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Detection of fecal blood by colloidal gold agglutination using an anti-human hemoglobin monoclonal antibody
Jourt~al of Immunological Methods, 153(1992) 185-192
185
© 1992ElsevierSciencePublishersB.V. All rights reserved 11022-1759/92/$05.110
JIM 1164P.2
Detection of fecal blood by colloidal gold agglutination using an anti-human hemoglobin monoclonal antibody Masahito Nagata a n d T o r u Tana'.,:a Central Research Laboratory. Godo Shusei Co.. Ltd.. 250, Nakahara. Kamihongo, Matsudo, Chiba271. Japan
(Received I May It~l, revisedreceived8 April 1992,accepted 8 April ITS2)
Monoclonal antibodies to human hemoglobin were produced and a colloidal gold agglutination method has been developed for detection of fecal occult blood. Since hemoglobin is composed of the tetramer, a2/32, a single monoclonal antibody-labeled colloidal gold can agglutinate with hemoglobin. The lowest detectable hemoglobin concentration was 0.5 p.g/ml. A total of 785 fecal samples were determined using colloidal gold agglutination and compared with latex agglutination. The colloidal gold agglutination detected blood in 75 samples, whereas latex agglutination detected blood in 76 samples, and among them 70 were positive in both methods. Overall agreement between the two methods was 98%. Key words: Colloidalgold agglutination;Monoclonalantibody;Hemoglobin;Fecal bhn)d
Introduction Methods for detecting blood in feces have been developed for use in screening for gastrointestinal bleeding, especially colorectal cancer. Chemical methods using guaiac or orthotoluidine as an indicator frequently give false-positive results because pseudoperoxidase activity is not specific for human hemoglobin (lllingworth, 1965; Jaffee et al., 1974; Ostrow et al., 1974). There is a fluorometric method, which is specific for hemederived porphyrin, but it requires cumbersome sample preparation (Ahlquist et al., 1985). Immunochemical methods, however, are specific for human hemoglobin (Barrows et al., 1978; Kim et al., 1985). Especially, enzyme immunoassay (EIA) Correspondence to: M. Nagata, Central Research Laboratory, Godo Shusei Co., Ltd., 250, Nakahara, Kamihongo, Matsudo, Chiba 271, Japan.
(Turunen et al., 1984), hemagglutination (Saito et al., 1984) and latex agglutination (Takeshita ct al., 1985) are commercially available. However, EIA involves time-consuming steps, and both hemagglutination and latex agglutination require skillful technique for reliable determination. Colloidal gold agglutination is a kind of particle agglutination. When antibody-labeled colloidal gold agglutinates with an antigen, there is a color change from red to gray. Colloidal gold is generally used as an immunospecific probe for electron microscopy (Falk and Taylor, 1971) and immunoblotting (Moeremans et al., 1984), but rarely for agglutination. Antibody-labeled colloidal gold and antigen agglutination was first described by Horisberger and Rosset (1977) and was applied to a human chorionic gonadotrophin assay (Leuvering et al., 1981). In this paper, the application of colloidal gold agglutination to detection of fecal blood using
186
human hemoglobin-specific monoclonal antibody is reported. Since hemoglobin is composed of the tetramer, 02/32, a single monoclonal antibodylabeled colloidal gold can agglutinate with the antigen.
Materials and methods
Hemoglobins Human hemoglobin Ao was prepared from fresh hemolysate according to the method of Williams and Tsay (1975). The a- and /3-globins were prepared by chromatography of heme-split hemoglobin on a CM-cellulose column in 8 M urea (Bucci, 1981). Bovine, sheep, goat, rabbit, pig and horse hemoglobin were purchased from Sigma (USA). Chicken hemolysate was used as hemoglobin preparation.
Production of monoclonal antibodies Mouse hybridomas producing anti-human hemoglobin were produced by the method of Oi and Herzenberg (1980), using human hemoglobin as the immunogen. The hybridomas were expanded as ascites tumors in mice, and antibodies were purified from the ascitic fluid by Affi-Gel protein A (Bio-Rad) chromatography. Antibody production was examined by an enzyme-linked immunosorbent assay (ELISA) using human hemoglobin-coated plates.
ELISA Hemoglobins at 1 /~g/well were used to coat 96-well microtiter plates. Without blocking, the antigen-coated wells were incubated with antibody. The plates were washed with 0.05 M phosphate-buffered saline (PBS) containing 0.05% Tween 20, and the antibody bound to hemoglobin was examined with peroxidase-labeled goat antimouse IgG and the chromogenic substrate, 2,2azinobis(3-ethyl benzothiazoline-6-sulfonic acidkliammonium according to the manufacturer's instructions (Bio-Rad). The absorbance at 405 nm was measured by a microtiter plate reader.
lsotyping of monoclonal antibodies The isotype of the antibody was determined by EIA using rabbit antiserum specific for mouse
IgG1, IgG2a, lgG2b, IgG3 and lgM, according to the manufacturer's instructions (Bio-Rad).
SDS-polyacrylamide manoblotting
electrophoresis and im-
Hemoglobins were electrophoresed on SDSPAGE using a 13.5% polyacrylamide gel. The separated proteins were electrophoretically transferred onto a nitrocellulose membrane, blocked with 3% gelatin, then incubated with purified antibody. After washing, the antibodies bound to the membrane were detected using peroxidaselabeled goat anti-mouse IgG and the chromogenic substrate, 3,3'-diaminobenzidine.
Preparation of colloidal gold Colloidal gold .was prepared by the citrate method (Frens, 1973). In brief, 100 ml of 0rnl% chloroauric acid was boiled, then 0.8 ml of 1% sodium citrate was rapidly added. The solution was boiled for 10 rain after the color changed, cooled, then made up to 100 ml. The absorbance of colloids indicated 1.1 at 540 nm in a 1 cm light path. The mean diameter of the colloid was examined using a laser submicron particle sizer NICOMP 370 (Pacific Scientific, USA).
Preparation of monoclonal antibody-labeled colloidal gold Monoclonal antibody, previously concentrated to 2-3 mg/ml and dialysed against 5 mM NaCI, was added to 100 ml of colloidal gold adjusted with 0.2 M K,CO a, After stirring for 1-2 min, 5 ml of 1% carbowax 20 M was added (Geoghegan and Ackerman, 1977). The gold-antibody complex was centrifuged at 12,000 × g for 20 min. The pellet was suspended in 20 mi of PBS containing 0.05% carbowax 20 M and stored at 4°C. The absorbance of labeled colloids indicated 5.5 at 540 nm. Polyclonal anti-human hemoglobin rabbit lgG (Capped was also labeled by the same procedure.
