta1–145. Blots indicated that some hCGßn
remains in this preparation, but the quantity is likely 
1%, based on
the HPLC profile. The hCGn preparation clearly contains non-nicked
hCGß (Fig. 1
). The integrated value of the hCGß peak was compared
with the total peaks of the alpha and beta subunits (nicked and
non-nicked); it was then calculated that this preparation contained
~20% non-nicked hCGß.
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mAb development and characterization
The development of mAbs to hCGn is challenging because of the
extensive structural homology between intact hCG and its nicked forms.
As immunogen, we used hCGn purified from a urine pool collected from an
hCG reference preparation (see Materials and Methods).
Six mAbs to hCGn (B601, B603, B604, B605, B607, and B610) were
selected, and each was characterized on the basis of its affinity and
specificity. Affinity constants were calculated as 5.3 x
108 L/mol for B601, 0.89 x 107 L/mol for
B603, 1.2 x 109 L/mol for B604, 1.4 x
109 L/mol for B605, 1.4 x 107 L/mol for
B607, and 4.6 x 107 L/mol for B610. All mAbs were
IgG1, 
isotype. Antibodies B601 and B605 recognized hCG and hCGn as
well as hLH but did not recognize hCGß, hCGßn, or hCGßcf.
Antibodies B604 and B610 bound to a common epitope present on all
hormones evaluated and could be used as universal second antibodies in
two-site assays for hCG/hLH. B603 and B607 preferred hCGn to intact hCG
(~17% cross-reactivity with hCG in our RIA), but also bind hLH as
well as hCGn. In addition, antibody B151 was also evaluated in the same
liquid phase RIA format for cross-reactivity with the normal-pregnancy
hCGn. Although B151 was developed to the choriocarcinoma hCGn (C5)
(29) and demonstrated a slightly higher affinity for C5 hCGn
than for the normal-pregnancy hCGn in our RIA assay format (Birken et
al., manuscript in preparation), this antibody provided the best
discrimination between pregnancy hCGn and intact hCG in our RIA format
(4–6% cross-reactivity with hCG and hCGßn; B151 affinity constant
for normal-pregnancy hCGn was calculated as 8.0 x 108
L/mol). It has an additional advantage over hCGn mAbs developed to
normal-pregnancy hCGn, in that it does not cross-react with hLH. Fig. 2
illustrates mAbs B151 and B604 binding with hCGn and related
molecules in a competitive liquid phase RIA where
[125I]hCGn was used as a tracer. These two antibodies
were determined to be the most specific combination for hCGn detection
in the IRMA format.
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Data obtained from the liquid phase competitive RIA suggest that nicking of normal-pregnancy hCG alters its structure so that epitopes common to intact hLH are exposed; however, this is not true for B151, which was developed to choriocarcinoma hCGn.
assay characterization
All antibodies, including B151, were checked in all possible
combinations that could provide the basis for construction of two-site
IRMAs. The best specificity for the normal-pregnancy hCGn was
demonstrated by an assay that used B151 as the capture antibody and
B604 for detection. The detection limit (least detectable dose, which
is defined as that concentration of hCGn corresponding to the zero
calibrator plus 2 SD) was <2 pmol/L. This assay was characterized by a
wide dynamic range (2–4000 pmol/L can be measured without the need for
dilution). The measured B151-B604 IRMA cross-reactivity with
normal-pregnancy hCG was 2.5%. Because hCGn is contaminated with 20%
non-nicked hCG, the true cross-reactivity probably lies close to 2%.
The cross-reactivity with hCGßn was 3.7% in the B151-B604 assay. For
all other checked analytes the cross-reactivity was <0.1%.
For hCG and hCGn, the intraassay coefficients of variation (CVs) were 5% and 12% for serum, and 6% and 7% for urine, respectively. The interassay CVs were 11% and 13% for serum, and 10% and 14% for urine, respectively.
recovery studies
hCGn recovery from hCG-free serum to which hCGn had been added
varied between 69% and 83%; hCGn from hCG-free urine to which hCGn
was added was 72%. The recovery in buffer was 78–94%, depending on
the hCGn concentration. The lower recoveries obtained in serum and
urine compared with buffer may reflect a matrix effect. The common
approach to diminishing this effect is sample dilution, which is not an
alternative available in this assay because of the low quantities of
hCGn present in specimens. As a consequence of the relatively low hCGn
concentrations compared with the non-nicked hCG concentrations we
observed in clinical specimens, determination of the analytical
recovery of hCGn in the presence of the non-nicked variant was an
essential component of assay validation. This experiment illustrated
that as the concentration of intact hCG in the sample increased, the
apparent concentrations of hCGn change dramatically (Fig. 3
A). In the case of pregnancy or choriocarcinoma samples with
very high concentrations of hCG, the 2–3% cross-reactivity of intact
hCG in the nicked assay can appear as a substantial amount of hCGn
because of the cross-reactivity with non-nicked hCG in the hCGn assay.
