parietal cell antigen-antibody reaction in pernicious anaemia and

Clin. exp. Immunol. (1969) 5, 141-153.

THE PARTICIPATION OF COMPLEMENT IN THEPARIETAL CELL ANTIGEN-ANTIBODY REACTION

IN PERNICIOUS ANAEMIA AND ATROPHICGASTRITIS

ELIZABETH JACOB AND G. B. JERZY GLASSSection of Gastroenterology and Gastroenterology Research Laboratory,

Department of Medicine, New York Medical College, New York

(Received 25 February 1969)

SUMMARY

Indirect evidence suggests that the parietal cell antibody circulating in the serumof pernicious anaemia patients is a complement fixing antibody. In this work, wehave presented direct evidence using an immunofluorescent technique, that theantigen-antibody union occurring in the gastric mucosa between this antibodyand the parietal cell antigen binds complement (C'). We have further adduced datato indicate that serum C' activity was decreased in more than one-third of ourpatients with pernicious anaemia and in one-fourth of those with advancedatrophic gastritis. Eighty-five per cent of the patients with lowered serum C' hadparietal cell antibody in the serum and some of them also had intrinsic factor anti-body.

These findings support the concept of the autoimmune mechanism in thedevelopment of the gastric atrophic lesion in a proportion of patients withpernicious anaemia and atrophic gastritis. This mechanism includes the participa-tion of complement in the antigen-antibody reaction at the parietal cell level.

INTRODUCTION

It is now generally agreed that the serum of the majority of patients with pernicious anaemiaand of a large proportion of patients with idiopathic atrophic gastritis contains an antibodywhich reacts against a particulate component of the cytoplasm of the gastric parietal cell.This antibody has been detected in vitro by the complement fixation test and by the immuno-fluorescent technique (Irvine et al., 1962; Taylor et al., 1962; Irvine, 1963).

Taylor et al. (1962), in a study of 143 patients with pernicious anaemia found a closecorrelation between the results of these two methods and have therefore concluded that thetwo methods were measuring the same antibody. Te Velde et al. (1966) adduced evidence,in vitro, that complement may be fixed at the parietal cell site when gastric mucosa ofpatients with atrophic gastritis and circulating parietal cell antibody is exposed to their

Correspondence: Dr G. B. Jerzy Glass, Gastroenterology Research Laboratory and Section of Gastro-enterology, New York Medical College, East 106th Street and Fifth Avenue, New York, N.Y. 10029, U.S.A.

141

142 Elizabeth Jacob and G. B. Jerzy Glass

autologous serum and fluoresceinated globulin fractions of rabbit antisera to the C'4 andC'3 components of human complement. This suggests that the parietal cell antibody isindeed a complement fixing antibody.

In the present study, we have attempted to determine, using an immunofluorescentmethod, whether the fixation of complement (C') in the antigen-antibody reaction occursalso in the parietal cell of the normal gastric mucosa. Furthermore, it might reasonably beexpected that serum C' activity would be lowered in pernicious anaemia patients and thosewith other forms of atrophic gastritis, if indeed C' is utilized in the immunological reactionin the gastric mucosa.

In the second part of our study, therefore, we assayed serum C' activity in a group ofpatients with pernicious anaemia and atrophic gastritis of various aetiology, as well as innormal controls, and correlated it with the presence or absence of parietal cell and intrinsicfactor antibodies in the serum and their titres.

MATERIAL AND METHODS

Material of casesThis consisted of fifty-one patients with atrophic gastritis of various aetiology and twenty

normal controls. Pertinent data are listed in Tables 1 and 2.The controls, apparently healthy individuals, included six males and fourteen females,

their ages ranging from 22 to 65 years. Their sera did not contain parietal cell antibody.Of the fifty-one patients, twenty-four had pernicious anaemia, sixteen idiopathic atrophic

