Immunological Characterization of Human Glomerular Basement Membrane Antigens K. HENRIKSEN Sc H. JENSEN Immunological Ijibi>r3tor>', University Qinic for Infectious Dueases, and Medical Department P, Division of Nephrology, Rigshospitalet, Denmark

Hcnriksen, K. it Jensen, H. Immunoiogtcal Characterization of Human Glomerular Basement Membrane Anti^ns. Scand. / . immunol. 4, 699-706. 1975. Normal human glomerular bast-ment membrane (H-GBM) was soliibili/ed by collagenase and subjected to crossed immunoelectrophoresis with rabbit antibodies ag3inst H.GBM. Se%'en precipitates appeared with the mobility of a, ^. ond y globulins. Only two of these precipitates might be spttific for GBM, since the other precipitates disappeared after absorption of the antiserum with liver and placenta. In normal human urine one precipitate, cross-reactinf} with one of the H-GBM precipitates, was found; tbis precipiute could also be demonstrated in human placenta and liver. K. Henriksen, Immunological Laboratory, 1842-9, Rigs has pitsieU M, Blegdamit'tf 3, 2200 Kebenhavn N. Denmark

Previously several investigators (6, 7, II, 16, 17) have studied the irnmunoreactive basement membrane antigens by means ot* double diffusion in gel and by means of the immunoelectrophoretic technique described by Grabar & Williams. Lately the antigen-antibody crossed immunoelectrophoretic technique has been applied to the study of different antigens—for instance, Candida albicans by Axelsen (2), non-plasma proteins in cerebrospinal fluid by Bock (4), and Pieudomonas aerugitiosa by Hoiby (8). Since crossed immunoelectrophoresis has advantages over double diffusion in gel and over classical immunoelectrophoresis, we have applied this mtrthoti to tbe study of coUagenasetreated human glomerular basement membrane. The glomerular basement membrane is composed of collagenous and non-collagenous glycoproteins (12). The use of collagenase to solubilize the glomerular basement membrane was based on the observation that the nephrotoxic component of the basement membrane

tpidtmiafdeling

is a non-collagen protein (9, 15, 16), Furthermore, the ultitnate purpose of this study is to investigate the urinar)- excretion of basement membrane fragments, which are glycoproteins devoid of collagen {*>). METHODS Preparation of human glomerular basement membrane (H-GBM) Normal kidneys obtained at autopsy and kidneys primarily destined for transplantation were used. Each kidne)' was perfused with 3-5 1 of cold sodium chloride. The giomerular basement membranes were isolated by a tecbnique described by Spiro (19) and modified by Westberg & Michael (26). The H-GBM preparation was stained with haematoxylin and eosin (no nuclear staining) and periodic acid-Schiff reagent (highly j-)ositive). Digestion was performed using highly purified coUagenase (.type CLSP-A) obtained from

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Worthington Biochemical Corp., New Jersey, as described by Spiro (20). Preparation oj rabbii glomerular basement membrane fR-GBM) Glomeruii were isolated using sieves with pore sizes of 91 ^"^ and 63 fim. Further procedures were the same as those applied to H-GBM. Preparation of human liver and placenta Human liver obtained at autopsy and placentas were mtnced with scissors and homogenized in isotonic smiiuni chloride in a blendor. After being washed, the material was resuspended in IM sodium chloride and sonicated with a Rapidis 300 ultrasonicator. After centrifugation at 2,000 !• at 4°C the sediment was washed three times in lM sodium chloride and four times in distilled water and finally tyophilized. Solubilization with collagenase was performed as described for H-GBM. Preparation of antisera lo H-GBM Six rabbit.s were immunized with either paniculate H-GBM (P-H-GBM) (8 mg) or collagenase-digcsted H-GBM (C-H-GBM) (1.5 and 2 mg, corresponding to anti-C-H-GBM I and anti-CNH-GBM II) in isotonic sodium chloride and were mixed with an equal volume of Freund's incomplete adjuvant. The animals were injetted with the mixture on days 0, 14, 28, and 42. The rabbits were bled on day 52. Every fourth week a further bleeding took place, the rabbits having received an injection of antigen mixture P-H-GBM (2 mg) or C-H-GBM (0.4 and 2 mg) 10 days earlier. The immunization procedure was performed by Dakopatts A/S, Copenhagen. Absorption of antisera The antisera were absorbed with human AB er)'throcytts ami normal human plasma (NHP) cross-linked by gkitaraldehyde, as described by

