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Radloimmunoassay of intestinal goblet cell mucin
ANALYTICAL
83, 657-665
BIOCHEMISTRY
Radioimmunoassay
(1977)
of Intestinal
Goblet Cell Mucin
J. F. FORSTNER, F. OFOSU, G. G. FORSTNER Research
Institute,
The Hospital for Toronto, Ontario,
Received
A double-antibody intestinal goblet cell
March
Sick Children, 555 University MSG IX8 Canada
21, 1977;
radioimmunoassay mucin. Labeling
accepted
July
21,
Avenue,
1977
was developed for the measurement of rat of the mucin was achieved by mild periodate
oxidation and sodium boro[W]hydride reduction using the technique of Van Lenten and Ashwell [(1971) J. Biol. Chem. 246, 1889-18941. Tracer amounts (0.5 ng) of labeled mucin were incubated with specific rabbit antibody in the presence secretions. 1: 10, v/v)
of unlabeled mucin standards or extracts of intestinal Normal rabbit serum and sheep antiserum to rabbit were used as a double-antibody system to precipitate
tissues or their IgG (in a ratio of the mucin-anti-
body complex. The radioimmunoassay proved to be suitable for measuring 3-80 ng of mucin in intestinal homogenates (soluble fraction). acid-precipitable glycoprotein (after solubilization and neutralization), and purer mucin fractions prepared by precipitation of homogenate supernatant solutions with cetyltrimethylammonium a mucin has been
bromide. To our measured by means
knowledge this is the first of a radioimmunoassay.
study
in which
In this paper we describe the development of a double-antibody radioimmunoassay for goblet ceil mucin (GCM) of rat small intestine. The impetus for developing this assay arose from the need to detect small amounts of mucin in intestinal tissues and their secretions during investigations of mucin synthesis and secretion. Studies of mucus production and secretion in a variety of organs have relied upon labeling of giycoproteins using precursor radioactive sugars, followed by either E/M radioautography of tissue sections (2-7) or cell fractionation and extraction techniques applied to tissue slices or organ cultures (7- 12). In some cases mucus secretion has been assessed by measuring carbohydrate “markers” of mucin, such as nondialyzab~e sulfate and fucose (13). But cellular giycoproteins other than mucins also incorporate radioactive precursors (12) and present themselves in nondiaiyzabie material. Since these contaminants are not easily removed and the yields of mucin resulting from various types of extraction are low, quantitation and identification of mucin products are often unreliable. An additional frustration arises from the characteristic polydispersity of size, charge, and composition of mucins (14), which makes their isolation in a homogeneous state extremely difficult. 657 Copyright b 1977 by Academic Press, Inc. AIt rights of reproduction in any form resewed.
ISSN oDo3-2697
658
FORSTNER,
OFOSU.
AND
FORSTNER
There is a need, therefore, to have an independent and quantitative technique for measuring low concentrations (micro- or nanograms) of mucin in tissues and secretions, without the requirement for extensive extraction or purification. In response to this need we have used the antigenic properties of intestinal GCM to construct a suitable assay in which mucin is identified and quantitated on the basis of its ability to bind to specific antibody. GCM from rat small intestine was previously isolated and characterized in this laboratory (15,16). An antibody developed in the rabbit was shown to give a single precipitin line in double immunodiffusion against purified GCM and the 30,OOOg supernatant fractions of intestinal homogenates (15). There was no reaction between the GCM antigen and rabbit antiserum to human IgA, secretory component, or a2 macroglobulin. There was also no cross-reaction with rat serum, rat intestinal brush-border membranes, or homogenates of normal human colon. Thus, the antibody appeared to be specific for rat GCM. As described below, the immunoassay we developed for GCM was tested on three different extracts prepared from rat intestinal slices after incubation in a modified Kreb’s buffer (11). The immunoassay was able to measure accurately nanogram concentrations of mucin in all three extracts. No crossreactions were observed with homogenates of several other rat tissues, rat milk or serum, canine trachea1 mucus, or human intestinal GCM. METHODS Preparation
0~ Antigen,
Labeled Antigen,
and Antibody
Preparations of goblet cell mucin (GCM) from rat small intestine and its antiserum from rabbits have been described in earlier reports (15,16). An immunoglobulin-rich fraction was prepared by precipitation of the serum with 40% ammonium sulfate. The pellet was washed twice, solubilized in, and dialyzed exhaustively against, 0.01 M Na,HP04NaH,PO* (2 H20) in 0.145 M NaCl, pH 7.0 (ie., phosphate-buffered saline, PBS). The final protein concentration was 20 mg/ml. This fraction was used as the antibody to GCM in all experiments. The technique of labeling GCM with tritiated borohydride was identical to that described for other glycoproteins by Van Lenten and Ashwell (1). The sodium boro[3H]hydride (Amersham/Searle) (100 mCi) had a specific activity of 156 mCi/mg. It was added to 116 pg (protein) of GCM which had been oxidized by 0.4 mM sodium periodate. After labeling, the [3H]GCM mixture was thoroughly dialyzed against distilled water and was applied to a Sepharose 4B column (1 x 40 cm), and 13H]GCM was eluted in the void volume with 0.1 M phosphate buffer, pH 7.0. The chromatographic characteristics of 13H]GCM were identical
ASSAY
OF INTESTINAL
GOBLET
CELL
MUCIN
659
to those of nonlabeled intact GCM described earlier (16). Specific activity of [3H]GCM was 2.7 x 10’ dpm/pg of GCM protein. Single-Antibody
Precipitution
of GCM