gastritis, four atrophic gastritis associated with gastric carcinoma, and in seven, the atrophicgastritis was localized to the gastric stump after subtotal gastrectomy. The patients in thepernicious anaemia group consisted of eight males and sixteen females, their ages rangingfrom 40 to 79 years. The diagnosis of pernicious anaemia was based on clinical criteria, aswell as on positive haematological, secretory, radioisotopic and immunologic findings (seeTable 1). Serum vitamin B12 levels were in the normal range, but most patients included inthis study had received vitamin B1 2 therapy at one time or the other prior to this study whichmade the B12 figures somewhat irrelevant.The patients included in the idiopathic atrophic gastritis group consisted of ten males and

six females, their ages ranging from 23 to 84 years. This group was characterized by thepresence of atrophic gastritis with or without intestinal metaplasia on gastric biopsy, theabsence of intrinsic factor antibody in the serum, a low or low normal radio-vitamin B12absorption pattern, and no gross haematologial abnormality (see Table 2). All thesepatients had histamine fast anacidity and a low output of intrinsic factor in the gastricjuice.

The subtotal gastrectomy group included two males and five females, their ages rangingfrom 35 to 71 years. The gastric resection was performed on six of these patients for duodenalulcer and in one for gastric ulcer.Of the four patients aged 56-67 years with atrophic gastritis associated with gastric

carcinoma, three were females.

Gastric biopsiesThese were performed with a Brandborg-Rubin modification of the Wood's tube, a

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Complement in pernicious anaemia and atrophic gastritis 145

Berry sheath on Eder-Palmer gastrocope, or with a Rubin's-Quinton hydraulic tube.In the patients with gastric carcinoma, surgical biopsies were obtained.

Gastric acid outputThis was estimated after the augmented histamine test, according to Card & Marks (1960).

Intrinsic factor output in the gastric juice obtained after augmented histamine stimulus andintrinsic factor antibody in the serum

This was estimated either by the charcoal technique of Ardeman & Chanarin (1963) asmodified in our laboratory by Yamaguchi & Glass (1967) or by the zirconium gel techniqueof Hansen, Miller & Tan (1966).

Intestinal absorption of radioactive vitamin B12Intestinal absorption of radioactive vitamin B12 was measured by the double label hepatic

uptake test (Weisberg & Glass, 1966).

Serum vitamin B12This was measured by a microbiological method using Lactobacillus leichmannii (Skeggs,

et al., 1950) in Cases 1-16 and Lactobacillus lactis (Cooperman, Drucker & Tabenkin,1951) in Cases 17-51.

Serum complement (C') activityThis was assayed according to the method of Lange, Wasserman & Slobody (1960), using a

sensitized sheep cell haemolysin system and guinea-pig complement standard. The serawere frozen at - 200C, immediately following collection, to prevent C' inactivation. Table3 lists the details of this method.

TABLE 3. Technique of serum complement assay

A. Serial serum dilutions and serial dilutions of standardguinea-pig complement are prepared (range 1:2 to 1: 256);end volume in each tube 0-2 ml

B. 0-2 ml buffered saline (pH 7-4) is addedC. 0-2 ml sensitized sheep cells is addedD. Incubation in water bath at 370C for 1 hrE. Centrifugation and reading for 50% haemolysis by com-

parison with haemolysed sheep cell colour standards (rangeof haemolysis 5-100%)

F. The serum which produced 50% haemolysis at the samedilution as the 1:8 standard guinea-pig complement is saidto contain one unit of complement

Immunofluorescent assay for parietal cell atibodyImmunofluorescent assay for parietal cell antibody in the serum was performed by


Coons' indirect technique, as applied to gastric mucosa by Taylor et al. (1962). The mucosalsections, 6 , thick, obtained from the fundic portion of human stomach removed at operationfor duodenal ulcer and/or from rat stomach, were incubated with two drops of the serumto be tested in a moist chamber at room temperature for 30 min and washed in two changesof Coons' buffer (pH 8 0) for 10 min. After removal of the excess fluid, the sections wereincubated for 30 min with fluorescein conjugated rabbit antiserum to human y-globulin(Hyland, Divison Travenol Laboratories Inc., Los Angeles, California; fluorescein proteinratio 5-7 mg fluorochrome/I g protein), washed in three changes of Coons' buffer for10 min, blotted dry except for section area, mounted in 5000 glycerol buffered with Coons'buffer (pH 8 0) and examined by ultraviolet microscopy within 1 hr using an OsramHBO-200 W high pressure vapour lamp and primary filter UV UG-1 and secondary filterBG-38. The fluorescein conjugated rabbit antiserum to human y-globulin G was absorbedwith acetone dried rabbit liver powder before use.The tests designed to demonstrate the in vitro fixation of C' in the parietal cell antigen-