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When the cross-reactivity factor is taken into account, it is possible
to obtain reasonably accurate measurements of hCGn only over a limited
range of values for the both hCGn and non-nicked hCG (Fig. 3A
). For
example, if hCGn is in the 300–400 pmol/L range and the corresponding
values for non-nicked hCG do not exceed 1760 pmol/L, then a reasonably
accurate determination of hCGn can be made. If non-nicked hCG
concentrations are in the range of 17 600 pmol/L, however, then the
hCGn concentration needs to be >800 pmol/L to be measurable. At
non-nicked hCG >17 600 pmol/L, the interference from the non-nicked
hCG in the hCGn assay becomes too large to provide a useful estimation.
Unfortunately, in our examination of clinical specimens thus far,
values of hCGn have been much below those required for their accurate
estimation by our assay.
An approach to this problem has been the use of scavengers to complex
the interfering analyte and neutralize its immunological activity
(30)(31). Our data illustrate that the
scavenger itself, unless absolutely specific, can bind hCGn and
hence change its apparent concentration (Fig. 3B
). This observation
is also supported by a slightly different assay format, i.e., B108 was
the detection antibody. The influence of different concentrations of
B109 scavenger antibody on apparent concentrations of hCGn in this type
of hCGn assay is shown on Fig. 4
B. The scavenger application decreases the recovered amount of
hCGn. Conversely, the scavenger does not totally eliminate the
influence of hCG on this assay (Fig. 4A
).
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When B109 is used as a scavenger for hCG in samples that contain both
hCGn and hCG (as occurs in clinical specimens), the resulting
equilibrium is complex. The presence of one immobilized mAb on the
plate (B151) and the second mAb (B109) together with both hCG and hCGn
in solution at the same time produced ambiguous results (Fig. 3
).
Because neither antibody is absolutely specific for either analyte,
both nicked and non-nicked hCG are in binding equilibrium with both the
immobilized antibody and the liquid phase scavenger antibody, although
with very different affinities. Consequently, the values obtained from
this assay format are not reliable. The final results are shown on Fig. 3B
. The degree of interference also depends on the concentration of
these analytes.
evaluation of hCGn and intact hCG in serum
and urine in various forms of clinical pregnancy
The hCGn content of clinical samples was evaluated with the
B151-B604 hCGn-specific assay. It should be kept in mind that these
results are reliable only within the relative ranges of hCGn and
non-nicked hCG within which cross-reactivity is not a problem. Values
in this range were never attained in any of these clinical specimens,
however. Thus, when interpreting the values, one must keep in mind that
they reflect extremely small amounts of hCGn compared with the much
larger quantities of non-nicked hCG present and cannot be construed as
representing absolute quantities. In none of the clinical specimens we
examined did we discover the quantities of hCGn found by other
investigators using indirect assay methodologies.
Urinary hCG and hCGn throughout the normal pregnancy.
The
median concentration and range of non-nicked hCG and hCGn throughout
normal pregnancy is illustrated in Table 1
. Both hormones have been normalized to urinary creatinine. It
is striking to note that difference between the values for non-nicked
hCG and hCGn is greater than three orders of magnitude. This wide
difference in concentration persists throughout pregnancy. Previous
reports have indicated that the extent of nicking both varied widely in
individual pregnancies (9) and generally increased as
the pregnancies progressed (32).
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We did not observe this increase. Indeed, the ratio of the median
concentrations of hCG to the hCGn in urine actually increased from 44
in the first trimester to >650 in the third trimester, suggesting that
the fraction of hCGn actually declined as the pregnancies progressed
(Table 1
). The interpretation of these results is clouded, however,
because their accuracy is compromised by the interference of the large
excess of non-nicked hCG. Because overall hCG concentrations are lower
in the third trimester than in the first, the effect we are observing
may be a result of the diminished effect of non-nicked hCG in the
assay.
Urinary hCG and hCGn in early normal pregnancy, ectopic pregnancy,
and spontaneous abortion from IVF patient samples.
Table 2
illustrates the median range of values, normalized to
creatinine, for the above listed pregnancy types at 2–4 weeks after
embryo transfer. In all types of IVF pregnancies, hCGn was either
nondetectable or present at such low concentrations that cross-reaction
cannot be ruled out as its source.
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Trophoblast disease serum and urine.
We examined 17 serum and
28 urine samples collected posttreatment from patients with
choriocarcinoma to determine whether hCGn, which has been reported to
be more prevalent in this condition (33), would be a more
sensitive marker for disease recurrence. Because of limited amounts of
sample, all of these specimens were run at a 1:10 initial dilution.