Colloidal gold agglutination Agglutination was performed in 96-well flat bottom microtiter plates. 50 /~l of hemoglobin diluted with the sample buffer, PBS containing 0.1% NaN 3 and 6% polyethylene glycol 6000 (PEG 6000), were mixed with 90/~i of antibody-
187 labeled colloidal gold. After incubation for 30 min at room temperature, the absorbance was measured at dual wavelength (540 nm/655 nm) using a microtiter plate reader with water as the blank. In inhibition experiments using MgCI2, 50 /tl of a mixture of hemoglobin and MgCI2, diluted with 0.05 M Tris-HCI buffer (pH 7.4), was pre-incubated for 5 min and reacted with the colloidal gold.
lo ~
Hb (vylml)
~04F
Latex agglutination OC-Hemodia (Eiken Kagaku, Japan) was used according to manufacturer's instruction. Briefly, 10 mg of feces was suspended in 2 ml of 0.2 M ammonium buffer (pH 8.2), then 100/LI of fecal suspension and 50 /~l of rabbit anti-human hemoglobin antibody-coated latex were mixed and rotated on the slide for 3 rain.
Gel filtration chromatography Human hemoglobin, dialyzed against 0.1 M Tris-HCI buffer containing 1 M MgCI2 (TBMg), was chromatographed on a Sephacryl S-100 column (2.5 x 80 cm) in the same buffer. Fractions of 2.5 ml were collected, and the reactivity of each to HH6422-1abeled colloidal gold was determined. Samples were diluted ten-fold with 0.1 M Tris-HC! buffer containing 0.15 M NaCI (TBS) and TBMg or not diluted. As a control, chromatography in the absence of 1 M MgCI2 was performed using TBS instead of TBMg.
Results
Screening of monoclonai antibodies Six mice were immunized, and monoclonal antibodies were selected by ELISA using human hemoglobin-coated plate. All 40 monocional antibodies intensely reacted with human hemoglobin by ELISA, but only three did not react with bovine, sheep, goat, rabbit, pig, horse and chicken hemoglobin. These three antibodies were all lgGl as determined by subclass analysis.
Labeling of antibodies to colloidal gold Mean diameters of colloidal gold varied in the range of 50-60 nm, but agglutination of labeled colloids with antigen was not affected by the
80
6b
70 pH
lb
80
Fig. I. Effect of labeling pH on agglulinatlon. 5/~g of HH6422 were labeled with I ml of colloidal gold at various pH, and
labeledcolloidswerereactedwithhumanhemoglobin.
diameters within this range. Three highly specific antibodies were labeled with colloidal gold. One antibody, named HH6422" agglutinated with ! /~g/ml of human hemoglobin within I h, and the other two required 2 h to agglutinate with 10 izg/ml of antigen even when the labeling pH was varied. Reactivity with hemoglobin increased as the labeling pH was lowered, but the colloids did not disperse in the solution after centrifugation if the pH was too low. Fig. 1 shows the correlation of labeling pH and agglutinationwith hemoglobin in HH6422-1abeling. As shown in Fig. 2. antibody was saturated at 3 /zg/ml of colloids when HH6422 was labeled at pH 6.4. Based upon the above results, HH6422 was labeled at pH 6.4 using 4 p.g/ml of colloids. The colloids labeled with rabbit anti-human hemoglobin lgG failed to agglutinate with the antigen.
Specificity of HH6422 HH6422 was highly specific for human hemoglobin as confirmed by ELISA (Fig. 3) and immunoblotting (Fig. 4), the latter of which revealed that HH6422 recognized the hemoglobin B-chain (Fig. 4).
188
[ 1o r
1
Hb (ug/mJ)
2
3
4
5
6
7
8
9
10
A 08
NO6 ~
O5 I
~
W
w
w
m
~
~ l l ~
glIB
02 0
I0 20
"~o--c~--o---o i
0
i
2 3 4 5 Ar,tubody ( pg/ml co~lo~ds) Fig. 2. Effect of labeling antibody concentradnn on agglutination. HH6422 was labeled with colloidal gold at various antibody concentration at pH 6.4, and labeled colloids were reacted with hemoglobin.
Reaction of HH6422 antibody-labeled colloidal gold with hemoglobin HH6422-1abeled colloidal gold that reacted with hemoglobin rapidly changed from red to gray. Fig. 5 s h o w s t h e a b s o r b a n c e s p e c t r u m o f t h e r e a c t i o n reLy.turn a f t e r a 30 r a i n i n c u b a t i o n . F o r 9 6 w e l l p l a t e s , t h e c o l o r c h a n g e in t h e c o l l o i d s
Fig. 4. lmmunobloning of hemoglobins, i p.g of human hemoglobin (lane !), human ~-globin (lane 2), human ,8-globin (lane 3), cow (lane 4), sheep (lane 5), goat (lane 6), rabbit (lane 7), pig (lane 8), horse (lane 9) and chicken (lane 10) hemoglobins were electrophoresed. Gels were stained with Coomassie blue (A), and the protein bands were detected by immunoblotting using ltH6422 (B).
20
1 bg/mO
0,2 %' 0
400
0 0
0001
O01
01
10
lO
Anhbodv ( pg/ml ) Fig. 3. Species specificity of HH6422 by ELISA. ELISA was performed using HH6422 as the first antibody. Microtiter wells were co, ted with human (o) and other hemoglobins ( ~, ) including o~,v. sheep, goat. rabbit, pig. horse and chicken.
I 500
I 600
700
800
Wave length ( nm } Fig. 5. Absorbanc¢ spectra of the reaction mixture. 1 ml of two-fold diluted HH6422-labeled colloidal gold was incubated with 0.5 ml of various concentrations of hemoglobin for 30 min. The absorbance spectrum was measured at a 1 cm light path.
189
08' K ) O ~ C ~ O ' ~
,06
Hb ( pg/r'nl ) ~-O-, OO
/
,~04 O-~
.
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.
.
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.
60
80
100
120
O
024
Reaction Time ( rain )
]
4
6
63
250
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Hemoglobin ( pg/ml )
Fig. 6. Time course of colloidal gold agglutination. HH6422labeled colloidswere incubated with variousconcentrationsof
Fig. 7. Dose-respon~ cu,'~,efor colloidal gold agglutination. HH6422-1abeled colloidswere incubated with serial two-fold
human hemoglobin.
dilutions of human hemoglobin.
was visually determined by the naked eye or in a spectrophotometer using a 96 well plate reader. PEG 6000 enhanced agglutination, but an excess caused non-specific agglutination even in the absence of antigen, therefore, 2% was an appropriate concentration in the reaction mixture. Fig. 6 shows the time-course of agglutination of labeled colloids with hemoglobin. Agglutination occurred rapidly, reaching completion after
30 min. The absorbance levels remained relatively stable for at least 2 h. Fig. 7 shows a dose-response curve for agglutination. The absorbance linearly decreased with increasing hemoglobin concentration up to 1 /~g/ml (reddish gray-color), in the range of 1-100 /~g/ml, labeled colloids completely reacted with hemoglobin, and the absorbance decreased below 0.2 (gray color). The agglutination gradually
10
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08
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, lO
'
04
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O8
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06 o4
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g O2
04 02
0 0 o 60 70 80 90 1OO 80 9O 100 F{actlon Number Fraettor, Number Fig. 8. Gel filtration chromatography of hemoglobin on Sephacryl S-100 in the absence (,4) and presence ( B ) of I M MgCI z. Protein (.), agglutination without dilution ( ,a, • ), I / I 0 dilution in TBS ( o ) and TBMg (e) were measured.
o
60
7O
ceased at the concentration above 100 /~g/ml because of prozone phenomenon and the redcolor of hemoglobin, however, agglutination was recognized at up to 1-2 mg/ml. The lowest detectable concentration was 0.5 p.g/ml by the naked eye and 0.2/zg/ml by the plate reader.