antibody reaction in the gastric mucosa were performed in a similar manner, with theexception that fluorescein conjugated anti-human C' (flc) serum was used instead of fluor-escein conjugated rabbit antiserum to human y-globulin. Control experiments were carriedout using normal serum and also normal and parietal cell antibody containing serum heatedto 56°C for 1 hr in order to inactivate the C' contained therein. The fluorescein conjugatedrabbit antiserum to human complement (ftc) was obtained from Dr K. Lange, RenalService, New York Medical College. It was prepared as follows: 1 mg of a highly purifiedpreparation of the Ihc component of C' (obtained from Dr Muller Eberhard, ScrippsLaboratory, La Jolla, California) is dissolved in 05 ml isotonic saline and 05 ml completeFreund's adjuvant. This suspension is injected subcutaneously into three to five young NewZealand female rabbits. The injections are repeated 7 days later and the titre of the anti-C'serum checked 10-20 days thereafter. If the titre is not greater than 1:32, 1 ml of an alumprecipitated preparation of the antigen is given in addition, intravenously, to the rabbits for4 consecutive days and this procedure is repeated until a good titre of anti-C' serum isobtained.The antiserum is conjugated with fluorescein isothiocyanate (Baltimore Biological

Laboratories) according to the method of Riggs, Loh & Eveland (1960).

RESULTS

Immunofluorescent studiesWhen sections of normal gastric mucosa were incubated with parietal cell antibody

containing serum, the addition of fluorescein labelled rabbit anti-human y-globulin serumproduced fluorescence of the parietal cells, as would be expected (Fig la). When both theparietal cell antibody containing serum and the fluorescein labelled anti-human y-globulinwere heated to 56°C for 1 hr in order to inactivate complement, and the same test wasrepeated, fluorescence of the parietal cells was still present (Fig. lb). This is in line withthe accepted notion that the union of the cellular antigen with the humoral antibody doesnot require the presence of C'.

Gastric mucosal sections were then incubated with serum containing parietal cell antibodyin the presence of C' contained in the same serum or supplied by adding fresh human serum.When the fluorescein labelled anti-human C' serum was added a bright fluorescence of the


'' t~~~~~~~~~*

FIG. 1. Fluorescence studies on human gastric parietal cells using the indirect Coons' technique.(a) Gastric mucosal section shows fluorescence of parietal cells after incubation with parietalcell antibody containing serum and fluorescein conjugated rabbit anti-human y-globulin serum.x 250. (b) Gastric mucosal section shows fluorescence of parietal cells after incubation with thesame parietal cell antibody containing serum and fluorescein conjugated rabbit anti-humany-globulin serum, both previously heated to 56 C for 1 hr. x 250.

parietal cells appeared (Fig. 2a). When the C' in the parietal cell antibody containing serumand normal serum added as a source of C' was inactivated by heating to 560C for 1 hr, thefluorescence was abolished (Fig. 2b). Control experiments, using normal serum instead ofthe parietal cell antibody containing serum also failed to show fluorescence, as shown in thesummarizing Fig. 3. These findings were reproduced four more times using a differentparietal cell antibody containing serum each time. The results indicate that following theparietal cell antigen-antibody union complement is bound in vitro to the parietal cell.

Serum complement studiesFig. 4 shows the serum C' activity in the fifty-one patients with pernicious anaemia and

atrophic gastritis of various aetiology and twenty controls. The presence or absence of

Elizabeth Jacob and G. B. Jerzy Glass

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FIG. 2. Fluorescence studies on human gastric parietal cells using the indirect Coons' technique.(a) Gastric mucosal section shows fluorescence of parietal cells when incubated with the sameparietal cell antibody containing serum and fluorescein conjugated anti-human complementserum. x 250. (b) Gastric mucosal section shows absence of fluorescence or parietal cells whenincubated with the same parietal cell antibody containing serum previously heated to 560C for1 hr and fluorescein conjugated anti-human complement serum. x 250.