In preliminary experiments we established that the rabbit antibody to GCM was capable of precipitating intact or unmodified GCM as follows: GCM was doubly labeled in viva by injecting rats intraperitoneally with 10 $Zi of D-[‘“Clglucosamine and 30 &i of [3H]threonine (New England Nuclear). After 3 hr the animals were sacrificed and small intestinal GCM was isolated as described before (15,16). The doubly labeled purified GCM (containing 10 pg of protein, 600 dpm of ‘“C, and 1200 dpm of “H) was then mixed with increasing volumes of rabbit anti-GCM antibody and was made up to a final volume of I ml with 0.15 M NaCI. The solutions were incubated at 37°C for 1 hr, then overnight at 4°C. After centrifugation for 20 min at 2000 rpm in a refrigerated International centrifuge, the supernatant was removed, and the pellet was washed once and then solubilized in 1% SDS at 65°C for 1 hr. Radioactivity was determined by counting the pellet, wash, and supernatant fractions separately in 10 ml of Aquasol (New England Nuclear) scintillation fluid in an lsocap (Searle) scintillation counter. Binding of radioactive antigen to antibody (ie., pellet radioactivity) was expressed for each isotope as the percentage of the total radioactivity. Background binding (ie., binding in the absence of antibody) was 2-5% and was subtracted from all binding values obtained using antibody. Double-Antibody
Rudioimmunoasscrg
Standard curves were derived from assays in which O-200 ng (protein) of unlabeled pure GCM was incubated for 30 min at 37°C with normal (nonimmune) rabbit serum (96 ~1 of a 1:lO dilution), rabbit antibody to GCM (40 ~1 of a I:100 dilution), and PBS. Labeled [3H]GCM (14,000 dpm, approximately 0.5 ng of protein) was added to each tube, and incubations were continued for 30 min. Sheep anti-rabbit IgG antiserum (100 ~1) (Joe DeRose and Associates, Toronto) was added to give a final total incubation volume of 0.5 ml. After standing for 48 hr at 4”C, samples were centrifuged at 2000 rpm for 15 min at 4”C, and radioactivity was counted in the supernatant, wash. and pellet to determine the percentage of [“H]GCM bound. Nonspecific binding in control incubations (ie., containing no antibody) ranged from 5 to 12%. Preparation
of Tissue Extructs for Rudioimmunoussuy
Small intestinal slices of rats (18 slices in 6 ml of buffer) were incubated for 3 hr at 37°C as described earlier (11). Tissues were separated
660
FORSTNER.
OFOSU.
AND
FORSTNER
from the media and were washed in 0.15 M NaCl, and three types of extracts were prepared. Supernfltant fiuctions. Slices (containing approximately 100 mg of protein) were homogenized in a Waring blender in 20 ml of 5 mM EDTA brought to pH 7.0 with NaOH, and the homogenate was centrifuged at 30,OOOg at 4°C for 30 min. The supernatant was dialyzed for 48 hr against four changes of distilled water and was lyophilized to a final volume of 5 ml. Samples of the incubation media were not homogenized, but otherwise were treated the same as tissue homogenates. The resulting soluble extracts are referred to as “supernatant fractions” of tissue or media. CTAB fructions. More purified fractions of GCM were prepared by subjecting tissue or medium supernatant fractions to precipitation with 0.1% cetyltrimethylammonium bromide (CTAB). After standing overnight at 4”C, the CTAB mixtures were centrifuged at 30,OOOg for 15 min, and the pellet was extracted by mixing it continuously with 4 ml of 0. I.5 M NaCI at room temperature for several hours. After recentrifugation, the soluble saline extract was used in radioimmunoassays. The saline extracts are referred to later as “CTAB fractions” of tissue or media. Acid pellet fructions. Aliquots (2 ml) of homogenates of either intestinal tissue or medium were precipitated by cold 10% trichloroacetic-1% phosphotungstic acid. After standing for 24 hr at 4°C the pellet was solubilized by mixing it with 1 ml of PBS and adding 0.4 N NaOH to bring it to pH 7.0-7.5. After appropriate dilution the solubilized pellet was used in the radioimmunoassay. Although these fractions contain all the proteins and glycoproteins of the tissues or medium, respectively, they are free of acid-soluble constituents, including many peptides and intermediates involved in glycoprotein biosynthesis (1 I). Cross-Reactivity of Other Tissue Extructs in the Radioimmunoassuy Supernatant fractions or acid pellet fractions of several rat tissues, including brain, heart, trachea, lung, liver, spleen, muscle, and kidney, were made in the same way as described above for the intestinal slices. In addition, normal rat serum, purified human small intestinal GCM (17), and canine tracheal mucus (kindly supplied by Dr. Alan Baker; Smith, Kline and French Laboratories, Philadelphia) were tested in the radioimmunoassay. Rat milk, as a source of secretory IgA, was kindly supplied for testing by Dr. Brian Underdown, Department of Medicine, University of Toronto, Canada. The concentration of protein in all the test samples ranged from 0. I to 200 pg. RESULTS Intact pure GCM (labeled with precursor [I-“C]glucosamine and 13H]threonine) was incubated with increasing amounts of rabbit antibody
ASSAYOFlNTESTlNALGOBLETCELLMUCiN
661
