Clinica Chimica Acta, 191 (1990) 211-220 Elsevier
One-step sandwich enzyme immunoassay for human laminin using monoclonal antibodies K. Iwata Fuji Chemicals Indtrstries Ltd., Takaoka, Toyama (Japan) (Received
Key words: Sandwich
13 June 1990; accepted
28 June 1990)
A one-step sandwich enzyme immunoassay (one step sandwich EIA) for human serum immunoreactive laminin was set up with a pair of monoclonal antibodies prepared against human placental laminin Pl fragment. The assay was characterized by carrying out two immunoreactions simultaneously, laminin Pl fragment reacting with both a monoclonal antibody as a solid phase and a horseradish peroxidaselabeled monoclonal antibody (Fab’) against human laminin Pl fragment as conjugate. Sensitivity of the immunoassay was 0.01 ng/well (0.5 pg/l), and linearity was obtained between 0.01-20 ng/well (0.5-l 000 pg/l). The levels of laminin in sera from normal individuals and patients with liver cirrhosis, hepatocellular carcinoma and primary biliary cirrhosis were 103 of 15 pg/l, 228 f 70 pg/l, 341 + 163 pg/l and 232 + 93 pg/l, respectively. Protein immunoblotting showed that the serum immunoreactive laminin measured by the assay was a fragment with rel mol mass of 200 kDa.
The major components of basement membranes are type IV collagen, laminin and heparan sulfate proteoglycan. Laminin is a non-collagenous glycoprotein with a rel mol mass of about 900 kDa and has a cross-like shape, consisting of three 200 kDa subunits and a 400 kDa subunit. It is assumed that laminin and heparan sulfate proteoglycan are bound to type IV collagen [1,2]. Laminin, as well as type IV collagen has been known to be involved in various clinical disorders, e.g., liver
* Correspondence Japan.
to: Dr. K. Iwata, Fuji Chemicals
0 1990 Elsevier Science Publishers
Ltd., 530 Chokeiji,
fibrosis [3-51, diabetes mellitus  and degradation of basement membranes accompanying tumor growth and metastasis [7,8]. Radioimmunoassays for laminin and 7-S collagen in serum were also developed using rabbit antisera [3,7,9]. We have reported that the central triple helix domain in human type IV collagen could be determined very simply with high sensitivity (40 pg) by using a one step sandwich enzyme immunoassay (one step sandwich EIA) with two kinds of monoclonal antibodies [lo]. In this study, we report the application of monoclonal antibodies specific for human laminin Pl fragment to a one step sandwich EIA for the simple and sensitive determination of human laminin with a short assay time. That is, the one step sandwich EIA performs the first and the second immunoreactions simultaneously and requires only 75 min. With this assay we evaluated the diagnostic application of i~unoreactive laminin in the sera of patients with liver disorders including primary biliary cirrhosis. Materials and methods
Bovine serum albumin (Fraction V, BSA) and ~-phenylenedi~e . HCl (OPD) were obtained from Sigma Chemical Company, St. Louis, MO, USA. Pepsin from porcine gastric mucosa and horseradish peroxidase (Grade 1, POD) were obtained from Boehringer Mannheim GmbH, Marmheim, FRG. A protein molecular weight standards, POD-labeled goat anti-mouse immunoglobulins and MonoAb-ID EIA Kit were obtained from Bethesda Research Laboratories, Inc., Gaithersburg, MD, USA, Organon Teknika Corporation, West Chester, PA, USA d Zymed Laboratories, Inc., San Francisco, CA, USA, respectively. Centricon & 10 and nitrocellulose filters (Trans-Blot transfer medium, 0.45 pm) were from Amicon Corporation, Danvers, MA, USA and Bio-Rad Laboratories, Richmond, CA, USA, respectively. 2 x 8 wells-Micro Well Module in frame and Falcon 96 well-flexible assay plates were obtained from Nunc, Denmark and Becton Dickinson Labware, Oxnard, CA, USA, respectively. CNBr-ac~vated Sepharose 4B and DEAE-Sephacel were obtained from Pharmacia Fine Chemicals AB, Uppsala, Sweden. Ultrogel AcA44 and Protein A-Cellulofine were obtained from LKB, Villeneuve la Garenne, France and Seikagakukogyo, Tokyo, Japan, respectively. N-( e-Maleimidocaproyloxy) succinimide (EMCS) and 3,3’-diaminobenzidine were obtained from Dojindo Laboratories, Kumamoto, Japan. Foetal bovine serum (FBS) and RPM1 1640 were obtained from Filtron Inc., Victoria, Australia and Flow Laboratories, Irvine, Scotland, respectively. 2-Aminoethanethiol * HCl and other chemicals were obtained from Wako Pure Chemical Industries, Ltd., Osaka, Japan. Sera of patients with liver disorders were used after diagnostic histological examination of biopsied specimens. Normal sera were obtained from individuals without any serological abno~a~ty of liver function. Each serum was frozen at -40°C until use and assayed within 3 mth. Buffers
The most frequently used buffers were a 30 mmol/l Na-phosphate buffer (A), pH 7.0, containing 0.1 mol/l NaCl, 10 g/l BSA and 10 mmol/l EDTA, and a 10
mmolJ1 Na-phosphate NaCl.