148


Normal serum

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FIG. 3. Indirect Coons' test on normal human gastric mucosa.

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carcinoma gastrectomy20 24 16 4 7

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FIG. 4. Serum C', parietal cell antibody and intrinsic factor antibody in seventy-one individuals.*, PCA positive; *, PCA and IFA positive; o, PCA and IFA negative. Broken line indicateslower limit of normal. The figures in the boxes represent the numbers of cases studied ineach group.


circulating parietal cell and intrinsic factor antibodies is also shown in Fig. 4. The range ofserum C' activity in our twenty controls was 1P3-3-9 units. Lange et al. (1960) have shown aserum C' activity range of 1 2-4-0 units by this technique in a group of 103 normal subjects.

In nine (370%) of the twenty-four patients with pernicious anaemia, the serum C' activitywas below the lower limit of normal, that is, below 1P3 units. In four (25%) of the sixteenpatients with idiopathic atrophic gastritis, and in one of the four patients with gastriccarcinoma, serum C' activity was also below the normal level. The mean, with standarddeviation of C' activity in the pernicious anaemia group was 1-7+0-87, and, in the controls,2-2 + 0-77. The difference was statistically significant at the 9500 level. In the group of sevenpatients with subtotal gastrectomy and atrophic gastritis, serum C' activity was normal.Of the fourteen patients in our study with lowered serum C' activity, twelve (85%) had

parietal cell antibody in the serum, nine of these being pernicious anaemia cases. Of theselatter nine patients, five had intrinsic factor antibody as well in the serum. Conversely,of the patients with neither type of circulating antibody, the vast majority had normalserum C' activity. However, it should be noted that eighteen patients in this study had normalserum C' activity in the presence of one or both types of serum antibody.

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FIG. 5. Titres of serum C' and intrinsic factor antibody in fifteen patients with perniciousanaemia. Broken line indicates lower limit of normal.

Fig. 5 compares the serum C' activity and the titres of intrinsic factor antibody in fifteenof our pernicious anaemia patients in whom both these studies were performed. Serum C'activity was below the lower limit of normal in four of the nine patients who did not haveintrinsic factor antibody in the serum. In the other six patients in whom the intrinsicfactor antibody was present, there was no correlation between the titre of this antibody andC' activity.

Fig. 6 compares the titres of serum parietal cell antibody and serum C' activity in twenty-nine of our patients in whom both these measurements were performed. No correlationbetween the two sets of values was observed, the correlation coefficient being 041.

Complement in pernicious anaemia and atrophic gastritis 1514 0

3002 o0o

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FIG. 6. Titres of serum C' and parietal cell antibody in twenty-nine patients with atrophicgastritis (including pernicious anaemia). o, PCA tested on human gastric mucosa; n, PCAtested on rat gastric mucosa. Broken line indicates lower limit of normal.

DISCUSSION

The results of our investigations using immunofluorescent techniques, would suggest that acomplement fixing antigen-antibody complex may form in the parietal cell following theunion of the parietal cell antigen with the parietal cell antibody. If, indeed, such a complexdoes form in the gastric parietal cell in pernicious anaemia, or other forms of atrophicgastritis, and if the complement sequence is initiated, this could result in complementinduced injury to the parietal cell. This, in turn, would lead to anacidity and secretory defectof intrinsic factor, resulting in vitamin B12 deficiency.Such a C' fixation has been demonstrated in the glomerular lesions in certain renal diseases

by Lachmann et al. (1962) and Lange et al. (1966). These investigators have observed a goodcorrelation between the extent of C' binding as demonstrated by immunological stainingand the levels ofserum C' and have suggested that in vivo C' binding may be a factor involvedin producing the low serum C' levels in these cases. The low C' activity in a proportion of ourpatients with pernicious anaemia and those with advanced atrophic gastritis may be due tosuch a C' binding by the parietal cell antigen-antibody complex. Contrary to the interpreta-tion of Te Velde et al. (1966), this would support the concept of the autoimmune mechanismin the development of some forms of atrophic gastritis.We did not, however, observe any correlation between the titres of the parietal cell anti-

body and the levels of serum C' activity in our patients. Furthermore, although 85% of ourpatients with lowered serum C' activity had parietal cell antibody in the serum, normalcomplement levels were also observed in several cases in the presence of circulating parietalcell antibody. Thus, perhaps in addition to the parietal cell antigen-antibody reaction,