as described in Methods. A maximum of 80% of both labels was precipitated with 0.6 ml of antibody (not shown). The identical precipitation of carbohydrate and protein labels indicated that the antibody was specific for the mucin and not for some minor contaminant in the preparation. To reduce the amount of antigen required and to increase its’ specific radioactivity to values suitable for use as a tracer in a radioimmunoassay, the GCM was labeled chemically (see Methods) using periodate and sodium boro[3H]hydride of high specific activity. Although this technique results in minor modifications of sialic acid (and possibly fucose residues) (I ,18), the resulting labeled GCM had the same remaining sugar and amino acid composition as did the intact GCM, behaved in a fashion identical to intact GCM in double immunodiffusion against the antibody to GCM, and cochromatographed with intact GCM on Sepharose 4B columns (not shown). The two GCM preparations were thus very similar. The labeled GCM had a specific activity of approximately 2.7 x 10’ dpm/pg of protein and, therefore, was deemed suitable for use in radioimmunoassays. A double-antibody system was developed using sheep anti-rabbit IgG antiserum and carrier nonimmune (normal) rabbit serum as outlined in Methods. The optimum ratio of rabbit serum (GCM antibody plus normal rabbit serum) to sheep anti-rabbit IgG was found to be 1:lO (v/v). In all subsequent experiments using the double-antibody technique, therefore, this ratio was used, and corrections were made for nonspecific binding (usually IO- 12%). Immunoprecipitation of [3H]GCM was carried out in the absence of unlabeled GCM to establish the volume of rabbit antibody required for subsequent immunoassay of nanogram quantities of GCM. Figure 1 shows that the maximum precipitation of label was 60%. It did not increase with the addition of higher volumes of either GCM antibody or anti-IgG antiserum. Since maximum precipitation of unmodified GCM by antibody had been found earlier to reach 80%, it was important to establish that, despite the apparent destruction of 20% of the binding potential of GCM, the labeled antigen was still suitable for use as a tracer in radioimmunoassay. To this end a standard curve (Fig. 2) was constructed by plotting percentage bound from incubations containing labeled GCM (14,000 dpm), GCM antibody (40 ~1 of a 1:lOO dilution, ie., enough to produce 40% binding), nonimmune rabbit serum (96 ~1 of 1:lO dilution), sheep antirabbit IgG antiserum (100 PI), and increasing amounts of non-radioactively labeled pure GCM. Two different batches of anti-GCM antibody diluted to the same final protein concentration (20 mg/ml) gave almost identical standard curves. The lower limit of reliable detection of GCM antigen was about 2 ng of protein. and the reliable range of binding was
662
FORSTNER.
OFOSU.
AND
0
I. lmmunoprecipitation
160
100 ANTIBODY
FIG.
FORSTNER
of [“H]GCM
0:lOO)
using
(ul)
the
double-antibody
technique.
lncuba-
tions of [3H]GCM (14,000 dpm) and rabbit sera were performed as described in Methods. Sheep anti-rabbit I& (100 ~1) was added to each incubation. Nonspecific binding (7- 10%) was subtracted in each case. Values represent the mean (? total range) obtained from four separate
incubations.
from 40 to 10%. The intra-assay variation for each value of percentage bound was &2.5%, and the interassay variation was 25%. Confirmation that the [3H]GCM and unlabeled GCM were bound with the same affinity by the antibody is provided by the slope of the standard curve. That is. with each doubling dose of unlabeled antigen added, the displacement of radioactive antigen from the antibody remains constant (4%). Eventually the labeled antigen is displaced completely by nonlabeled antigen. These findings indicate that the affinity of the antibody is identical for both labeled and unlabeled antigen (19), thereby validating the use of [“H]GCM in the assay.
UNLABELED
FIG. 2. Double-antibody radioimmunoassay Unlabeled GCM (0- 150 ng of protein) was antibody (40 as described curves binding
f
~1). in
MUCIN
of rat incubated
normal rabbit serum (96 ~1). Methods. Values represent
standard deviations. with no unlabeled GCM
(ng
protein)
intestinal goblet with [3H]GCM
PBS, and the mean
The highest value added. Semilogarithmic
on
cell mucin (GCM). (14,000 dpm), GCM
sheep anti-rabbit IgG of 16 independent
the plot.
y
axis
represents
(100 ~1) standard specific
ASSAY
OF
INTESTINAL
GOBLET
intes;gnal
frgtions
2 unlabeled
3. Radioimmunoassay supernatant,
I:10
300
'
0
FIG. (O---O):
663
MUClN
(Ul 1 100
1
CELL
of GCM dilution
muan
* I IO 20 30 (np protein)
in intestinal (O---O);
fractions. Acid pellet. I:100 dilution and CTAB. no dilution (A---A)
fractions were prepared as described in Methods, from rat intestinal slices and media after 3 hr of incubation at 37°C. Increasing volumes of each fraction were used in the immunoassay instead of purified GCM standards. A standard curve (solid line) is included in the figure
to show
that displacement
by the cruder
intestinal
fractions
followed
a parallel
course.
The K,,,,,,. of the antibody was calculated as the reciprocal of the unlabeled antigen concentration (20 ng) at 20% bound (ie., at half of the total binding in the absence of unlabeled GCM). Since GCM has a molecular weight of 2 x IO” and contains 12% protein by weight (l6), the K,,,,,,., is calculated to be 5 x 10” liters/mol. Several mucin extracts derived from rat small intestine were used in the radioimmunoassay in order to confirm that they behaved in a manner similar to that of purified intact GCM. After incubation of intestinal slices for 3 hr at 37°C extracts of tissues and media were prepared as described under Methods. Acid pellet fractions of tissues or media represent crude extracts of GCM from which only acid-soluble constituents have been removed. Supernatant fractions represent highspeed soluble components from which insoluble mucin and nuclear and cellular particulate material have been removed. CTAB fractions represent more purified soluble mucin extracts prepared from supernatant fractions. Figure 3 shows that all three fractions used in appropriate volumes gave parallel displacement of radioactive GCM from its antiserum, indicating that the antigenically active compounds in the fractions are structurally and immunologically similar to the purified GCM used in the standard curve. The radioimmunoassay was useful, therefore, in detecting small amounts of intracellular or secreted GCM from intestinal slices. Quantitative recovery of purified standard GCM added in varying amounts to the cruder fractions was 100%. Using the radioimmunoassay data and
664
FORSTNER.