06
Reaction with hemoglobin dimer Colloidal gold agglutination is based on the assumption that the hemoglobin molecule is composed of a tetramer which shows a bivalent antigen. The monovalent aft dimer, therefore should not agglutinate with labeled colloids. In order to confirm this assumption, hemoglobin was chromatographed on a column of Sephacryl S-100 in the presence of 1 M MgCI 2, which causes hemoglobin to completely dissociate into the dimer subunits (Kirshner and Tanford, 1964). As shown in Fig. 8A, the apparent molecular weight of hemoglobin in TBS was 48,000, which agglutinated with labeled colloids. In the presence of 1 M MgCI z, the hemoglobin peak shifted to the dimer, and the apparent molecular weight was 28,000, which did not agglutinate without dilution (Fig. 8B). However, agglutination readily occurred when fractions were diluted with TBS. On the other hand, eluted [~actions in the absence of 1 M MgCI 2 barely agglutinated when diluted with TBMg (Fig. 8A). In Fig. 8A, agglutination in the peak was weaker than around the [ ~ak due to the prozone phenomenon, because terl-fold dilutions were sufficiently agglutinated. Aaditionally, hemoglobin in 1 M MgCI 2 was dialyzed against TBS and gel-chromatographed. The elution profile and agglutination pattern were the same as that of hemoglobin not treated with 1 M MgCl 2 (data not shown).
Inhibition of agglutination by MgClz Agglutination, after preincubation for 5 min, was interfered by MgCI 2 in a dose-dependent manner, especially at concentrations above 0.2 M (Fig. 9). In order to determine whether this was due to inhibition of the antibody-antigen reaction, ELISA and another colloidal gold agglutination assay were performed in the presence of 1 M MgCl z. In ELISA, the first reaction between hemoglobin-coated wells and free HH6422 antibody was not affected by 1 M MgCl 2 (data not
~ 04 <
0
02
04
0.8
16 31
62 125
v "2_5 50
100
Hemoglobin (iJg/ml)
Fig. 9. Inhibition of colloidal gold agglutination by MgCI 2.
Hemoglobin and MgCI2 were preincubated for 5 rain and agglutinated with HH6422-1abeledcolloids.Concentrationsof MgCI2 in samples: I M (o), 0.5 M (<>),0.25 M (l:3), 0.12 M (v), 0.06 M(A), and 0 M (e). shown). Also, agglutination of anti-human albumin monoclonal antibody-labeled colloidal gold with human albumin was not inhibited by 1 M MgCl 2 (unpublished data). It appeared that inhibition of agglutination by MgCl 2 was due to dissociation of the tetramer into the dimer, because neither antibody-antigen reaction nor colloidal gold agglutination against the monomeric antigen was entirely inhibited by 1 M MgCl 2.
Applicatic'~ to fecal clinical samples In colloide.l gold agglutination, 50 mg of feces was suspended in 2.5 ml of sample buffer. The lowest detectable concentration of hemoglobin was 0.025 m g / g stool. The color derived from feces was so pale in this fecal content that the color did not affect to either absorbance or judgement by the naked eye. Initial absorbance of the reaction mixture in normal colored feces was 0.8-0.9, whereas absorbance in dark colored feces enough higher, did not exceed 1.0. Recovery studies using fecal suspensions revealed that the absorbance of reaction mixture, even in dark colored feces, decreased below 0.6 when the sample contained hemoglobin at levels above 0.5 p.g/ml. However, dark or red colored feces, such as tar
TABLE 1 COMPARISON OF COLLOIDAl, GOLD AGGLUTINATION AND LATEX AGGLUTINATIONIN FECAL SAMPLES The X2 test with the Yates correction was not significant. Coll. Gold / Latex + -
+ 70 6
5 704
75 710
76
709
785
feces and that containing a large amount of blood, often contained hemoglobin at levels above 2 mg/ml, which showed the prozone phenomenon. To avoid false-negative reactions due to this, smaller sample volumes were also assayed. A comparison with latex agglutination was performed in 785 fecal samples collected for clinical diagnosis. The lowest detectable concentration in latex agglutination was 0.04 m g / g stool. As summarized in Table I, colloidal gold agglutination gave 75 positive results whereas latex agglutination gave 76. Among them, 70 samples were positive in both assays, and overall agreement between the two methods was 98%.