D

Elizabeth Jacob and G. B. Jerzy Glass

various other factors have to be considered as well, to explain the lowering of serum C'activity observed in some of our patients with pernicious anaemia and atrophic gastritis:

(1) One such factor could be the utilization of C' in the antigen-antibody reaction occurringbetween intrinsic factor and intrinsic factor antibody in the gastric mucosa or in the gastro-intestinal lumen. However, no correlation was observed in our study between the titres ofthis antibody and C' activity in the serum.

(2) Antibodies associated with the immunoglobulins contained in the lymphocytes andplasma cells and infiltrating the gastric mucosa of patients with atrophic gastritis mayperhaps utilize C' at the target site and thereby lead to a lowering of serum C'. Irvine et al.(1965), have suggested that such cell bound antibody carried to the target organ by lympho-cytes and plasma cells may be of more fundamental importance in the pathogenesis ofatrophic gastritis than circulating antibody. The present study, however, showed serum C'activity to be normal in the group of patients with atrophic gastritis following subtotalgastrectomy, and in those with atrophic gastritis without intestinal metaplasia, both ofwhich groups often had marked lymphocytic infiltration and a high content of IgA in thegastric mucosa (Brus et al., 1968).

(3) Complement may be utilized in the antigen-antibody reactions occurring in extra-gastric tissues and thus its decrease may not be related to the gastric atrophic lesion. How-ever, none of our patients were known to have glomerulonephritis, systemic lupus erythema-tosus, or any other disease which might be expected to lower serum C'.

(4) A diminished rate of synthesis of complement proteins may lead to low serum C'.However, in pernicious anaemia, no direct evidence is available to date which wouldsupport this possibility.

(5) Finally, one has to consider the possible existence of a complement inactivating factorin the serum which might lead to low serum C'. Pickering, Gewurz & Good (1968) havedemonstrated such a factor in the serum of patients with acute and some forms of chronicglomerulonephritis which, according to these authors, may be partially responsible for thelow serum C' levels in these patients. Nothing is known as yet about the possible occurrenceof such an abnormality in pernicious anaemia.

Thus, our findings support the concept of the autoimmune mechanism in the developmentof the gastric atrophic lesion in a proportion of patients with pernicious anaemia andatrophic gastritis. This mechanism includes the participation of complement in the antigen-antibody reaction at the parietal cell level.

ACKNOWLEDGMENTS

We thank Dr H. J. Hansen, Department of Biochemical Research, Tuoro Infirmary,New Orleans, Louisiana, for making available blood samples from sixteen perniciousanaemia patients and for providing us with the results of some of the tests performed onthese patients. Our thanks are also due to several members of our Gastroenterology ResearchLaboratory for their collaboration in these studies: to Dr N. Tanaka for serum vitamin B12assays; to Dr H. Siegel, for performing the gastric suction biopsies; to Mrs B. Towey andMrs E. Markley for valuable technical assistance. Dr K. Lange, Mr M. Semar and Mr P.Burgos, from the Renal Diseases Section, Department of Medicine, New York MedicalCollege, have given us valuable advice and technical instruction on serum complement

152


assay. Dr J. M. Cooperman, from the Hematology and Nutrition Laboratory, Departmentof Pediatrics, New York Medical College, has generously co-operated in the setting up of thevitamin B12 assays. Dr S. P. Rothenberg, from the Hematology Section, Department ofMedicine, New York Medical College, has made available blood samples and some labora-tory results on two pernicious anaemia patients included in this study.

This work has been supported in part by Research Grants-in-Aid AM-00068 and AM-09701 from the National Institute of Arthritis and Metabolic Diseases, and CA-08251from the National Cancer Institute, National Institutes of Health, United States PublicHealth Service.

