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Thyroid Study Unit, Department of Medicine, University of Chicago, Chicago, Illinois 60637
Address all correspondence and requests for reprints to: Leslie J. DeGroot, M.D., Thyroid Study Unit, Mail Code 3090, University of Chicago, 5841 South Maryland Avenue, Chicago, Illinois 60637.
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Abstract |
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A 33 x 33 matrix solid-liquid mAb inhibition assay was carried out to probe the rCT molecule. Five different clusters of mAb were distinguished and interpreted as reflecting five distinct antigenic regions on the surface of the rCT molecule.
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Introduction |
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A high level of CT and heterogeneous forms are found in the plasma of patients with MTC. Elevated CT is occasionally found in patients with nonthyroid tumors (e.g. lung carcinoma) and even in patients with nonmalignant disease (e.g. chronic pulmonary disease and renal failure) (6). However, elevated circulating immunoreactive CT is always considered to be a marker for MTC.
The amino acid sequences of CTs from 8 species are known. Rat CT (rCT) shares 93.75% structural homology with hCT (4), with only 2 amino acid differences in their amino acid sequences. Because of the low mol wt of CT, it has been difficult to raise mAbs to CT of high quality. Only a few laboratories succeeded in producing mAbs to hCT, and no mAbs to rCT have been reported. To our knowledge, up until now all reported antibodies to rCT or hCT cross-react with hCT or rCT. There are no studies of the antigenic differences between rCT and hCT, and no studies in which their antigenic surfaces were mapped. The availability of more than 40 mAb to rCT prompted us to study in detail the antigenic difference between rCT and hCT, and the number and relative spatial distribution of antigenic epitopes on the surface of the rCT molecule. In the present work, we describe 1) an efficient immunization/fusion protocol, 2) characterization of the mAbs to rCT and a study of antigenic differences between rCT and hCT, and 3) mapping of the antigenic surface of the rCT molecule.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Media and cell lines
The cell line P3-NS1/1-Ag4–1 (P3) was provided by Dr. Jose
Quintans (University of Chicago, Chicago, IL). Cultures were maintained
in RPMI 1640 (Life Technologies, Grand Island, NY) and supplemented
with 20% FBS (Life Technologies), 100 U/ml penicillin G sodium (Life
Technologies), and 100 µg/ml streptomycin sulfate (Life
Technologies). Hybrids were selected in the same medium supplemented
with 1 x 10-3 M hypoxanthine, 4 x
10-7 M aminopterin, and 1.6 x
10-5 M thymidine (HAT; Life Technologies).
Immunization, hybridization, and production of the anti-rCT mAb
Female BALB/c mice (4 weeks old; Jackson Laboratory, Bar Harbor,
ME) were immunized with 10 µg rCT equivalent conjugate in a 1:1
emulsion with Freund’s complete adjuvant (Sigma Chemical Co., St.
Louis, MO) and 0.01 M PBS. A volume of 50 µl was injected
into hind footpads (day 0). Thirteen days later, a secondary
immunization was given with the same amount of KLH-rCT in Freund’s
incomplete adjuvant (Sigma). Twenty-seven days later, a third
immunization was given. Three days after the last immunization, spleen
and popliteal lymph nodes were removed, and single cell suspensions
were separately prepared. Lymphocytes and splenocytes were fused with
P3 myloma cells using the 50% polyethylene glycol (PEG; Life
Technologies) method at a ratio of 3 lymphocytes to 1 P3 cell (8, 9).
Fused cell suspension was distributed into 96-well plates (Costar,
Cambridge, MA) and incubated at 37 C in 5% CO2. Hybridomas
were selected with HAT-selective medium. Hybrid growth was routinely
checked in all wells. Supernatants were tested for antibodies to rCT
after 10–15 days using an enzyme-linked immunosorbent assay (ELISA)
method (see below). Positive hybridomas were routinely cloned at least
twice. Cloned hybridomas were injected ip into pristane
(2,6,10,14-tetramethylpentadecane, Sigma)-primed BALB/c mice. The
resulting ascites was harvested from each mouse and tested for the
presence of anti-rCT antibody.