Avremeas & Ternynck (1). In some cases the antisera were absorbed with lyophilized NNP (80 mg/ml) instead of insolubilized NHP. The antisera against C-H-GBM (anti-C-H-GBM) were further absorbed with collagenase, 400800 units/ml antiserum. The mixture was incubated for 1 h at room temperature and for about 20 h at 4°C with constant stirring. After centrifugation at 20,000 ^ for 20 min the antiserum was removed. In some experiments the antisera were also absorbed with lyophilized P-H-GBM, 10 mg ml; lyophilized placenta, 40 + 40 mg/ml in two stages; or lyophilized liver, -10 + 40 mg/ ml in two stages, following the same procedure as described above.

Ehftion of ttnti'H-GBM antibodies from rabbit kidneys Two rabbits were killed 5 months after the first injection of H-GBM Anti-H-GBM antibodies were eluted from the kidney as described by Boesken et at. (6). The eluates obtained were concentrated, using Visking 23/32 in. dialysis tubes, to a protein concentration of 10 mg/ml.

Illedrophoretic technicfues Crossed immunoelectrophoresis with and without intermediate gel was carried out as described earlier (22, 25), using \*'c agarose gel (thickness of gel, 1 mm) in barbital buffer, pH 8.6, ionic strength 0.02. Tlie first-dimension electrophoresfs of C!-H-GBM (3.5 mg-ml), collagenase-digested R-GBM (C-R-GBM), collagenase-digested placenta (C-placenta) {6 mg' ml and 30 mgml), and collagenase-digested liver (C-liver) (6 mg,ml and 30 mg.ml), NHP undiluted, NHP with a protein concentration of 330 mg/ml, and NHP diluted 1:4 - » 1:32, concentrated normal human urine (NHU) (protein concentration, 22 mg/ml, corresponding to a 200-fold concentration), and a collagenase solution were run at IO°C, applying 10 V/cm for 1 h; 4-10 ^1 of the above-

Glomerular Basement Membrane Anligens

mentioned dilutions were used. The seconddimension electrophoreses were run at 10°C, applying 3 V/cm for 20 h. Tandetri-crossed imm//>ioelectrop/}ore.fis was

performed as described by Krcll (13)-

Gel diffusion Double diffusion was carried out in 1% agaroie in barbital buffer, pH 8.6, ionic strength 0.02. Wells 4 mm in diameter were punched at a distance of 4 mm. Two X 10 fd of anti-H-GBM, C-H-GBM (2 mg/ml), C-pIacenta (6 mg/ml), and C-liver (6 mg/ml) were applied.

Immunofluorescent techniques Fluorescein isothiocj-anate (FITC)-conjugated swine anti-rabbit serum IgG (heavj' and light chains) was purchased from Dakopatts A/S. The indirect immunofluorescent technique was employed, using normal human kidney as substrate. Kidney Crj-ostat sections were thrice washed and immersed in phosphatebuffered saline (PBS) for 15 min. The sections were incubated with anti-H-GBM for 30 min at room temperature, followed by washing and immersion in PBS twice for 10 min, and were finally stained with FITC-conjugated swine anti-rabbit serum IgG for 30 min (dilution, 1:25). Controls included treatment of the kidney sections with normal rabbit: serum before staining and direct staining of the sections with the conjugated antiserum. The antisera were titrated, using the foHowing dilutions: 1:2, 1:4, 1:8, 1:128, 1:256, 1:512. 1:1.024, 1:2,048, and i:4,096. The end[x>int titer was taken as the highest dilution with a distinct linear fluorescence on the glomerular basement membrane. The indirect immunofluorescent technique was also employed, using normal human leukocytes as substrate, with anti-H-GBM in the first layer and FITC-conjugated swine antirabbit serum IgG in the second layer.