Discussion Colloidal gold agglutination, which differs from other particle agglutination methods, can detect antigen by a color change. The reaction is single step, and positive tests are clearly distinguishable from negative tests by the color. Using 96 well titer plates, large numbers of samples can be determined within 60 min. This method is based on the assumption that hemoglobin is a tetramer. Gel chromatography in the presence of 1 M MgCI 2 suggested that the hemoglobin dimer did not agglutinate with HH6422-1abeled colloids. Forms of hemoglobin in feces are unknown. However, even if hemoglobin is in the dimer form in f6ces, it would readily associate into the tetramer when suspended in low salt solution at a neutral pH. It is possible that fecal hemoglobin is complexed with haptoglobin and does not react with HH6422-1abeled colloids. In fact, haptoglobin inhibited colloidal
gold agglutination (data not shown). However, the haptoglobin concentration in feces is probably negligible since blood levels are 200 times lower than that of hemoglobin. HH6422, a monoclonal antibody selected for colloidal gold agglutination, was highly specific for human hemoglobin. Human hemoglobinspecific monoclonal antibodies have been reported (Stamateyannopoulos et al., 1981; Krco et al., 1985). However, these monoclonal antibodies were not used for diagnostic hemoglobin determination. The sensitivity for human hemoglobin of colloidal gold and latex agglutination 'OC-Hemodia' is 0 . 5 / ~ g / m l and 0.1 / t g / m l respectively. However, the sensitivity per feces weight of colloidal gold was equal to that of latex agglutination (0.04 m g / g feces) by adjusting the sampling volumes. Good agreement between colloidal gold and latex agglutination has been obtained in fecal blood detection (Table !). The detection limits of chemical methods, such as guaiac tests, are as low as 1-3 m g / g feces (Barrows et al., 1978; Turunen et al., 1984; Saito et al., 1985). These tests are not specific for human hemoglobin, because they react with animal blood and many nonhemoglobin substances and there is interference by substances in stools such as ascorbic acid (lllingworth, 1965; Jaffe et a~.~ 1974; Ostrow e t a ; . , 1974). In fact, the guaiac test gave a higher positive rate ( P < 0.01), presenting that the 38 (40%) and 10 (11%) of 94 fecal samples were positive in guaiac test and colloidal gold agglutination, respectively (data not shown). This higher rate may be caused by using fecal specimens without restriction of foods and drugs. Concentrations of fecal hemoglobin from normal volunteers or cut-off values in immunochemical methods depend on sensitivity. These reported values are as follows: 0.3 mg/g of feces in radial immunodiffusion (Barrows et al., 1978), 0.1 m g / g of feces in immunoprecipitin (Kim et al., 1985), and 0.01-0.05 m g / g of feces in EIA (Turunen et al., 1984). In heme-derived Imrphyrin fluorometry fecal hemoglobin in samples from healthy volunteers were less than 2 m g / g of stool (Ahlquist et ah, 1985). This value is much higher than that of the negative tests in immunochemical methods. The hemoglobin concentra-
192 tion may decrease due to degradation by digestive enzymes and colonic flora during intestinal transit or fecal storage. However, porphyrin fluorometry may overestimate the concentration of hemoglobin in feces, because heine, degraded h e m e and heine-derived porphyrins are m e a s u r e d as hemoglobin. Moreover, the origins o f the porphyrins may not be limited to hemoglobin. Therefore, cut-off values for fecal blood detection should differ from a m o n g methods. The advantages o f colloidal gold agglutination are specificity for h u m a n hemoglobin removing dietary restrictions and ease of j u d g e m e n t without technical skill. This m e t h o d using 96 well plates and a plate reader is suitable for mass screening of colorectal cancer. F u r t h e r studies are required to clinically evaluate this method.
Acknowledgements We wish to thank Miss Yoko l n a m o r i for production o f monocional antibodies and Mr. Ryo t a r o Higashiyama, Sanritu Soubu Clinical Diagnosis Center, for supply of fecal specimens.
References Ahlquist. D.A., McGill, D.B., Schwartz, S.. Taylor, W.F. and Owen, R.A. (1985) Fecal blood levels in health and disease/A study using HemoQuant. New Engl. J. Mad. 312, 1422. Barrows, G.H., Burton, R.M., Jarren, D.D., Russell, G.G., Alford. M.D. and Songster, C.L. (1978) lmmunochemical detection of human blood in feces. Am. J. Clin. Pathol. 09, 342. Bucci, E. (1981) Preparation of isolated chains of human hemoglobin. Methods Enzymol. 76, 97. Faulk, W.P. and Taylor. G.M. (1971) An immunocolloid method for the electron microscope. Immun~hemistry 8, 1081. Frens, G. (1973) Controlled nucleation for the regulation on the particle size in monodisperse gold suspensions. Nature Phys. Sci. 241.20. Ge," ~gan, W.D. and Ackerman. G.D. (1977) Adsorption of horseradish peroxidase, ovomucoid and anti-immunogIobulin to colloidal gold for the ~ndirect detection of concanavalin A, whe[~ germ agglutinin and goat anti-human intmuooglobuiin G on cell surfaces at the electron microscopic level: A new method, theory and application. J. Hi~tochem. Cy~ochem. 25, 1187.
Horisberger, M. and Rosset, J. (1977) Colloidal gold, a useful marker for transmission and scanning electron microscopy. J. Histochem. Cytochem. 25, 295. Illingworth, D.G. (1965) Influence of diet on occult blood tests. Gut 6, 595. Jaffee, R.M., Kasten, B., Young, M.D. and MacLowry, N. (1974) False negative stool occult blood tests caused by ingestion of aseorbic acid (Vitamin C). Ann. Intern. Mad. 83, 824. Kim, Y.D., Nolan, J.M., Malkin, A., Barch, D. and Tomita, J.T. (1985) A qualitative agar gel immunoprecipitin (lP) test for detection of fecal occult human hemoglobin, Clin. Chim. Acta 152, 175. Kirshner, A.G. and Tanford, C. (1964) The dissociation of hemoglobin by inorganic salts. Biochemistry3, 291. Krco, C.J., Gorzynsky,T.J. and Beito, T. (1985) Characterization of a monoclonal antibody that recognizes a determinant unique to human haemoglobin /]-chain. J. Immunogenet. 12, 197. Leuvering, J.H.W., Thai, P.J.1t.M., Waart, M.V.D. and Schuurs, A.H.W.M. (1981) A sol particule agglutination assay for human chorioni'c gonadotrophin. J. ImmunoL Methods 45, 183. Moeremans, M,, Daneels, G., Dijck, A.V., Langanger, G. and May, J.D. (1984) Sensitive visualization of antigen-antibody reactions in dot and blot immune overlay assayswith immunogold and immunogold/silver staining. J. Immunol. Methods 74, 353. Oi, V.T. and Herzenberg, L.A. (1980) Immunoglobulin-produc~ng hybrid cell lines. In: B.B. Mishell and S.M. Shiigi (Eds.), Selected Methods in Cellular Immunology. W.H. Freeman, San Francisco, p. 351. Ostrow, J.D., Mulvaney, C.A., Hansel, J.R. and Rhodes, R.S. (1974) Sensitivity and reproducibility of chemical tests for fecal occult blood with an emphasis on false-positive reactions. Am. J. Dig. Dis. 18, 930. Saito, H., Tsutida, S., Kakizaki, R.. Fukusi, M., Sano, M., Aisawa, N., Munakata, A. and Yoshida, Y. (1984) An immunological fecal occult blood test for mass screening of colorectal cancer by reversed passive hemagglutination (RPHA). Nippon Syokakibyo Gakkaizassi 81, 2831 (in Japanese). Stamatoyannopoulos, G., Farquhar, M., Lindsley, D., Brice, M., Papayannopoulou, Th., Nute, P.E., Serjeant, G.R. and Lehamaun, H. (1981) M:,pping of antigenic sites on human haemoglobin by means of mouoclonal antibodies and haemoglobin variants. Lancet ii, 952. Takeshita, T., Horiguchi, J., Kinoshita, T., Kubota, Y., Chen. P.-C., Katsumata. S., Horimukai, F., Miyaoka. M., Matsumoto. E., Saito, 3". a~ld Ashizawa, S. (1985) Latex agglutination test for fecal occult blood. Daityokomonshi 38, 780 (in Japanese). Turunen, M.J., Liewendahl, K.. Partanen, P. and Adlercreutz, H. (1984) Immunologicaldetection of faecal occult blood in colorectal cancer. Br. J. Cancer 49, 141. Williams, R.C. and "rsay. K.-Y. (1975) A convenient chromatographic method for the preparation of human hemoglobin. Anal. Biochem. 54, 137.