Screening assays for anti-rCT antibody and determination of Ig
Chain class. Screening was routinely carried out by antibody
capture ELISA. To prepare the solid phase CT, a stock solution of rCT
was diluted to 2.0 µg/ml in PBS. One hundred microliters were added
to the wells of 96-well plates, and the plates were incubated for
24 h at 4 C. The supernatants were discarded, and the plates were
washed twice using PBS. Two hundred microliters of 1% BSA were added
to each well for 12 h at 4 C for blocking. Plates were washed five
times with distilled water and kept at 4 C.
For screening, 100 µl culture supernatant were added and incubated 45 min at RT. The supernatants was discarded, and 100 µl diluted horseradish peroxidase-conjugated goat antimouse IgG antiserum (Sigma) were added. After 45-min incubation at RT, the supernatants were discarded, and 100 µl developing solution (0.4 mg/ml o-phenylenediamine in 0.1 M citrate buffer, pH 4.5) were added and incubated for 15 min. After each step, the plates were washed five times with distilled water. The OD450 was measured in an ELISA reader.
Determination of the Ig chain class of mAbs was carried out using the same procedure as that described above, with modifications. After the first incubation, alkaline phosphatase-conjugated goat antimouse IgM, IgG, IgG1, IgG2a, IgG2b, or IgG3 (Fisher Scientific, Pittsburgh, PA) was added. P-Nitro-phenyl phosphate solution (1 mg/ml in Tris buffer, pH 9.8) served as developing solution, and the OD405 was measured.
Radioiodination of rCT and binding of mAbs with labeled rCT in
liquid phase
Lactoperoxidase (Sigma)-directed radioiodination was performed
at pH 5 in 0.1 M sodium acetate as previously described
(10). One microgram of rCT, 1 µg lactoperoxidase, and 0.5 mCi
sodium125I (ICN Pharmaceuticals, Irvine, CA) were added
sequentially. Two microliters of freshly prepared 0.03%
H2O2 (J. T. Baker Chemical Co., Phillipsburg,
NJ) were added to start the reaction. The reaction was terminated at 10
min by adding 200 µl 0.005 M NaHSO3 (pH 7.0).
The labeled rCT was separated from unreacted iodide by gel filtration.
The specific radioactivity of [125I]rCT was 850 ±
150 Ci/mmol, as calculated from the estimated recovery of rCT, the
purity of rCT, and the known incorporation of 125I.
The binding of mAbs with rCT in liquid phase was carried out using
125I-labeled rCT. One hundred microliters of mAb were
incubated with 
6 fmol (100 µl) labeled rCT overnight at 4 C. One
hundred microliters of 1% normal mouse serum were added as carrier
protein. The specific immunocomplex was precipitated by adding goat
antimouse IgG and PEG (mol wt, 6000; final concentration, 3%). The
sample was centrifuged at 4 C for 25 min, and radioactivity in the
pellet was determined.
Affinity of mAb
The dilution of mAb that half-maximally bound approximately 6
fmol [125I]rCT was determined for each mAb. The
concentration of mAb thus determined was used as a working solution.
One hundred microliters of working solution were incubated with 100
µl 125I-labeled rCT (6 fmol) and 100 µl of various
concentrations of unlabeled rCT solution for 24 h at 4 C. Normal
mouse serum, goat antimouse IgG Ab, and PEG (final concentration, 3%)
were added for precipitation. The specific immunocomplex was sedimented
at 3000 x g for 25 min at 4 C, supernatant was
discarded, and radioactivity in the pellet was determined. The results
were analyzed by Scatchard plot to determined binding affinity.
Cross-reaction with hCT
Cross-reaction of mAbs with hCT was studied using solid phase
hCT. The preparation of solid phase hCT was the same as that of solid
phase rCT. The same procedure and conditions were used as those in the
screening method.