OFOSU.
AND
FORSTNER
the known dilutions and starting values of tissue protein, it was catcutated that the GCM protein of small intestinal homogenates (ie., the GCM in the acid pellet fractions) accounted for about 0% 1% by weight of the total tissue protein. No displacement occurred with samples of rat serum, rat milk. or supernatant or acid pellet fractions of homogenates of rat kidney. brain, heart, muscle, trachea, spleen, lung, and liver (containing 0. I-200 pg protein per assay). Purified human small intestinal GCM and canine trachea! mucus samples were similarly without effect. Thus, the radioimmunoassay appears to be specific for rat intestinal GCM. DISCUSSION The preceding experiments indicate that rat GCM can be measured in nanogram concentrations by a double-antibody radioimmunoassay using “H-labeled GCM in tracer amounts. To our knowledge this is the first time that a mucus gtycoprotein of any animal has been measured by an immunoassay technique. The assay is sensitive, precise, and easy to perform. It represents an advantage over other methods of measuring mucin because it provides specific data on relatively crude extracts of small amounts of tissue or incubation medium, obviating the need to purify the mucin before quantitation. A maximum of 80% of intact GCM was precipitated by specific antibody. whereas only about 60% of the “H-labeled antigen could be precipitated. even though the affinity of the antibody for the two preparations of antigen was the same. This discrepancy in binding is not fully understood. Possibly the labeling technique “destroyed” 20% of the antigen in some fashion, thus preventing its participation in the assay. Alternatively, technical factors such as the extreme dilution of antigen and antibody associated with the double-antibody assay may be responsible (19). They were not solved by adding more of the second antibody, because when this was done the background (nonspeci~c) binding became prohibitively high (>20%). Fortunately, reduced binding was not a serious problem since the standard curve using the [:‘H]GCM preparation proved to be extremely accurate in detecting GCM in intestinal fractions. Specificity of the radioimmunoassay for rat small intestinal mucin was tested by including rat serum, milk. several rat tissues, canine trachea! mucus, and human GCM in the assay. None of the samples displaced [“HIGCM from the GCM antibody, despite the fact that up to 200 pg (protein) of material was used. These findings help to confirm that the rabbit antibody was formed specifically in response to rat GCM and that the other organs tested do not contain cross-reactive materials. The radioimmunoassay of rat mucin has wide potential applications in the field of mucus synthesis, secretion, and tuminat degradation,
ASSAY
OF INTESTINAL
GOBLET
CELL
665
MUCtN
since it could be used as a means of measuring the effects of many pharmacologic and pathologic compounds, including drugs, carcinogens, and bacterial and dietary products. Calculation from our data suggests that the actual amount of rat mucosal tissue necessary for the determination of GCM concentration is not more than 2 mm2 (equivalent to a concentration of 3-4 mg of total tissue protein). Thus, the potential also exists for application of a similar mucin radioimmunoassay in human intestinal biopsy tissue. Since we have recently isolated human small intestinal CCM and have developed a rabbit anti-GCM antibody to it (17). it should now be possible to develop a radioimmunoassay for human GCM. ACKNOWLEDGMENTS The authors are grateful to Dr. B. Zimmerman. Department of Immunology. and Dr. Lilian Lee, Department of Biochemistry, The Hospital for Sick Children, Toronto, for their advice on the development of the radioimmunoas~y. Expert technical assistance was given by Mrs. C. Feng and Dr. R. Qureshi. Financial assistance was provided by the Canadian
Cystic
Fihrosis
Foundation
and the
Medical
Research
Council
of Canada.
REFERENCES I. 7. 3. 4.
Van Lenten. Neutra, M.. Neutra. M.. Bennett, C.
L.. and Ashwell. G. (1971) J. Biol. Chent. and Leblond. C. P. (1966) .I. Cell Biol. 30, and Leblond, t. P. I 1966) /. C&l Bid. 30, (19701 j. Cell Biol. 45, 668-673.
246, 1889-1894. 119-136. 137-150.
5. 6. 7.
Bennett. G.. Leblond. C. P., and Haddad, A. (1974) J. Cd/Bid. 60, 258-284. Sturgess, J., and Reid. L. (1972) E.rp. Mol. Putho/. 16, 362-368. MacDermott. R. P.. Donaldson, R. M.. and Trier, J. S. (1974) J. C/in.
Invest.
54, 545-554. 8. Ellis. 9. Boat,
D. B.. and Stahl, T. F., Kteinerman,
(1974)
Amer.
Rev.
G. H. (1973) ~;~~~~z~~f~z. J. 136, 837-844. J. I., Carlson, D. M.. Maloney, W. H..
Rrsp.
Dis.
and
L. W.
Matthews,
110, 428-441.