185
© 1992ElsevierSciencePublishersB.V. All rights reserved 11022-1759/92/$05.110
JIM 1164P.2
Detection of fecal blood by colloidal gold agglutination using an anti-human hemoglobin monoclonal antibody Masahito Nagata a n d T o r u Tana'.,:a Central Research Laboratory. Godo Shusei Co.. Ltd.. 250, Nakahara. Kamihongo, Matsudo, Chiba271. Japan
(Received I May It~l, revisedreceived8 April 1992,accepted 8 April ITS2)
Monoclonal antibodies to human hemoglobin were produced and a colloidal gold agglutination method has been developed for detection of fecal occult blood. Since hemoglobin is composed of the tetramer, a2/32, a single monoclonal antibody-labeled colloidal gold can agglutinate with hemoglobin. The lowest detectable hemoglobin concentration was 0.5 p.g/ml. A total of 785 fecal samples were determined using colloidal gold agglutination and compared with latex agglutination. The colloidal gold agglutination detected blood in 75 samples, whereas latex agglutination detected blood in 76 samples, and among them 70 were positive in both methods. Overall agreement between the two methods was 98%. Key words: Colloidalgold agglutination;Monoclonalantibody;Hemoglobin;Fecal bhn)d
Introduction Methods for detecting blood in feces have been developed for use in screening for gastrointestinal bleeding, especially colorectal cancer. Chemical methods using guaiac or orthotoluidine as an indicator frequently give false-positive results because pseudoperoxidase activity is not specific for human hemoglobin (lllingworth, 1965; Jaffee et al., 1974; Ostrow et al., 1974). There is a fluorometric method, which is specific for hemederived porphyrin, but it requires cumbersome sample preparation (Ahlquist et al., 1985). Immunochemical methods, however, are specific for human hemoglobin (Barrows et al., 1978; Kim et al., 1985). Especially, enzyme immunoassay (EIA) Correspondence to: M. Nagata, Central Research Laboratory, Godo Shusei Co., Ltd., 250, Nakahara, Kamihongo, Matsudo, Chiba 271, Japan.
(Turunen et al., 1984), hemagglutination (Saito et al., 1984) and latex agglutination (Takeshita ct al., 1985) are commercially available. However, EIA involves time-consuming steps, and both hemagglutination and latex agglutination require skillful technique for reliable determination. Colloidal gold agglutination is a kind of particle agglutination. When antibody-labeled colloidal gold agglutinates with an antigen, there is a color change from red to gray. Colloidal gold is generally used as an immunospecific probe for electron microscopy (Falk and Taylor, 1971) and immunoblotting (Moeremans et al., 1984), but rarely for agglutination. Antibody-labeled colloidal gold and antigen agglutination was first described by Horisberger and Rosset (1977) and was applied to a human chorionic gonadotrophin assay (Leuvering et al., 1981). In this paper, the application of colloidal gold agglutination to detection of fecal blood using
186
human hemoglobin-specific monoclonal antibody is reported. Since hemoglobin is composed of the tetramer, 02/32, a single monoclonal antibodylabeled colloidal gold can agglutinate with the antigen.
Materials and methods
Hemoglobins Human hemoglobin Ao was prepared from fresh hemolysate according to the method of Williams and Tsay (1975). The a- and /3-globins were prepared by chromatography of heme-split hemoglobin on a CM-cellulose column in 8 M urea (Bucci, 1981). Bovine, sheep, goat, rabbit, pig and horse hemoglobin were purchased from Sigma (USA). Chicken hemolysate was used as hemoglobin preparation.
Production of monoclonal antibodies Mouse hybridomas producing anti-human hemoglobin were produced by the method of Oi and Herzenberg (1980), using human hemoglobin as the immunogen. The hybridomas were expanded as ascites tumors in mice, and antibodies were purified from the ascitic fluid by Affi-Gel protein A (Bio-Rad) chromatography. Antibody production was examined by an enzyme-linked immunosorbent assay (ELISA) using human hemoglobin-coated plates.
ELISA Hemoglobins at 1 /~g/well were used to coat 96-well microtiter plates. Without blocking, the antigen-coated wells were incubated with antibody. The plates were washed with 0.05 M phosphate-buffered saline (PBS) containing 0.05% Tween 20, and the antibody bound to hemoglobin was examined with peroxidase-labeled goat antimouse IgG and the chromogenic substrate, 2,2azinobis(3-ethyl benzothiazoline-6-sulfonic acidkliammonium according to the manufacturer's instructions (Bio-Rad). The absorbance at 405 nm was measured by a microtiter plate reader.
lsotyping of monoclonal antibodies The isotype of the antibody was determined by EIA using rabbit antiserum specific for mouse
IgG1, IgG2a, lgG2b, IgG3 and lgM, according to the manufacturer's instructions (Bio-Rad).
SDS-polyacrylamide manoblotting
electrophoresis and im-
Hemoglobins were electrophoresed on SDSPAGE using a 13.5% polyacrylamide gel. The separated proteins were electrophoretically transferred onto a nitrocellulose membrane, blocked with 3% gelatin, then incubated with purified antibody. After washing, the antibodies bound to the membrane were detected using peroxidaselabeled goat anti-mouse IgG and the chromogenic substrate, 3,3'-diaminobenzidine.
Preparation of colloidal gold Colloidal gold .was prepared by the citrate method (Frens, 1973). In brief, 100 ml of 0rnl% chloroauric acid was boiled, then 0.8 ml of 1% sodium citrate was rapidly added. The solution was boiled for 10 rain after the color changed, cooled, then made up to 100 ml. The absorbance of colloids indicated 1.1 at 540 nm in a 1 cm light path. The mean diameter of the colloid was examined using a laser submicron particle sizer NICOMP 370 (Pacific Scientific, USA).
Preparation of monoclonal antibody-labeled colloidal gold Monoclonal antibody, previously concentrated to 2-3 mg/ml and dialysed against 5 mM NaCI, was added to 100 ml of colloidal gold adjusted with 0.2 M K,CO a, After stirring for 1-2 min, 5 ml of 1% carbowax 20 M was added (Geoghegan and Ackerman, 1977). The gold-antibody complex was centrifuged at 12,000 × g for 20 min. The pellet was suspended in 20 mi of PBS containing 0.05% carbowax 20 M and stored at 4°C. The absorbance of labeled colloids indicated 5.5 at 540 nm. Polyclonal anti-human hemoglobin rabbit lgG (Capped was also labeled by the same procedure.