CT immunohistochemistry
Ten 5-µm sections of each human MTC tissue were mounted onto
Superfrost Plus slides (Fisher Scientific), baked at 60 C for 1 h,
cleared in xylene, and hydrated through a descending alcohol series to
distilled water. For microwave antigen retrieval, the hydrated tissue
sections were placed in a coplin jar containing 0.01 M
citrate buffer, pH 7.0, inside a Samsung 1.5-ft3, 900-watt
microwave. The tissue sections were heated for 2 min on the high
setting, followed by 13 min at 20% power. The heated sections were
allowed to cool for 15 min and washed in running water. After
retrieval, endogenous peroxidase activity was blocked by treating the
sections with 3% hydrogen peroxide in methanol for 20 min. Two tissue
sections from each case were then incubated overnight at 4 C with mAbs
that cross-react with hCT. One slide from each case was treated in a
similar fashion using normal mouse serum Ig (Ventana Medical Systems,
Tucson, AZ) as the negative control. The immunohistochemical staining
was performed on a Ventana Gen System that uses an indirect strepavidin
biotin system conjugated with horseradish peroxidase for detecting the
immunocomplex and diaminobenzidine as substrate for localization. The
immunostained sections were counterstained with hematoxylin, dehydrated
through an ascending alcohol series, cleared, and coverslipped. A
positive reaction was indicated by the accumulation of a brown reaction
product within the cytoplasm of the cell.
Competition for binding of different antibodies
To investigate whether the determinants of the rCT molecule
recognized by mAbs differed, the binding of 125I-labeled
rCT on mAb-coated plates was measured with various concentrations of
the same or other mAbs. Solid phase mAb was prepared by incubating 50
µg of the mAb in coating buffer (10 mM PBS, pH 7.2) for
24 h at 4 C. The solid phase was then blocked with 1% BSA for
2 h at RT, washed, and incubated for 2 h at 37 C with an
125I-labeled rCT-second mAb complex that had been
preincubated overnight at 4 C, the solid phase mAb was washed, and the
bound radioactivity was determined in a 
-counter. The bound
radioactivity using the same solid phase mAb incubated with free
labeled rCT served as the binding control. The decreased binding of the
radioactive mAb-Ag complex to the solid phase mAb represents the degree
of epitopic proximity of the pair of antibodies.
Prediction of antigenic regions
Analysis of hydrophilicity and prediction of antigenic regions
from the primary structure of rCT were made by the Lasergene-Protean
program, biocomputing software for the Macintosh, which helps to
analyze and predict protein characteristics from primary sequence data
(DNASTAR, Madison, WI). The positive values are interpreted as
corresponding to the immunogenic regions.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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summarizes the results obtained from 4
separate fusions. Two fusions used lymphocytes from popliteal lymph
nodes, and 2 fusions used splenocytes. One hundred and ninety-one
culture supernatants were positive for anti-rCT mAb. Fifty-seven
monoclones that displayed significant binding at a high dilution of
supernatant in the ELISA system were selected. Fusions of popliteal
lymphocytes appeared to give a higher percentage of positive hybrids.
Forty-four of 57 stable clones came from the fusion of popliteal
lymphocytes, and only 13 specific clones came from the fusion of
splenocytes.
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summarizes some of the characteristics of the
mAbs. All of the mAbs were IgG type, 20 mAbs were of IgG1 subclass, 14
mAbs were of the IgG2a subclass, and 14 mAbs were of the IgG2b
subclass. None was of the IgG3 subclass.
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). We cannot calculate the affinity of some
mAbs using Scatchard plots of rCT binding, because these mAbs do not or
only slightly react with liquid phase 125I-labeled rCT.
Some mAbs show the same IgG type, similar Ka
value, and the same pattern of cross-reaction with hCT. It is possible
that they are the same mAb, because some stable hybridomas came from
same original positive well. Table 3 shows the binding
activity of mAbs with 125I-labeled rCT in liquid phase.
Some mAbs do not react or show slight binding activity with liquid
phase [125I]rCT (Table 3), indicating that a different
epitopic conformation exists in the rCT molecule in the liquid phase
compared to that in the solid phase. A similar phenomenon was reported
for mAbs to hCT (11).