buffer (B), pH 7X), containing
1 g/l BSA and 0.1 mol/l
Lamiain Pi &agmeM was prepared from hwx3n pZaCexSaas desctikd by Ristefi and Timpl [llf. Type I, III, IV, V and VI collagens were isolated by salt precipitation from pepsin digests of human placental tissues according to the method of Mayne et al. [X2) with some modifications f13]. Long form 7-S domain of type IV coffagen was prepared from the pepsin ~lubi~~ed type IV colIagen as described by Biszeli et al, ff4j.
Two &week-old female BALB/c mice were i~~~~~ with 60 $g of purified laminin emulsified with an equal volume of Freund’s complete adjuvant. After 15 and 50 days, they received booster injections of 60 ir;g each of the ~~~o~en in sdine (154 mmol/l NaCl}. On the 3rd day after the Iast i~traveno#s ~~j~tio~~ the spleen was removed and the spleen cells were isolated for fusion. The hybridization, subsequent culturing, and cloning of hybrids were carried out as described by Oi and Herzenberg . Spleen cells and mouse myeloma cdls (P3-NS-I) were mixed in a ratio uf 5 to 1 in RP&G I&@ medium and were ~~~~d~~d in 50% ~~~~~~~~~n~ I Ceils f6 x 10’) wse ~~~~~~~~~ into each tve3f d 9&d ~a~-bott~m~ g$KZd tissue culture plates and were cultured in RPMI 1640 medium supplemented with 15% (v/v) FBS containing hypoxanthine, aminopterin and thymidine in an atmosphere of ~~u~dified 7% CO2 in air at 37YZ. Anti-labia of spent media was tested in 96-well flat-bottomed microtitration plates by the ELISA according to the method of Rennard et al. [Xf. Ce#s secretirmgrno~~~o~~ an~~b~~~ were cultured in wells of a transfer plate (Z&I-welleat-b~~torn~ plate) using mouse ~yrn~y~es as feeders. Wells of the transfer plate that showed high reactivity were identified, and cells forming plaques were cloned by the limiting dilution method in 96-well flat-bottomed tissue culture plates. Each hybridoma obt~ned was grown in a flask, and was injected in~rap~~ton~ly into ~~st~~e-treat~d BALB/c mice for ascites ~r~~~~~ (1 x 30’ cells per mouse]. The a&tic fluids were harvested l-2 weeks later. A&bodies (IgCl) in as&c fluids were purified by ammonium sulfate fractionation, followed by DEAE-Sephacel and Ultrogel AcA44 column chromatographies or a protein A-Cellulofine column c~orn~~~~~~phy, accordirrg to the method of Obata et al. [Xl]* The amount of antibodies was determined by taking extinction atwfficient to be 14.0 f1’Q
Purified mouse moarrrclonal antibodies were digested with pepsin from porcine mucosa to obtain F(ab’)z as described by Hamaguchi @tal. . F(ab’), obtained was reduced with ~-~~~~e~ane~ol~ and the Fab’-POD conjugate was prepared by the method of Hasbida et & I191using E&KS as if ~~e~~d~ co~~~~d for the ~onju~a~~on of Fab’ to POD through thiol groups in the hinge region.