Colloidal gold agglutination Agglutination was performed in 96-well flat bottom microtiter plates. 50 /~l of hemoglobin diluted with the sample buffer, PBS containing 0.1% NaN 3 and 6% polyethylene glycol 6000 (PEG 6000), were mixed with 90/~i of antibody-
187 labeled colloidal gold. After incubation for 30 min at room temperature, the absorbance was measured at dual wavelength (540 nm/655 nm) using a microtiter plate reader with water as the blank. In inhibition experiments using MgCI2, 50 /tl of a mixture of hemoglobin and MgCI2, diluted with 0.05 M Tris-HCI buffer (pH 7.4), was pre-incubated for 5 min and reacted with the colloidal gold.
lo ~
Hb (vylml)
~04F
Latex agglutination OC-Hemodia (Eiken Kagaku, Japan) was used according to manufacturer's instruction. Briefly, 10 mg of feces was suspended in 2 ml of 0.2 M ammonium buffer (pH 8.2), then 100/LI of fecal suspension and 50 /~l of rabbit anti-human hemoglobin antibody-coated latex were mixed and rotated on the slide for 3 rain.
Gel filtration chromatography Human hemoglobin, dialyzed against 0.1 M Tris-HCI buffer containing 1 M MgCI2 (TBMg), was chromatographed on a Sephacryl S-100 column (2.5 x 80 cm) in the same buffer. Fractions of 2.5 ml were collected, and the reactivity of each to HH6422-1abeled colloidal gold was determined. Samples were diluted ten-fold with 0.1 M Tris-HC! buffer containing 0.15 M NaCI (TBS) and TBMg or not diluted. As a control, chromatography in the absence of 1 M MgCI2 was performed using TBS instead of TBMg.
Results
Screening of monoclonai antibodies Six mice were immunized, and monoclonal antibodies were selected by ELISA using human hemoglobin-coated plate. All 40 monocional antibodies intensely reacted with human hemoglobin by ELISA, but only three did not react with bovine, sheep, goat, rabbit, pig, horse and chicken hemoglobin. These three antibodies were all lgGl as determined by subclass analysis.
Labeling of antibodies to colloidal gold Mean diameters of colloidal gold varied in the range of 50-60 nm, but agglutination of labeled colloids with antigen was not affected by the
80
6b
70 pH
lb
80
Fig. I. Effect of labeling pH on agglulinatlon. 5/~g of HH6422 were labeled with I ml of colloidal gold at various pH, and
labeledcolloidswerereactedwithhumanhemoglobin.
diameters within this range. Three highly specific antibodies were labeled with colloidal gold. One antibody, named HH6422" agglutinated with ! /~g/ml of human hemoglobin within I h, and the other two required 2 h to agglutinate with 10 izg/ml of antigen even when the labeling pH was varied. Reactivity with hemoglobin increased as the labeling pH was lowered, but the colloids did not disperse in the solution after centrifugation if the pH was too low. Fig. 1 shows the correlation of labeling pH and agglutinationwith hemoglobin in HH6422-1abeling. As shown in Fig. 2. antibody was saturated at 3 /zg/ml of colloids when HH6422 was labeled at pH 6.4. Based upon the above results, HH6422 was labeled at pH 6.4 using 4 p.g/ml of colloids. The colloids labeled with rabbit anti-human hemoglobin lgG failed to agglutinate with the antigen.
Specificity of HH6422 HH6422 was highly specific for human hemoglobin as confirmed by ELISA (Fig. 3) and immunoblotting (Fig. 4), the latter of which revealed that HH6422 recognized the hemoglobin B-chain (Fig. 4).
188
[ 1o r
1
Hb (ug/mJ)
2
3
4
5
6
7
8
9
10
A 08
NO6 ~
O5 I
~
W
w
w
m
~
~ l l ~
glIB
02 0
I0 20
"~o--c~--o---o i
0
i
2 3 4 5 Ar,tubody ( pg/ml co~lo~ds) Fig. 2. Effect of labeling antibody concentradnn on agglutination. HH6422 was labeled with colloidal gold at various antibody concentration at pH 6.4, and labeled colloids were reacted with hemoglobin.
Reaction of HH6422 antibody-labeled colloidal gold with hemoglobin HH6422-1abeled colloidal gold that reacted with hemoglobin rapidly changed from red to gray. Fig. 5 s h o w s t h e a b s o r b a n c e s p e c t r u m o f t h e r e a c t i o n reLy.turn a f t e r a 30 r a i n i n c u b a t i o n . F o r 9 6 w e l l p l a t e s , t h e c o l o r c h a n g e in t h e c o l l o i d s
Fig. 4. lmmunobloning of hemoglobins, i p.g of human hemoglobin (lane !), human ~-globin (lane 2), human ,8-globin (lane 3), cow (lane 4), sheep (lane 5), goat (lane 6), rabbit (lane 7), pig (lane 8), horse (lane 9) and chicken (lane 10) hemoglobins were electrophoresed. Gels were stained with Coomassie blue (A), and the protein bands were detected by immunoblotting using ltH6422 (B).
20
1 bg/mO
0,2 %' 0
400
0 0
0001
O01
01
10
lO
Anhbodv ( pg/ml ) Fig. 3. Species specificity of HH6422 by ELISA. ELISA was performed using HH6422 as the first antibody. Microtiter wells were co, ted with human (o) and other hemoglobins ( ~, ) including o~,v. sheep, goat. rabbit, pig. horse and chicken.
I 500
I 600
700
800
Wave length ( nm } Fig. 5. Absorbanc¢ spectra of the reaction mixture. 1 ml of two-fold diluted HH6422-labeled colloidal gold was incubated with 0.5 ml of various concentrations of hemoglobin for 30 min. The absorbance spectrum was measured at a 1 cm light path.
189
08' K ) O ~ C ~ O ' ~
,06
Hb ( pg/r'nl ) ~-O-, OO
/
,~04 O-~
.
20
.
.
40
005
.
60
80
100
120
O
024
Reaction Time ( rain )
]
4
6
63
250
IOOO
Hemoglobin ( pg/ml )
Fig. 6. Time course of colloidal gold agglutination. HH6422labeled colloidswere incubated with variousconcentrationsof
Fig. 7. Dose-respon~ cu,'~,efor colloidal gold agglutination. HH6422-1abeled colloidswere incubated with serial two-fold
human hemoglobin.
dilutions of human hemoglobin.
was visually determined by the naked eye or in a spectrophotometer using a 96 well plate reader. PEG 6000 enhanced agglutination, but an excess caused non-specific agglutination even in the absence of antigen, therefore, 2% was an appropriate concentration in the reaction mixture. Fig. 6 shows the time-course of agglutination of labeled colloids with hemoglobin. Agglutination occurred rapidly, reaching completion after
30 min. The absorbance levels remained relatively stable for at least 2 h. Fig. 7 shows a dose-response curve for agglutination. The absorbance linearly decreased with increasing hemoglobin concentration up to 1 /~g/ml (reddish gray-color), in the range of 1-100 /~g/ml, labeled colloids completely reacted with hemoglobin, and the absorbance decreased below 0.2 (gray color). The agglutination gradually
10
10
?