REFERENCES

ARDEMAN, S. & CHANARIN, I. (1963) A method for the assay of human gastric intrinsic factor and for thedetection and titration of antibodies against intrinsic factor. Lancet, ii, 1350.

BRUS, I., SIEGEL, H., YAMAGUCHI, N. & GLASS, G.B.J. (1968) Immunoglobulins IgA and IgG in gastricmucosa of patients with atrophic gastritis and pernicious anemia. Scand. J. Gastroent. 3, 43.

CARD, W.I. & MARKS, I.N. (1960) The relationship between the acid output of the stomach following'maximal' histamine stimulation and the parietal cell mass. Clin. Sci. 19, 147.

COOPERMAN, J.M., DRUCKER, R. & TABENKIN, B. (1951) Microbiological assays for vitamin B12: A cyanideenhancement effect. J. biol. Chem. 191, 135.

HANSEN, H.J., MILLER, O.N. & TAN, C.H. (1966) Assay of the autohumoral antibody that neutralizes thevitamin B12 combining site of intrinsic factor in serum from patients with pernicious anemia. Amer.J. clin. Nutr. 19, 10.

IRVINE, W.J. (1963) Gastric antibodies studied by fluorescence microscopy. Quart. J. exp. Physiol. 48, 427.IRVINE, W.J., DAVIES, S.H., DELAMORE, I.W. & WILLIAMS, A.W. (1962) Immunological relationship between

pernicious anaemia and thyroid disease. Brit. med. J. ii, 454.IRVINE, W.J., DAVIES, S.H., TEITELBAUM, S., DELAMORE, I. W. & WYNN WILLIAMS, A. (1965) The clinical

and pathological significance of gastric parietal cell antibody. Ann. N. Y. Acad. Sci. 124, 657.LACHMANN, P.J., MULLER-EBERHARD, H.J., KUNKEL, H.G. & PARONETTO, F. (1962) The localization of

in vivo bound complement in tissue sections. J. exp. Med. 115, 63.LANGE, K., TRESER, G., SAGEL, I., Ty, A. & WASSERMAN, E. (1966) Routine immunohistology in renal

diseases. Ann. intern. Med. 64, 25.LANGE, K., WASSERMAN, E. & SLOBODY, L.B. (1960) The significance of serum complement levels for the

diagnosis and prognosis ofacute and subacute glomerulonephritis and lupus erythematosus disseminatus.Ann. intern. Med. 53, 636.

PICKERING, R.J., GEWURZ, H. & GOOD, R.A. (1968) Complement inactivation by serum from patients withacute and hypocomplementemic chronic glomerulonephritis. J. Lab. clin. Med. 72, 298.

RIGGS, J.L., LOH, P.C. & EVELAND, W.C. (1960) A simple fractionation method for preparation of fluoresceinlabelled gamma globulin. Proc. Soc. exp. Biol. (N. Y.), 105, 655.

ROTHENBERG, S.P. (1966) A radioimmuno assay for human intrinsic factor. J. Lab. clin. Med. 67, 879.SKEGGS, H.R., NEPPLE, H.M., VALENTIK, K.A., HUFF, J.W. & WRIGHT, L.D. (1950) Observations on the use

of Lactobacillus leichmannii 4797 in the microbiological assay of vitamin B12. J. biol. Chem. 184, 211.TAYLOR, K.B., Rorrr, I.M., DONIACH, D., COUCHMAN, K.G. & SHAPLAND, C. (1962) Autoimmune pheno-

mena in pernicious anaemia: Gastric antibodies. Brit. med. J. ii, 1347.TE VELDE, K., HOEDEMAEKER, P.J., ANDERS, G.J.P.A., ARENDS, A. & NIEWEG, H.O. (1966) A comparative

morphological and functional study of gastritis with and without autoantibodies. Gastroenterology, 51,138.

WEISBERG, H. & GLASS, G.B.J. (1966) A rapid quantitative method for measuring intestinal absorption of

vitamin B12 in man using a double label hepatic uptake test. J. Lab. clin. Med. 68, 163.YAMAGUCHI, N. & GLASS, G.B.J. (1967) The determination of intrinsic factor in gastric secretory analysis.

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parietal cell antigen-antibody reaction in pernicious anaemia and