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AB er)'throq'tes were washed four times in isotonic sodium chloride, and one drop of the er)'throc7tes was incubated for 30 min at room temperature with one drop of anti-H-GBM. The suspension was wa.shed four times in PBS and incubated for 30 min at room temperature with FITC-conjugated swine anti-rabbit serum IgG. After being washed, the erythroc7tes were studied for fluorescence.

Normai human urines were collected and concentrated as earlier described (10). RESULTS Immunoelectrophoresis By means of crossed inimiinoelectrophoresis with C-H-GBM as antigen and unabsorbed anti-H-GBM in the second-dimension gel, several precipitates were demonstrated. Some of these precipitates showed identit)' in tandemcrossed immunoelectrophoresis with NHP and collagenase solution. Anti-C-H-GBM absorbed with NHP and collagenase and anti-P-H-GBM absorbed with NHP showed no reaction with concentrated NHP, undiluted NHP, NHP in dilution 1:32, or with collagenase solution. The precipitates were unchanged by absorption with AB erythrocytes. In the following anti-H-GBM always refers to antisera absorbed with collagenase or NHP, or both. Crossed immunoelectrophoresis with C-HGBM as antigen and anti-C-H-GBM I as antibody demonstrated seven precipitates in the reference gel (Fig. 1). Fig. 2 shows the reaction between C-H-GBM and anti-C-H-GBM II. The relative heights of the precipitates differed from those obtained by anti-C-H-GBM I, resulting in a fusion between precipitates 2 and 3. Tlie identity of the different praipitates was demonstrated by application of one of the antibodies to the reference gel and the other to the intermediate gel. With anti-P-H-GBM only 3-4 precipitates could be demonstrated (no. l - 2 - 3 ( - 4 ) ) . The relative height of the precipitates varied not only with the amount and character of the

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Fig. 1. Crossed immunoelectrophoresis of 4 ^l human glomerular basement membrane A with antiserum against human glomerulur basement membrane (antiC-H-GBM 1) in tbe reference gel. Tbe precipitates are marked witb numbers. The intermediate gel contained antibody to human serum protein and demonstrated the contamination of the H-GDM preparation with serum proteins and the mobility of H-GBM antigens compared to albumin (arrow). (For tbis and subsequent figures, first-dimension electrophoresis was performed witb anode to the right and second-dimension electropboresis with the anode at the top.)

antigen used for immunization but also with the time of the bleeding. The precipitates were reproducible in five different preparations of normal H-GBM but with variations in the relative height. All the precipitates were unchanged by addition of rabbit antibodies to human serum, normal buman urine protein, human fibrinogen, and /32-microglobulin (purchased from Dakopatts A/S) to the intermediate gel. The GBM precipitates were similar whether anti-GBM was absorbed with insolubilized or lyophilized NHP. Tandem-crossed immunoclcctrophoresis with C-H-GBM and NHP as antigens did not change the pattern demonstrated in Figs. 1 and 2. In contrast, tan dem-crossed immunoelectrophoresis with C-H-GBM and concentrated normal human urine as antigens showed that one

Fig. 2. Crossed immunoelectropboresis of 4 ^(.1 human glomerular basement membrane A against antiserum to human glomerular basement membrane (anti-CH-GBM 11). No antiserum was included in the intermediate gel. The precipitates are marked with numbers.

precipitate developed between anti-H-GBM and concentrated urine and that this precipitate cross-reacted with precipitate 1. C%H-GBM did not react with rabbit kidney eluate in crossed immunoelectrophoresis. Tandem-crossed immunoelectrophoresis with C-placenta and C-H-CiBM as antigens demonstrated three placental precipitates identical to no. 1, 2, and 3. With C-Iiver as antigen, precipitate I was clearly demonstrated, although in a smaller amount than with C-H-GBM. This is shown in Fig. 3, where five times as much C-liver as C-H-GBM was used. Also a very faint line along the base line was seen, and when as much as 8 fi\ of 30 mg/ml C-liver (approx. 20 times as much liver (w/v) as GBM) was used, precipitates 2 and 3 were also demonstrated. Absorption of anti-H-GBM with placenta abolished all precipitates except no. 6 and 7. Precipitate I disappeared after absorption of anti-H-GBM with liver powder. Furthermore,

GlomermUr Basement Memhtane AntigtMj

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Tahlc I. Antibody-titers to buman glomeruUr buement membrane (H-GBM) before and ifter absorption with human glomerular basement membrane, placenta, and liver. Indirect immunofluorescent staining Titers after ibsorption with 0 Anti-C-HGBM* Anti-P-H-GBM

2,048 1,024

H GBM Placenta 2,048

Liver 2,048 1,024

• Anti-P-H GBM and anti-C-H-GBM designate antibodies to, respectively, particuUtc and colk^nasetrcatcd normal buman glomcrular basement membrane.