08
~ 4~
, lO
'
04
lo
g o4
08
o6~ 02
O8
}o6
06 o4
B,
08 &
o6,2
g O2
04 02
0 0 o 60 70 80 90 1OO 80 9O 100 F{actlon Number Fraettor, Number Fig. 8. Gel filtration chromatography of hemoglobin on Sephacryl S-100 in the absence (,4) and presence ( B ) of I M MgCI z. Protein (.), agglutination without dilution ( ,a, • ), I / I 0 dilution in TBS ( o ) and TBMg (e) were measured.
o
60
7O
ceased at the concentration above 100 /~g/ml because of prozone phenomenon and the redcolor of hemoglobin, however, agglutination was recognized at up to 1-2 mg/ml. The lowest detectable concentration was 0.5 p.g/ml by the naked eye and 0.2/zg/ml by the plate reader.
06
Reaction with hemoglobin dimer Colloidal gold agglutination is based on the assumption that the hemoglobin molecule is composed of a tetramer which shows a bivalent antigen. The monovalent aft dimer, therefore should not agglutinate with labeled colloids. In order to confirm this assumption, hemoglobin was chromatographed on a column of Sephacryl S-100 in the presence of 1 M MgCI 2, which causes hemoglobin to completely dissociate into the dimer subunits (Kirshner and Tanford, 1964). As shown in Fig. 8A, the apparent molecular weight of hemoglobin in TBS was 48,000, which agglutinated with labeled colloids. In the presence of 1 M MgCI z, the hemoglobin peak shifted to the dimer, and the apparent molecular weight was 28,000, which did not agglutinate without dilution (Fig. 8B). However, agglutination readily occurred when fractions were diluted with TBS. On the other hand, eluted [~actions in the absence of 1 M MgCI 2 barely agglutinated when diluted with TBMg (Fig. 8A). In Fig. 8A, agglutination in the peak was weaker than around the [ ~ak due to the prozone phenomenon, because terl-fold dilutions were sufficiently agglutinated. Aaditionally, hemoglobin in 1 M MgCI 2 was dialyzed against TBS and gel-chromatographed. The elution profile and agglutination pattern were the same as that of hemoglobin not treated with 1 M MgCl 2 (data not shown).
Inhibition of agglutination by MgClz Agglutination, after preincubation for 5 min, was interfered by MgCI 2 in a dose-dependent manner, especially at concentrations above 0.2 M (Fig. 9). In order to determine whether this was due to inhibition of the antibody-antigen reaction, ELISA and another colloidal gold agglutination assay were performed in the presence of 1 M MgCl z. In ELISA, the first reaction between hemoglobin-coated wells and free HH6422 antibody was not affected by 1 M MgCl 2 (data not
~ 04 <
0
02
04
0.8
16 31
62 125
v "2_5 50
100
Hemoglobin (iJg/ml)
Fig. 9. Inhibition of colloidal gold agglutination by MgCI 2.
Hemoglobin and MgCI2 were preincubated for 5 rain and agglutinated with HH6422-1abeledcolloids.Concentrationsof MgCI2 in samples: I M (o), 0.5 M (<>),0.25 M (l:3), 0.12 M (v), 0.06 M(A), and 0 M (e). shown). Also, agglutination of anti-human albumin monoclonal antibody-labeled colloidal gold with human albumin was not inhibited by 1 M MgCl 2 (unpublished data). It appeared that inhibition of agglutination by MgCl 2 was due to dissociation of the tetramer into the dimer, because neither antibody-antigen reaction nor colloidal gold agglutination against the monomeric antigen was entirely inhibited by 1 M MgCl 2.
Applicatic'~ to fecal clinical samples In colloide.l gold agglutination, 50 mg of feces was suspended in 2.5 ml of sample buffer. The lowest detectable concentration of hemoglobin was 0.025 m g / g stool. The color derived from feces was so pale in this fecal content that the color did not affect to either absorbance or judgement by the naked eye. Initial absorbance of the reaction mixture in normal colored feces was 0.8-0.9, whereas absorbance in dark colored feces enough higher, did not exceed 1.0. Recovery studies using fecal suspensions revealed that the absorbance of reaction mixture, even in dark colored feces, decreased below 0.6 when the sample contained hemoglobin at levels above 0.5 p.g/ml. However, dark or red colored feces, such as tar
TABLE 1 COMPARISON OF COLLOIDAl, GOLD AGGLUTINATION AND LATEX AGGLUTINATIONIN FECAL SAMPLES The X2 test with the Yates correction was not significant. Coll. Gold / Latex + -
+ 70 6
5 704
75 710
76
709
785
feces and that containing a large amount of blood, often contained hemoglobin at levels above 2 mg/ml, which showed the prozone phenomenon. To avoid false-negative reactions due to this, smaller sample volumes were also assayed. A comparison with latex agglutination was performed in 785 fecal samples collected for clinical diagnosis. The lowest detectable concentration in latex agglutination was 0.04 m g / g stool. As summarized in Table I, colloidal gold agglutination gave 75 positive results whereas latex agglutination gave 76. Among them, 70 samples were positive in both assays, and overall agreement between the two methods was 98%.