IO. Lawford, C. R.. and Schachter. H. (1967) Crlrurd. 1. Biochem. 45, 507-517. Il. Lukie. B. E.. and Forstner. C. G. (1972) Biochirn. Biophys. Ada 261, 353-364. 12. Forstner. G. G. (1970) .I. Bid. Chem. 245. 3584-3592. 13. Lopez-Vidriero. M. T., and Reid, L. f 1977) irr Modern Problems in Pediatrics, Mucus Secretions and Cystic Fibrosis, Proceedings of a Conference Kimberley, Ontario, October 74-27. 1976. S. Karger, Base1 (in press). 14. Creeth, J. M.. Ramakrishnan. B., Donald. A. S. R.. and Morgan. W. T.
Bioc~llrm. 1.5. Forstner.
J. 143,
IC9-
J.. Taichman.
16. Forstner, J. F.. I154- 1166. 17. Jabbal. I.. Kells.
19: at
J. (1974)
170. N..
Jabbal. D.
Vol. held
Kalnins. I..
I. C..
and Forstner.
V.. and Forstner. Forstner. G..
G. and
G. (1973)J.
G.
Forstner,
(1973)
Ccl//Sci.
Crrnrrd.
.I. (1976)
J. C~rrrrtf.
12,585-602. 51,
Biochem.
J. Bj~f~~~.
54,707-716. 18. Jabbal. I., Forstner, G., Forstner, 558-571. 19. Hunter, W. M. (1973) i/t Handbook 2nd ed..
Vol.
I, pp.
17-l
to 17-35,
J., and
Kells.
D.
I. C.
11975)
of Experimental Immunology, Blackwell Scientific, Oxford.
Ancrl. (Weir.
Biochem. D.
M.,
69, ed.).
83, 657-665
BIOCHEMISTRY
Radioimmunoassay
(1977)
of Intestinal
Goblet Cell Mucin
J. F. FORSTNER, F. OFOSU, G. G. FORSTNER Research
Institute,
The Hospital for Toronto, Ontario,
Received
A double-antibody intestinal goblet cell
March
Sick Children, 555 University MSG IX8 Canada
21, 1977;
radioimmunoassay mucin. Labeling
accepted
July
21,
Avenue,
1977
was developed for the measurement of rat of the mucin was achieved by mild periodate
oxidation and sodium boro[W]hydride reduction using the technique of Van Lenten and Ashwell [(1971) J. Biol. Chem. 246, 1889-18941. Tracer amounts (0.5 ng) of labeled mucin were incubated with specific rabbit antibody in the presence secretions. 1: 10, v/v)
of unlabeled mucin standards or extracts of intestinal Normal rabbit serum and sheep antiserum to rabbit were used as a double-antibody system to precipitate
tissues or their IgG (in a ratio of the mucin-anti-
body complex. The radioimmunoassay proved to be suitable for measuring 3-80 ng of mucin in intestinal homogenates (soluble fraction). acid-precipitable glycoprotein (after solubilization and neutralization), and purer mucin fractions prepared by precipitation of homogenate supernatant solutions with cetyltrimethylammonium a mucin has been
bromide. To our measured by means
knowledge this is the first of a radioimmunoassay.
study
in which
In this paper we describe the development of a double-antibody radioimmunoassay for goblet ceil mucin (GCM) of rat small intestine. The impetus for developing this assay arose from the need to detect small amounts of mucin in intestinal tissues and their secretions during investigations of mucin synthesis and secretion. Studies of mucus production and secretion in a variety of organs have relied upon labeling of giycoproteins using precursor radioactive sugars, followed by either E/M radioautography of tissue sections (2-7) or cell fractionation and extraction techniques applied to tissue slices or organ cultures (7- 12). In some cases mucus secretion has been assessed by measuring carbohydrate “markers” of mucin, such as nondialyzab~e sulfate and fucose (13). But cellular giycoproteins other than mucins also incorporate radioactive precursors (12) and present themselves in nondiaiyzabie material. Since these contaminants are not easily removed and the yields of mucin resulting from various types of extraction are low, quantitation and identification of mucin products are often unreliable. An additional frustration arises from the characteristic polydispersity of size, charge, and composition of mucins (14), which makes their isolation in a homogeneous state extremely difficult. 657 Copyright b 1977 by Academic Press, Inc. AIt rights of reproduction in any form resewed.
ISSN oDo3-2697
658
FORSTNER,
OFOSU.