Fig. 3. Tandem-crossed immunoelectrophoreiis witb intermediatf gel. Antigens; 10 ^I liver (left) and 4 ii.1 human glomerutjr basement membrane B (right). Antibodies: intermediate gei, antUerum against whole human serum; reference gel. antiserum against human gtumerulur basement membrane (antt-C-H-GBM 11). The arrow indicates the ctoss-reactJng precipitate (no. I).

a very slight augmentation in the heights of precipitates 2 and 3 was seen. No precipitates were demonstrated between C-H-GBM and .inti-H-GBM absorbed with H-GBM,

Rabbit C-GBM No precipitates appeared with either anti-HGBM at any time during the immunization or with eluate of the killed rabbit's kidneys.

Gel diffusion Anti-C-H-GBM showed three precipitin lines with C-H-GBM, two of which fused with the two lines formed with C-placenta. Absorption with liver powder did not change this pattern. A ver)' faint band was formed between C-liver and anti-C-H-CiBM. Wben anli-C-H-GBM was absorbed with placenta, the reactions with C-

placenta and C-liver disappeared but the precipitin line specific for C-H-GBM was still present. Immunofluorescent studies Anti-AB activity was demonstrated in andH-GBM but disappeared after absorption with AB erythroc)'tes, No antinuclear activity could be demonstrated on the leukocj'tes. When the antisera absorbed with NHP and AB er^'throcytes were applied to normal human kidney sections, a sharp linear fluorescence was observed on glomerular. peritubular, and other vascular basement membranes, but no cellular stiining was seen. The results of application of serial dilutions of anti-H-GBM before and after absorption with H-GBM, placenta, and liver are listed in Table I. No difference in titers of anti-C-HGBM before and after absorption with placenta was observed. However, the linear fluorescence of the extraglomerular basement membranes disappeared more rapidly after absorption with placenta (end-point titer, 256) than without absorption with placenta (end-point titer, 2,048). Absorption of anti-P-H-GBM witli placenta diminished slightly the end-point titer of the fluorescence on glomerular basement membranes.

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Absorption of anti-H-GBM with liver did not in any way change the pattern. After treatment of human kidney sections with rabbit kidney eluate a linear pattern was seen (eadpoint titer, 4).

DISCUSSION Seven antigenic determinants were demonstrated in the non-collagenous portion of the normal human tilomeriilar basement membrane, in which the nephritogenic antigen (s) is localized (8, 16). These antigens had the electrophoretic mobility of n, ^, and y globulin. The relative quantity of the different antigens evidenced by the height of the precipitates varied among different preparations of normal H-GBM. The above-mentioned seven antigens are probably a minimum figure, since rabbit and man share closely related GBM antigens (15, 21). Even if rabbits may produce antibodies against their own antigens (23, 24), these antibodies are most likely rapidly removed from the circulation by the basement membranes in the host. This is in agreement with the fact that we were not able to demonstrate precipitating antibodies against rabbit GBM at any time during the immunization. A combination of anti-H-GBM from animals not sharing the same common antigens with man may reveal more antigenic determinants in H-GBM. Furthermore, in the effort to obtain the cleanest possible H-GBM preparation, water-soluble antigens may be lost, since Naruse & Shibata (18) were able to extract soluble GBM antigens from rats by the use of ultrasonic treatment alone. Fewer precipitates could be demonstrated by anti-P-H-GBM than by anti-C-H-GBM. This could imply that collagenase treatment exposed new antigenic determinants. However, all antiH-GBM activity was absorbed by particulate HGBM, as demonstrated by both the electrophoretic and fluorescent techniques. Lung, placenta, liver, and other organs share antigens with the kidney, and Baxter & Goodman i'i') produced nephrotoxic serum nephritis