Discussion Colloidal gold agglutination, which differs from other particle agglutination methods, can detect antigen by a color change. The reaction is single step, and positive tests are clearly distinguishable from negative tests by the color. Using 96 well titer plates, large numbers of samples can be determined within 60 min. This method is based on the assumption that hemoglobin is a tetramer. Gel chromatography in the presence of 1 M MgCI 2 suggested that the hemoglobin dimer did not agglutinate with HH6422-1abeled colloids. Forms of hemoglobin in feces are unknown. However, even if hemoglobin is in the dimer form in f6ces, it would readily associate into the tetramer when suspended in low salt solution at a neutral pH. It is possible that fecal hemoglobin is complexed with haptoglobin and does not react with HH6422-1abeled colloids. In fact, haptoglobin inhibited colloidal
gold agglutination (data not shown). However, the haptoglobin concentration in feces is probably negligible since blood levels are 200 times lower than that of hemoglobin. HH6422, a monoclonal antibody selected for colloidal gold agglutination, was highly specific for human hemoglobin. Human hemoglobinspecific monoclonal antibodies have been reported (Stamateyannopoulos et al., 1981; Krco et al., 1985). However, these monoclonal antibodies were not used for diagnostic hemoglobin determination. The sensitivity for human hemoglobin of colloidal gold and latex agglutination 'OC-Hemodia' is 0 . 5 / ~ g / m l and 0.1 / t g / m l respectively. However, the sensitivity per feces weight of colloidal gold was equal to that of latex agglutination (0.04 m g / g feces) by adjusting the sampling volumes. Good agreement between colloidal gold and latex agglutination has been obtained in fecal blood detection (Table !). The detection limits of chemical methods, such as guaiac tests, are as low as 1-3 m g / g feces (Barrows et al., 1978; Turunen et al., 1984; Saito et al., 1985). These tests are not specific for human hemoglobin, because they react with animal blood and many nonhemoglobin substances and there is interference by substances in stools such as ascorbic acid (lllingworth, 1965; Jaffe et a~.~ 1974; Ostrow e t a ; . , 1974). In fact, the guaiac test gave a higher positive rate ( P < 0.01), presenting that the 38 (40%) and 10 (11%) of 94 fecal samples were positive in guaiac test and colloidal gold agglutination, respectively (data not shown). This higher rate may be caused by using fecal specimens without restriction of foods and drugs. Concentrations of fecal hemoglobin from normal volunteers or cut-off values in immunochemical methods depend on sensitivity. These reported values are as follows: 0.3 mg/g of feces in radial immunodiffusion (Barrows et al., 1978), 0.1 m g / g of feces in immunoprecipitin (Kim et al., 1985), and 0.01-0.05 m g / g of feces in EIA (Turunen et al., 1984). In heme-derived Imrphyrin fluorometry fecal hemoglobin in samples from healthy volunteers were less than 2 m g / g of stool (Ahlquist et ah, 1985). This value is much higher than that of the negative tests in immunochemical methods. The hemoglobin concentra-
192 tion may decrease due to degradation by digestive enzymes and colonic flora during intestinal transit or fecal storage. However, porphyrin fluorometry may overestimate the concentration of hemoglobin in feces, because heine, degraded h e m e and heine-derived porphyrins are m e a s u r e d as hemoglobin. Moreover, the origins o f the porphyrins may not be limited to hemoglobin. Therefore, cut-off values for fecal blood detection should differ from a m o n g methods. The advantages o f colloidal gold agglutination are specificity for h u m a n hemoglobin removing dietary restrictions and ease of j u d g e m e n t without technical skill. This m e t h o d using 96 well plates and a plate reader is suitable for mass screening of colorectal cancer. F u r t h e r studies are required to clinically evaluate this method.
Acknowledgements We wish to thank Miss Yoko l n a m o r i for production o f monocional antibodies and Mr. Ryo t a r o Higashiyama, Sanritu Soubu Clinical Diagnosis Center, for supply of fecal specimens.
References Ahlquist. D.A., McGill, D.B., Schwartz, S.. Taylor, W.F. and Owen, R.A. (1985) Fecal blood levels in health and disease/A study using HemoQuant. New Engl. J. Mad. 312, 1422. Barrows, G.H., Burton, R.M., Jarren, D.D., Russell, G.G., Alford. M.D. and Songster, C.L. (1978) lmmunochemical detection of human blood in feces. Am. J. Clin. Pathol. 09, 342. Bucci, E. (1981) Preparation of isolated chains of human hemoglobin. Methods Enzymol. 76, 97. Faulk, W.P. and Taylor. G.M. (1971) An immunocolloid method for the electron microscope. Immun~hemistry 8, 1081. Frens, G. (1973) Controlled nucleation for the regulation on the particle size in monodisperse gold suspensions. Nature Phys. Sci. 241.20. Ge," ~gan, W.D. and Ackerman. G.D. (1977) Adsorption of horseradish peroxidase, ovomucoid and anti-immunogIobulin to colloidal gold for the ~ndirect detection of concanavalin A, whe[~ germ agglutinin and goat anti-human intmuooglobuiin G on cell surfaces at the electron microscopic level: A new method, theory and application. J. Hi~tochem. Cy~ochem. 25, 1187.
Horisberger, M. and Rosset, J. (1977) Colloidal gold, a useful marker for transmission and scanning electron microscopy. J. Histochem. Cytochem. 25, 295. Illingworth, D.G. (1965) Influence of diet on occult blood tests. Gut 6, 595. Jaffee, R.M., Kasten, B., Young, M.D. and MacLowry, N. (1974) False negative stool occult blood tests caused by ingestion of aseorbic acid (Vitamin C). Ann. Intern. Mad. 83, 824. Kim, Y.D., Nolan, J.M., Malkin, A., Barch, D. and Tomita, J.T. (1985) A qualitative agar gel immunoprecipitin (lP) test for detection of fecal occult human hemoglobin, Clin. Chim. Acta 152, 175. Kirshner, A.G. and Tanford, C. (1964) The dissociation of hemoglobin by inorganic salts. Biochemistry3, 291. Krco, C.J., Gorzynsky,T.J. and Beito, T. (1985) Characterization of a monoclonal antibody that recognizes a determinant unique to human haemoglobin /]-chain. J. Immunogenet. 12, 197. Leuvering, J.H.W., Thai, P.J.1t.M., Waart, M.V.D. and Schuurs, A.H.W.M. (1981) A sol particule agglutination assay for human chorioni'c gonadotrophin. J. ImmunoL Methods 45, 183. Moeremans, M,, Daneels, G., Dijck, A.V., Langanger, G. and May, J.D. (1984) Sensitive visualization of antigen-antibody reactions in dot and blot immune overlay assayswith immunogold and immunogold/silver staining. J. Immunol. Methods 74, 353. Oi, V.T. and Herzenberg, L.A. (1980) Immunoglobulin-produc~ng hybrid cell lines. In: B.B. Mishell and S.M. Shiigi (Eds.), Selected Methods in Cellular Immunology. W.H. Freeman, San Francisco, p. 351. Ostrow, J.D., Mulvaney, C.A., Hansel, J.R. and Rhodes, R.S. (1974) Sensitivity and reproducibility of chemical tests for fecal occult blood with an emphasis on false-positive reactions. Am. J. Dig. Dis. 18, 930. Saito, H., Tsutida, S., Kakizaki, R.. Fukusi, M., Sano, M., Aisawa, N., Munakata, A. and Yoshida, Y. (1984) An immunological fecal occult blood test for mass screening of colorectal cancer by reversed passive hemagglutination (RPHA). Nippon Syokakibyo Gakkaizassi 81, 2831 (in Japanese). Stamatoyannopoulos, G., Farquhar, M., Lindsley, D., Brice, M., Papayannopoulou, Th., Nute, P.E., Serjeant, G.R. and Lehamaun, H. (1981) M:,pping of antigenic sites on human haemoglobin by means of mouoclonal antibodies and haemoglobin variants. Lancet ii, 952. Takeshita, T., Horiguchi, J., Kinoshita, T., Kubota, Y., Chen. P.-C., Katsumata. S., Horimukai, F., Miyaoka. M., Matsumoto. E., Saito, 3". a~ld Ashizawa, S. (1985) Latex agglutination test for fecal occult blood. Daityokomonshi 38, 780 (in Japanese). Turunen, M.J., Liewendahl, K.. Partanen, P. and Adlercreutz, H. (1984) Immunologicaldetection of faecal occult blood in colorectal cancer. Br. J. Cancer 49, 141. Williams, R.C. and "rsay. K.-Y. (1975) A convenient chromatographic method for the preparation of human hemoglobin. Anal. Biochem. 54, 137.