AND
FORSTNER
There is a need, therefore, to have an independent and quantitative technique for measuring low concentrations (micro- or nanograms) of mucin in tissues and secretions, without the requirement for extensive extraction or purification. In response to this need we have used the antigenic properties of intestinal GCM to construct a suitable assay in which mucin is identified and quantitated on the basis of its ability to bind to specific antibody. GCM from rat small intestine was previously isolated and characterized in this laboratory (15,16). An antibody developed in the rabbit was shown to give a single precipitin line in double immunodiffusion against purified GCM and the 30,OOOg supernatant fractions of intestinal homogenates (15). There was no reaction between the GCM antigen and rabbit antiserum to human IgA, secretory component, or a2 macroglobulin. There was also no cross-reaction with rat serum, rat intestinal brush-border membranes, or homogenates of normal human colon. Thus, the antibody appeared to be specific for rat GCM. As described below, the immunoassay we developed for GCM was tested on three different extracts prepared from rat intestinal slices after incubation in a modified Kreb’s buffer (11). The immunoassay was able to measure accurately nanogram concentrations of mucin in all three extracts. No crossreactions were observed with homogenates of several other rat tissues, rat milk or serum, canine trachea1 mucus, or human intestinal GCM. METHODS Preparation
0~ Antigen,
Labeled Antigen,
and Antibody
Preparations of goblet cell mucin (GCM) from rat small intestine and its antiserum from rabbits have been described in earlier reports (15,16). An immunoglobulin-rich fraction was prepared by precipitation of the serum with 40% ammonium sulfate. The pellet was washed twice, solubilized in, and dialyzed exhaustively against, 0.01 M Na,HP04NaH,PO* (2 H20) in 0.145 M NaCl, pH 7.0 (ie., phosphate-buffered saline, PBS). The final protein concentration was 20 mg/ml. This fraction was used as the antibody to GCM in all experiments. The technique of labeling GCM with tritiated borohydride was identical to that described for other glycoproteins by Van Lenten and Ashwell (1). The sodium boro[3H]hydride (Amersham/Searle) (100 mCi) had a specific activity of 156 mCi/mg. It was added to 116 pg (protein) of GCM which had been oxidized by 0.4 mM sodium periodate. After labeling, the [3H]GCM mixture was thoroughly dialyzed against distilled water and was applied to a Sepharose 4B column (1 x 40 cm), and 13H]GCM was eluted in the void volume with 0.1 M phosphate buffer, pH 7.0. The chromatographic characteristics of 13H]GCM were identical
ASSAY
OF INTESTINAL
GOBLET
CELL
MUCIN
659
to those of nonlabeled intact GCM described earlier (16). Specific activity of [3H]GCM was 2.7 x 10’ dpm/pg of GCM protein. Single-Antibody
Precipitution
of GCM
In preliminary experiments we established that the rabbit antibody to GCM was capable of precipitating intact or unmodified GCM as follows: GCM was doubly labeled in viva by injecting rats intraperitoneally with 10 $Zi of D-[‘“Clglucosamine and 30 &i of [3H]threonine (New England Nuclear). After 3 hr the animals were sacrificed and small intestinal GCM was isolated as described before (15,16). The doubly labeled purified GCM (containing 10 pg of protein, 600 dpm of ‘“C, and 1200 dpm of “H) was then mixed with increasing volumes of rabbit anti-GCM antibody and was made up to a final volume of I ml with 0.15 M NaCI. The solutions were incubated at 37°C for 1 hr, then overnight at 4°C. After centrifugation for 20 min at 2000 rpm in a refrigerated International centrifuge, the supernatant was removed, and the pellet was washed once and then solubilized in 1% SDS at 65°C for 1 hr. Radioactivity was determined by counting the pellet, wash, and supernatant fractions separately in 10 ml of Aquasol (New England Nuclear) scintillation fluid in an lsocap (Searle) scintillation counter. Binding of radioactive antigen to antibody (ie., pellet radioactivity) was expressed for each isotope as the percentage of the total radioactivity. Background binding (ie., binding in the absence of antibody) was 2-5% and was subtracted from all binding values obtained using antibody. Double-Antibody
Rudioimmunoasscrg
Standard curves were derived from assays in which O-200 ng (protein) of unlabeled pure GCM was incubated for 30 min at 37°C with normal (nonimmune) rabbit serum (96 ~1 of a 1:lO dilution), rabbit antibody to GCM (40 ~1 of a I:100 dilution), and PBS. Labeled [3H]GCM (14,000 dpm, approximately 0.5 ng of protein) was added to each tube, and incubations were continued for 30 min. Sheep anti-rabbit IgG antiserum (100 ~1) (Joe DeRose and Associates, Toronto) was added to give a final total incubation volume of 0.5 ml. After standing for 48 hr at 4”C, samples were centrifuged at 2000 rpm for 15 min at 4”C, and radioactivity was counted in the supernatant, wash. and pellet to determine the percentage of [“H]GCM bound. Nonspecific binding in control incubations (ie., containing no antibody) ranged from 5 to 12%. Preparation
of Tissue Extructs for Rudioimmunoussuy
Small intestinal slices of rats (18 slices in 6 ml of buffer) were incubated for 3 hr at 37°C as described earlier (11). Tissues were separated
660
FORSTNER.
OFOSU.