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). The cross-reaction was also studied by immunohistochemistry
using human MTC samples. As shown in Fig. 1, the
selected mAbs show a strong positive cell staining of hMTC tissue.
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. Complete reciprocal inhibition was interpreted as
epitope identity or proximity, and absence of inhibition was
interpreted as interaction of the pair of mAbs with remote epitopes.
Partial inhibition was postulated as being due to binding of the mAb to
epitopes sufficiently close to each other to induce some steric
hindrance. It must be noted that a change in the structure of rCT
induced by mAb binding cannot be ruled out in the case of reciprocal
inhibition. Analysis of the results allowed us to distinguish five
clusters of mAb reactions, reflecting the presence of five antigenic
regions on the surface of the rCT molecule.
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. Only one
hydrophilic region was found on the molecule (Fig. 2A). It was
localized around residues 11–24. The antigenic regions around residues
1–4 and 14–22 could be distinguished on the molecule (Fig. 2B). The
predicted regions may not be consistent with the experimental data due
to the folding of the molecule or conformational change after
conjugation.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The binding capacity of the mAbs to rCT in solution was evaluated using 125I-labeled rCT. Several mAbs preferentially react with solid phase rCT, but do not react with liquid phase 125I-labeled rCT. One possibility for this phenomenon is that in our experiments, an epitope including lysine 18 was altered by the labeling procedure. Another possibility is that when the free rCT is immobilized to solid phase, the molecular conformation may be changed, and some new epitopes may be induced. It has been reported that screening by ELISA with an immobilized antigen selects mAbs that might not be able to bind the free form of the antigen (13, 14). The binding activity study also showed that less than 50% of 125I-labeled rCT molecules are immunoprecipitated by mAbs, suggesting significant heterogeneity of iodinated rCT molecules.
We found that different epitopes exist in rCT and hCT. This suggests that Leu16 and Ser26 participate in the formation of epitopes on the molecular surface of rCT. To our knowledge, this is the first report of different epitopes between rCT and hCT.
Several mAbs were used in immunohistochemical studies of human MTC samples. All selected cross-reactive mAbs showed strongly positive cell staining in human MTC samples. It is possible to use these cross-reactive mAbs to differentiate or confirm MTC in thyroid tumors (6).
To enumerate the epitopes on the rCT molecule, the mAbs were used as described in previous studies (15, 16) in liquid phase/solid phase mAb inhibition assays in a 33 x 33 matrix. This allowed us to systematically scan the antigenic surface of rCT. To avoid artifacts, rCT was in solution and not bound to solid phase, which could lead to nonrandom conformation and epitope masking. A two-step consecutive assay protocol was used. In the first incubation, a large amount of mAb was used to bind a limited amount of liquid phase 125I-labeled rCT to make sure that 125I-labeled rCT was completely captured by the liquid phase mAb. Although it is more appropriate to maintain the native structure of antigens for precise mapping, rCT has been labeled with 125I throughout this study. As stated above, after iodination with 125I, the epitopic conformation including lysine 18 may be changed. Furthermore, there are two cystines in the first seven-amino acid sequence of the N-terminal position of rCT. The two cystines naturally make a cyclic structure with a disulfide bridge. When we raised mAb to rCT, a KLH-rCT conjugate was used as immunogen. No cyclic structure exists in this conjugate. This may limit probing using the mAbs, because some mAb may preferentially react with rCT without a cyclic structure.
At least 5 distinct epitopic regions were identified on the 32-amino acid peptide. The epitope number is more than that predicted from the analysis of antigenic regions of rCT. This may be due to folding of the molecule or to differences between its 3-dimensional structure and the amino acid sequence (12). Antigenic mapping of human insulin (51 amino acids; mol wt, 6000) identified up to 18 epitopes (17), suggesting a similar phenomenon. The mAbs will allow study of the biological activity of different epitopes.
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Acknowledgments |
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Received September 23, 1996.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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