This increased the limit of detectability to 7 pmol/L in serum and 8
pmol/L in urine (the least detectable doses for these assays were 0.7
and 0.8 pmol/L, respectively). There were only small amounts of
non-nicked hCG detected in the sera; hCGn was not detectable in any of
these specimens. In the urine, only 2 of 28 specimens had substantial
concentrations of hCG (587 and 18 715 pmol/L). Only the latter
positive specimen contained a detectable amount of hCGn, again at a
concentration (150 pmol/L) that could not preclude cross-reaction as
the source.
Matched blood and urine specimens in early clinically normal
pregnancy.
Nine matched blood and urine specimens collected at
5–6 weeks gestational age were evaluated for hCGn content (data
incorporated in Table 1
). In the blood, seven of nine samples had
detectable hCGn, ranging from 0.1% to 4.6% of the corresponding value
for intact, non-nicked hCG. However, although high values of hCG were
associated with increased values of hCGn, there was no significant
correlation between the two numbers. In the corresponding urine
samples, there were only three samples positive for hCGn; all three
corresponded to specimens in which the highest values for non-nicked
hCG were present.
Down syndrome and control normal urine for the same gestational
age.
Nine urine samples from Down syndrome patients were tested in
B109-B108 and B151-B604 assays and compared with 99 normal-pregnancy
samples matched for gestational age (Table 3
and Fig. 5
). Statistical analysis revealed that the distributions of hCG
and hCGn were highly skewed, even after extreme values were excluded.
To improve the distribution, log-transformation was used. There was
significant correlation between the gestational age (in weeks) and
log(hCG) and log(hCGn) among non-Down syndrome subjects
(r = -0.28, P = 0.006; and
r = -0.39, P = 0.0001, respectively)
and a correlation of a similar magnitude but lacking significance among
Down syndrome subjects (r = -0.39, P =
0.34; and r = -0.23, P = 0.55,
respectively); this is mainly attributable to the small sample size in
the Down syndrome group. The Fisher z-test revealed no
significant difference in the gestational age (in weeks) between the
two groups: for log(hCG), z = -0.17, P
= 0.865; and for log(hCGn), z = 0.25, P
= 0.802. To examine the difference of log(hCG) and log(hCGn) between
the two experimental groups, we used a generalized additive model,
which permits adjustment for the effect of gestational age through a
nonlinear smoothing function. Although the concentrations of both hCG
(P = 0.0057) and hCGn (P = 0.0009) were
significantly higher in the Down syndrome group than in the
normal-pregnancy group, it is impossible to state with certainty that
this change in hCGn represents a true increase or is just a reflection
of the increased amounts of non-nicked hCG.
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Because hCGn was expressed at such low concentrations relative to the intact molecule in other forms of pregnancy, extensive statistical analysis on pregnancy categories other than Down syndrome was not performed.
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Conclusions |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionConclusionsReferences |
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Nicking of normal-pregnancy hCG appears to expose hCG epitopes common to hLH. This effect was not observed in mAb B151 raised against choriocarcinoma-derived hCG (C5), which was 100% nicked.
It appears that only small quantities of hCGn are present in most clinical specimens. An assay of virtually perfect specificity is therefore required to accurately measure these concentrations. However, the use of scavenger antibodies to increase the specificity of our assay, although somewhat effective, also introduced other interferences into the measurement, again producing compromised results.
An alternative methodology, that of making a measurement with an assay that is indifferent to nicking ("total hCG"), subtracting from it the hCG concentration found in an assay which does not measure hCGn ("non-nicked hCG"), and assigning the value of the difference to hCGn is risky in the absence of an independent criterion for ascertaining the accuracy of the initial assumption.
It is well-documented that hCG exists in multiple isoforms that change throughout pregnancy. For the subtractive approach to be valid, the two assays chosen must have the same measuring characteristics vis a vis all of these isoforms except hCGn, a requirement that may not always be fulfilled and is extremely difficult, if not impossible, to demonstrate. We illustrated this observation recently in a study of hCG expression in early pregnancy and early pregnancy loss, where we showed that the difference between total hCG and non-nicked hCG does not represent hCGn but rather multiple hCG isoforms recognized differentially, at least in early pregnancy specimens, by the two hCG assays we used (34).
The main finding with respect to the application of our hCGn specific assay is that the values for hCGn in clinical specimens were too small to be measured reliably in the presence of large quantities of non-nicked hCG. We clearly failed to detect hCGn in the quantities described by other investigators using indirect assay techniques (12)(14). Our results, with an assay that probably has the best specificity practically achievable, remain unsatisfactory. Our findings clearly show that some additional separation technique must be applied to the clinical specimens before they can be assayed in our system. We have shown that an in situ separation, as exemplified by the scavenger approach, is not satisfactory. Unfortunately, the partial purification afforded by chromatography is not applicable to the analysis of anything but small numbers of samples.