in rats with rabbit antibodies directed against these organs. However, lung and placenta were superior to other organs in their ability to absorb and induce formation of nephrotoxic antibodies, When sufficient amounts of Cplacenta and C-liver were used, three precipitates were observed to cross-react with H-GBM in crossed immunoelectrophoresis. However, all C-H-GBM precipitates except no. 6 and 7 disappeared after absorption with placenta, but absorption with liver resulted in the disappearance of precipitate 1 alone and a slight elevation of precipitates 2 and 3- Thus there may exist both a qualitative and a quantitative difference between all the three organs concerning tlie non-collagenous basement membrane antigens. The results of reaction between H-GBM and anti-C-H-GBM absorbed with placenta in gel diffusion, crossed immunoelectrophoresis, and immunofluorescence indicated the presence of antigenic determinants specific for GBM and not found in placenta and liver. However, this difference may be due to the use of more highly purified basement membrane material in the H-GBM preparation. Hawking & Milgrom (7) also demonstrated a glomcnilarspecific antigen with sheep anti-H-GBM by means of gel diffusion. The eluate from the killed rabbits' kidneys did not react with C-H-GBM or C-R-GBM cither in gel diffusion or in crossed immunoelectrophoresis. It is likely that only a small amount of antibody was bound at the time of killing (in agreement with the low fluorescence titer of the eluate). It is also possible that the eluate, however strong, never reacts with collagenase-trented GBM (6). To show that none of the demonstrated GBM precipitates were due to a plasma protein and an inadequate absorption, the reaction of NHP in various concentrations in tandem-crossed immunoelectrophore.sis with C-HGBM against anti-H-GBM absorbed with NHP was examined, with negative results. However, this finding does not exclude the possibilit}' that a protein is present at low levels in plasma but in higher concentration in the kidney and thereby contaminates the GBM preparation and

Clomemlar Basement Membrane Antigens

elicits antibody production. It would be difficult to decide whether such a protein was a plasma protein sui generis or a basement membrane protein. As reported by others ( H , 17), we also found soluble BM-antigenic material in normal human urine. One precipitate (no. I) crossreacted with H-GBM and had the mobility of an tt globulin. The urine was concentrated for analysis, but further enrichment by chromatography or preparative electrophoresis was not attempted. The basement membrane antigen found in concentrated normal human urine may be derived from GBM alone, but since it is present in liver (and probably other organs as well), it is possible that trace amounts enter the circulation and are preferentially concentrated and excreted by the kidney.

ACXNOWLEDGEMENTS Tliis work was supported by Kobmand i Odense Johann og Hanne Weimanns legat, Kong Christian X Fund, Ingenior Snren Alfred Andersens legat, Ebba Celinders legat, and grant 512-3648 from the Danish State Medical Research Council.

REFERENCES 1. Avremcas, S. & Ternynck. T. The cross-linking of proteins with jflutarnldchydc and its use for the preparation of immunoadsorbents. Immunechemistry (S, 53. 1969. 2. Axelsen, N. H. Antijjen-antibody crossed immunoelectrophoresis (Laurcll) applied to the study of the antigenic structure of Candida albicam. Infection and Immunity 4. 52'i. 197i. 5. Baxter. J. H. & Goodman. H. C. Nephrotojtic seserum nephritis in rats. 1. Distribution and specificity of the antigen responsible for the production of nephrotoxic antibodies. /. exp. Med. 104. -167, 1956. 4, Bock, E. Non-plasma proteins in cerebrospinal fluid, pp. 119-12^ in Axelsen. N. H.. Krwll, J. & Weckc. B. (eds.) A Manujl of QuantiSalire Immunoelectrophoresis. Methods and Applications. Universitetsforlaget, Oslo. 1973. 5. Boesken, W. H. & Hammer, D. K. Purification and chemical characterization of u basement mem-