AND
FORSTNER
from the media and were washed in 0.15 M NaCl, and three types of extracts were prepared. Supernfltant fiuctions. Slices (containing approximately 100 mg of protein) were homogenized in a Waring blender in 20 ml of 5 mM EDTA brought to pH 7.0 with NaOH, and the homogenate was centrifuged at 30,OOOg at 4°C for 30 min. The supernatant was dialyzed for 48 hr against four changes of distilled water and was lyophilized to a final volume of 5 ml. Samples of the incubation media were not homogenized, but otherwise were treated the same as tissue homogenates. The resulting soluble extracts are referred to as “supernatant fractions” of tissue or media. CTAB fructions. More purified fractions of GCM were prepared by subjecting tissue or medium supernatant fractions to precipitation with 0.1% cetyltrimethylammonium bromide (CTAB). After standing overnight at 4”C, the CTAB mixtures were centrifuged at 30,OOOg for 15 min, and the pellet was extracted by mixing it continuously with 4 ml of 0. I.5 M NaCI at room temperature for several hours. After recentrifugation, the soluble saline extract was used in radioimmunoassays. The saline extracts are referred to later as “CTAB fractions” of tissue or media. Acid pellet fructions. Aliquots (2 ml) of homogenates of either intestinal tissue or medium were precipitated by cold 10% trichloroacetic-1% phosphotungstic acid. After standing for 24 hr at 4°C the pellet was solubilized by mixing it with 1 ml of PBS and adding 0.4 N NaOH to bring it to pH 7.0-7.5. After appropriate dilution the solubilized pellet was used in the radioimmunoassay. Although these fractions contain all the proteins and glycoproteins of the tissues or medium, respectively, they are free of acid-soluble constituents, including many peptides and intermediates involved in glycoprotein biosynthesis (1 I). Cross-Reactivity of Other Tissue Extructs in the Radioimmunoassuy Supernatant fractions or acid pellet fractions of several rat tissues, including brain, heart, trachea, lung, liver, spleen, muscle, and kidney, were made in the same way as described above for the intestinal slices. In addition, normal rat serum, purified human small intestinal GCM (17), and canine tracheal mucus (kindly supplied by Dr. Alan Baker; Smith, Kline and French Laboratories, Philadelphia) were tested in the radioimmunoassay. Rat milk, as a source of secretory IgA, was kindly supplied for testing by Dr. Brian Underdown, Department of Medicine, University of Toronto, Canada. The concentration of protein in all the test samples ranged from 0. I to 200 pg. RESULTS Intact pure GCM (labeled with precursor [I-“C]glucosamine and 13H]threonine) was incubated with increasing amounts of rabbit antibody
ASSAYOFlNTESTlNALGOBLETCELLMUCiN
661
as described in Methods. A maximum of 80% of both labels was precipitated with 0.6 ml of antibody (not shown). The identical precipitation of carbohydrate and protein labels indicated that the antibody was specific for the mucin and not for some minor contaminant in the preparation. To reduce the amount of antigen required and to increase its’ specific radioactivity to values suitable for use as a tracer in a radioimmunoassay, the GCM was labeled chemically (see Methods) using periodate and sodium boro[3H]hydride of high specific activity. Although this technique results in minor modifications of sialic acid (and possibly fucose residues) (I ,18), the resulting labeled GCM had the same remaining sugar and amino acid composition as did the intact GCM, behaved in a fashion identical to intact GCM in double immunodiffusion against the antibody to GCM, and cochromatographed with intact GCM on Sepharose 4B columns (not shown). The two GCM preparations were thus very similar. The labeled GCM had a specific activity of approximately 2.7 x 10’ dpm/pg of protein and, therefore, was deemed suitable for use in radioimmunoassays. A double-antibody system was developed using sheep anti-rabbit IgG antiserum and carrier nonimmune (normal) rabbit serum as outlined in Methods. The optimum ratio of rabbit serum (GCM antibody plus normal rabbit serum) to sheep anti-rabbit IgG was found to be 1:lO (v/v). In all subsequent experiments using the double-antibody technique, therefore, this ratio was used, and corrections were made for nonspecific binding (usually IO- 12%). Immunoprecipitation of [3H]GCM was carried out in the absence of unlabeled GCM to establish the volume of rabbit antibody required for subsequent immunoassay of nanogram quantities of GCM. Figure 1 shows that the maximum precipitation of label was 60%. It did not increase with the addition of higher volumes of either GCM antibody or anti-IgG antiserum. Since maximum precipitation of unmodified GCM by antibody had been found earlier to reach 80%, it was important to establish that, despite the apparent destruction of 20% of the binding potential of GCM, the labeled antigen was still suitable for use as a tracer in radioimmunoassay. To this end a standard curve (Fig. 2) was constructed by plotting percentage bound from incubations containing labeled GCM (14,000 dpm), GCM antibody (40 ~1 of a 1:lOO dilution, ie., enough to produce 40% binding), nonimmune rabbit serum (96 ~1 of 1:lO dilution), sheep antirabbit IgG antiserum (100 PI), and increasing amounts of non-radioactively labeled pure GCM. Two different batches of anti-GCM antibody diluted to the same final protein concentration (20 mg/ml) gave almost identical standard curves. The lower limit of reliable detection of GCM antigen was about 2 ng of protein. and the reliable range of binding was
662
FORSTNER.
OFOSU.
AND
0
I. lmmunoprecipitation
160
100 ANTIBODY
FIG.
FORSTNER
of [“H]GCM
0:lOO)
using
(ul)
the
double-antibody
technique.
lncuba-
tions of [3H]GCM (14,000 dpm) and rabbit sera were performed as described in Methods. Sheep anti-rabbit I& (100 ~1) was added to each incubation. Nonspecific binding (7- 10%) was subtracted in each case. Values represent the mean (? total range) obtained from four separate
incubations.
from 40 to 10%. The intra-assay variation for each value of percentage bound was &2.5%, and the interassay variation was 25%. Confirmation that the [3H]GCM and unlabeled GCM were bound with the same affinity by the antibody is provided by the slope of the standard curve. That is. with each doubling dose of unlabeled antigen added, the displacement of radioactive antigen from the antibody remains constant (4%). Eventually the labeled antigen is displaced completely by nonlabeled antigen. These findings indicate that the affinity of the antibody is identical for both labeled and unlabeled antigen (19), thereby validating the use of [“H]GCM in the assay.
UNLABELED
FIG. 2. Double-antibody radioimmunoassay Unlabeled GCM (0- 150 ng of protein) was antibody (40 as described curves binding
f
~1). in
MUCIN
of rat incubated
normal rabbit serum (96 ~1). Methods. Values represent
standard deviations. with no unlabeled GCM
(ng
protein)
intestinal goblet with [3H]GCM
PBS, and the mean
The highest value added. Semilogarithmic
on
cell mucin (GCM). (14,000 dpm), GCM
sheep anti-rabbit IgG of 16 independent
the plot.
y
axis
represents
(100 ~1) standard specific
ASSAY
OF
INTESTINAL
GOBLET
intes;gnal
frgtions
2 unlabeled
3. Radioimmunoassay supernatant,
I:10
300
'
0
FIG. (O---O):
663
MUClN
(Ul 1 100
1
CELL
of GCM dilution
muan
* I IO 20 30 (np protein)
in intestinal (O---O);
fractions. Acid pellet. I:100 dilution and CTAB. no dilution (A---A)
fractions were prepared as described in Methods, from rat intestinal slices and media after 3 hr of incubation at 37°C. Increasing volumes of each fraction were used in the immunoassay instead of purified GCM standards. A standard curve (solid line) is included in the figure
to show
that displacement
by the cruder
intestinal
fractions
followed
a parallel
course.