Our results present a conundrum. The existence of hCGn has been established by several investigators, including ourselves, using isolation of hCGn from urine and sequence analysis (9)(10). Having unequivocally established its existence, however, we do not know its origin and are unable to measure it with our assay, except rarely in some patients with a malignancy in which there is no overwhelming amount of non-nicked hCG present to interfere.
However, the hCGn preparations that were characterized by primary structural analysis were all from nonsterile urine collections. It is possible that microbial growth in the urine produced additional protease activity. In fact, a recent report demonstrates that microbial action in serum is the origin of hCGn and hCGßn (30). Microbial proteases are also a likely source of nicking in the reference hCG preparations, such as CR 127, that are derived from Diosynth crude commercial hCG (10). The peptide bond cleavage found at ß44–45 is not usually found in urine isolated from individual healthy pregnant women (10)(11)(12)(29). This implies a bacterially generated protease activity (10)(11)(12), It is known that loop 2 of hCGß is highly sensitive to peptide cleavage because it is completely exposed to solvent (35) and is easily cleaved by trypsin (36) or elastase (10).
Another possibility might be that the assay for hCGn is subject to some matrix interference present in serum or urine. However, our recovery studies of hCGn added to serum or urine, although not quantitative, nevertheless demonstrate that authentic hCGn added to either serum or urine, is recoverable in our assay. In summary, using our newly developed direct immunometric assay for hCGn, we cannot confirm previously published reports of significant quantities of hCGn produced during normal pregnancies. We must, therefore, raise the question of whether the observed presence of peptide bond cleavages in the highly protease-sensitive loop 2 region are caused by microbial growth during the collection and processing of urine.
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Acknowledgments |
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Footnotes |
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1 6 IBM personal computer version of program distributed by P. Munson, Laboratory of Theoretical and Physical Biology, National Institute of Child Health and Human Development, Bethesda, MD 20892 in 1993.
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionConclusionsReferences |
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  C. M. Sturgeon, P. Berger, J.-M. Bidart, S. Birken, C. Burns, R. J. Norman, U.-H. Stenman, and on behalf of the IFCC Working Group on hCG Differences in Recognition of the 1st WHO International Reference Reagents for hCG-Related Isoforms by Diagnostic Immunoassays for Human Chorionic Gonadotropin Clin. Chem., August 1, 2009; 55(8): 1484 - 1491. [Abstract] [Full Text] [PDF]
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 S.F. de Medeiros and R.J. Norman Human choriogonadotrophin protein core and sugar branches heterogeneity: basic and clinical insights Hum. Reprod. Update, January 1, 2009; 15(1): 69 - 95. [Abstract] [Full Text] [PDF]
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 R. McChesney, A. J. Wilcox, J. F. O'Connor, C. R. Weinberg, D. D. Baird, J. P. Schlatterer, D.R. McConnaughey, S. Birken, and R. E. Canfield Intact HCG, free HCG {beta} subunit and HCG {beta} core fragment: longitudinal patterns in urine during early pregnancy Hum. Reprod., April 1, 2005; 20(4): 928 - 935. [Abstract] [Full Text] [PDF]
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 M. Maccarrone, T. Bisogno, H. Valensise, N. Lazzarin, F. Fezza, C. Manna, V. Di Marzo, and A. Finazzi-Agro Low fatty acid amide hydrolase and high anandamide levels are associated with failure to achieve an ongoing pregnancy after IVF and embryo transfer Mol. Hum. Reprod., February 1, 2002; 8(2): 188 - 195. [Abstract] [Full Text] [PDF]
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 M. Maccarrone, H. Valensise, M. Bari, N. Lazzarin, C. Romanini, and A. Finazzi-Agro Progesterone Up-Regulates Anandamide Hydrolase in Human Lymphocytes: Role of Cytokines and Implications for Fertility J. Immunol., June 15, 2001; 166(12): 7183 - 7189. [Abstract] [Full Text] [PDF]
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P. Mock, G. Kovalevskaya, J. F. O'Connor, and A. Campana Choriocarcinoma-like human chorionic gonadotrophin (HCG) and HCG bioactivity during the first trimester of pregnancy Hum. Reprod., October 1, 2000; 15(10): 2209 - 2214. [Abstract] [Full Text] [PDF]
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| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
, hCG; 
, hCGn; 
, hCGß; 
, hCGßn; 
,
hCGßcf; 
, hLH.
, hCGn + 1.76 nmol/L hCG; 
, hCGn + 17.6 nmol/L hCG;
, hCGn + 176 nmol/L hCG; 
, 0 mg/L; 
, 50 mg/L; 
, 100 mg/L.