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brane glycoprotfin present in the urine of nephritic rabbits. Happf-Seyltn Z. physiol. Chem. 353, !429. 1972. 6. Boesken. W. H.. Meinhart. K.. Rentsch. 1. & Hammer, D. K. Characterization of basement membrane antigens present in the urine of normal and nephritic rabbits. Int. Arch. Allergy 43, 2J2, 1972. 7. Hawking, K. M. 8c Milgrom. F. Basement membrane antigens combinin^t with antisera to human j;lomcrular basement membrane and lymphocytes. ht. Arch. Allergy 43, 641. 1972. 8. Hoiby. N. Be Axetscn, N. H. Identification and quantitation of precipitins against pseudomous aeruginosa in patients with cystic fibrosis by means of crossed immunoelectropboresis with in. termediate gel. Acta {>.uh. microhiol. scand. 81. 298. 197 V 9. Huang, p. & Kalant, N. Isolation and characterization of aniigenic components of rat glomerular basement membrane. Canad. J. Biochem. 46, 1523. 1968. 10. Jensen, H. & Henriksen. K. Proleinuria in nonrenal infectious diseases. Actj med. scand. t96, 75, 1974. 11. Kefalides, N. A. Chemical properties of basement membranes. Int. Rer. exp. Path. 10. I. 1971a. 12. Kefaiides. N. A. Structure and biosynthesis of basement membranes. Int. Rer. conned. Tissue Res. 6. 6?,, 1973 13. Kroll, J. Tandem-crossed immunoelectrophoresis, pp. 57-59 in Axelsen. N. H.. Kroll, J. & Weeke. B. (eds.) A Manual of Quantilatit'e Immunoelectrophoresis. Methods und Applications. Universitctsforlaget, Oslo, 197 3. H. Lerner. R. A. & Dixon, F. J. The induction of acute glonicrulont'phritis in rabbits with soluble antigens isolated from homologous and autologous urine. /. Immunot. 100. 1277, 1968. n . Markowitz. A. S. Interactions of aniiglomerular basement-membrane antisera. Immunology .?. 117, 1960. 16. Marquardt. H., Wilson, C. B. & Dixon, F. J. Isolation and immunological characterization of human glomerular basement membrane antigens. Kidney hit- 3. ^7, 197^. 17. McPhaul. J. J. & Dixon, F. J. Immunoreactive basement membrane antigens in normal human urine and serum. /. trxp. Med. 7.16, 1}9>, 1969. 18. Naruse. T. & Sbibato, S. Mechanical extraction of the water-soluble antigen that induces nephrotoxic antiserum from rat glomerular basement membrane. Immunology 22, 925, !972. 19. Spiro. R. G. Studies on the renal j;tomenilar basement membrane. Preparation and chemical composition. /. hiol. Chem. 242. 1915. 1967. 20. Spiro. R. G. Studies on the renal glomerular basement membrane. Nature of the carbohydrate

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units and their attachment to the peptide portion. /. hiol. Chem. 242, I92J. 1967. 21. Steblay. R. V. Some immunoiogic properties of buman glomerular basement membrane. IV. Immediate or delayed nephritis induced in rabbits by sheep antihuman glomerular basement membrane sera. /. Immunol. 9^, 517. 1965. 22. Svendsen. P. J. & Axelsen. N. H. A modified antigen-antibody crossed eloctrophoresis characterizing tbe specificity and titre of human precipitins against Candida alhicans. J. immunol. Methods ;. 169, 1972.

Received 28 April 1975 Received in revised form 8 August 1975

25. Unanue, E. R. & Dixon, F. J. Experimental allergic glomerulonephritis induced in the rabbit witb heterologous renal anigens. / . exp. Med. 125, t49, 1967. 24. Unanue, V.. R.. Dixon. F. J. & Feldman, I. D. Experimental allergic glomerulonepbritis induced in tbe rabbit with homologous renal antigens. / . exp. Med. 125. I6J, 1966. 25. Weeke. B. Crossed immunoelectropboresis. pp. 47-56 in Axelsen, N. H., Krdll. J. & Weeke. B. (eds.) A Manual of Quantitative Immunoelectrophoresis. Methods and Applications. Universitetsforlaget, Oslo, 1973. 26. Westberg, N. G. & Michael, A. F. Human glomerular basement membrane. Preparation and composition. Biochemistry 9, 3837, 1970.

Immunological characterization of human glomerular basement membrane antigens.

Immunological Characterization of Human Glomerular Basement Membrane Antigens K. HENRIKSEN Sc H. JENSEN Immunological Ijibi>r3tor>', University Qinic...
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