The K,,,,,,. of the antibody was calculated as the reciprocal of the unlabeled antigen concentration (20 ng) at 20% bound (ie., at half of the total binding in the absence of unlabeled GCM). Since GCM has a molecular weight of 2 x IO” and contains 12% protein by weight (l6), the K,,,,,,., is calculated to be 5 x 10” liters/mol. Several mucin extracts derived from rat small intestine were used in the radioimmunoassay in order to confirm that they behaved in a manner similar to that of purified intact GCM. After incubation of intestinal slices for 3 hr at 37°C extracts of tissues and media were prepared as described under Methods. Acid pellet fractions of tissues or media represent crude extracts of GCM from which only acid-soluble constituents have been removed. Supernatant fractions represent highspeed soluble components from which insoluble mucin and nuclear and cellular particulate material have been removed. CTAB fractions represent more purified soluble mucin extracts prepared from supernatant fractions. Figure 3 shows that all three fractions used in appropriate volumes gave parallel displacement of radioactive GCM from its antiserum, indicating that the antigenically active compounds in the fractions are structurally and immunologically similar to the purified GCM used in the standard curve. The radioimmunoassay was useful, therefore, in detecting small amounts of intracellular or secreted GCM from intestinal slices. Quantitative recovery of purified standard GCM added in varying amounts to the cruder fractions was 100%. Using the radioimmunoassay data and
664
FORSTNER.
OFOSU.
AND
FORSTNER
the known dilutions and starting values of tissue protein, it was catcutated that the GCM protein of small intestinal homogenates (ie., the GCM in the acid pellet fractions) accounted for about 0% 1% by weight of the total tissue protein. No displacement occurred with samples of rat serum, rat milk. or supernatant or acid pellet fractions of homogenates of rat kidney. brain, heart, muscle, trachea, spleen, lung, and liver (containing 0. I-200 pg protein per assay). Purified human small intestinal GCM and canine trachea! mucus samples were similarly without effect. Thus, the radioimmunoassay appears to be specific for rat intestinal GCM. DISCUSSION The preceding experiments indicate that rat GCM can be measured in nanogram concentrations by a double-antibody radioimmunoassay using “H-labeled GCM in tracer amounts. To our knowledge this is the first time that a mucus gtycoprotein of any animal has been measured by an immunoassay technique. The assay is sensitive, precise, and easy to perform. It represents an advantage over other methods of measuring mucin because it provides specific data on relatively crude extracts of small amounts of tissue or incubation medium, obviating the need to purify the mucin before quantitation. A maximum of 80% of intact GCM was precipitated by specific antibody. whereas only about 60% of the “H-labeled antigen could be precipitated. even though the affinity of the antibody for the two preparations of antigen was the same. This discrepancy in binding is not fully understood. Possibly the labeling technique “destroyed” 20% of the antigen in some fashion, thus preventing its participation in the assay. Alternatively, technical factors such as the extreme dilution of antigen and antibody associated with the double-antibody assay may be responsible (19). They were not solved by adding more of the second antibody, because when this was done the background (nonspeci~c) binding became prohibitively high (>20%). Fortunately, reduced binding was not a serious problem since the standard curve using the [:‘H]GCM preparation proved to be extremely accurate in detecting GCM in intestinal fractions. Specificity of the radioimmunoassay for rat small intestinal mucin was tested by including rat serum, milk. several rat tissues, canine trachea! mucus, and human GCM in the assay. None of the samples displaced [“HIGCM from the GCM antibody, despite the fact that up to 200 pg (protein) of material was used. These findings help to confirm that the rabbit antibody was formed specifically in response to rat GCM and that the other organs tested do not contain cross-reactive materials. The radioimmunoassay of rat mucin has wide potential applications in the field of mucus synthesis, secretion, and tuminat degradation,
ASSAY
OF INTESTINAL
GOBLET
CELL
665
MUCtN
since it could be used as a means of measuring the effects of many pharmacologic and pathologic compounds, including drugs, carcinogens, and bacterial and dietary products. Calculation from our data suggests that the actual amount of rat mucosal tissue necessary for the determination of GCM concentration is not more than 2 mm2 (equivalent to a concentration of 3-4 mg of total tissue protein). Thus, the potential also exists for application of a similar mucin radioimmunoassay in human intestinal biopsy tissue. Since we have recently isolated human small intestinal CCM and have developed a rabbit anti-GCM antibody to it (17). it should now be possible to develop a radioimmunoassay for human GCM. ACKNOWLEDGMENTS The authors are grateful to Dr. B. Zimmerman. Department of Immunology. and Dr. Lilian Lee, Department of Biochemistry, The Hospital for Sick Children, Toronto, for their advice on the development of the radioimmunoas~y. Expert technical assistance was given by Mrs. C. Feng and Dr. R. Qureshi. Financial assistance was provided by the Canadian
Cystic
Fihrosis
Foundation
and the
Medical
Research
Council
of Canada.
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