Preparation of monoclonal antibody-coated microplates According to the method of Obata et al. [lo] with some modifications, each of the wells of the 2 x 8 wells-MicroWell Module in frame were coated at 4’C for 24 h with 100 pl purified monoclonal antibody (0.1 g/l) of clone 22”3BlO in 0.1 mol/l Na-phosphate buffer, pH 7.5, containing 15 mmol/l NaN, by physical adsorption and washed twice with saline. The microplate was then blocked at 4’C for 24 h with buffer B and washed in saline just before use. One-step sandwich EIA method A 20 yl portion of laminin Pl fragment as standard solution or serum was mixed with 100 ~1 mouse monoclonal antibody, Fab’ (clone HL-4H3)-POD conjugate (1 mg/l) in buffer A in the Falcon 96 wells-flexible assay plate. A 100~~1portion of the solution was then added to each of the wells coated with monoclonal antibody, the microplate was left for 60 min at room temperature without shaking (immunoreaction). After washing the microplate twice with saline, the POD activity bound to the microplate was assayed for 15 min at room temperature in a solution (100 ~1) of 0.1 mol/l citric acid-O.2 mol/l Na-phosphate buffer, pH 5.0, containing 46 mmol/l OPD and 0.02% hydrogen peroxide (v/v). The reaction was stopped by adding 100 ~1 sulfuric acid (1 mol/l) and the absorbance at 492 nm was measured in a microplate reader (Toso Mode1 MPR-A4, Tokyo, Japan). The concentration of laminin was calculated from the standard curve. Preparation of protein-Sepharose 43 BSA (4.5 mg) and a mouse monoclonal antibody of clone 22-3BlO (0.5 mg) were coupled to CNBr-activated Sepharose 4B (1 g) according to the instructions of Pharmacia Fine Chemicals AB. Affinity-purification of laminin in serum Serum (2.5 ml) from a patient with liver cirrhosis or normal sernm (8 ml) was applied to a mouse monoclonal antibody (IgG) of clone 22-3BlO-bound Sepharose 4B column (gel, 3.5 ml) which was equilibrated with 0.1 mol/l Na-phosphate buffer, pH 8.3. The laminin was eluted with 0.1 mol/l glycine-HCl buffer, $I 2.5. The eluate (4.0 ml) was i~~iately neutralized with 0.1 ml of 3 01/l Tris-HCl buffer, pH 8.0 and concentrated to about 200 ~1 using Centricon & 10 for i~unoblotting. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting According to the method of Laemmli and Favre  with slight modification, SDS-PAGE was carried out on a mini-slab gel (6.4 X 8.9 X 0.1 cm) formed by 35 g/l acrylamide and 5 g/l agarose S with the running buffer containing 20 g/l SDS and 0.025 mol/l Tris-0.192 mol/l glycine buffer, pH 8.5. After electrophoresis the protein were transferred onto nitrocellulose filter. The filter was then soaked in 5 mg/l POD-labeled mouse monoclonal antibody (IgG) of clone HL-4H3 at 20°C for 2 h. Staining was performed in a 0.01% hydrogen peroxide (v/v) system with 0.56 mmol/l 3,3’-~~nobe~dine f21].
Production of monoclonal antibodies Sixteen hybridoma cell lines secreting monoclonal antibodies against laminin Pl fragment were selected by ELISA from a total 4 cloning (Table I). According to isotype assay of each monoclonal antibody using a MonoAb-ID EIA Kit. All the light chains were the K type but the heavy chains were variable as follows: 14 clones, yl clone (22-lEl), y2a and 1 clone (HL-SAlO), (Y.
standard laminin curue by one step sandwich EIA system The sensitivity of the assay for standard laminin was 0.01 ng/well, calculated as the concentration which is two standard deviations (2 SD) above the zero standard. The linearity was from 0.01-20 ng/well by combination of a monoclonal antibody (clone 22-3BlO) as a solid phase and a POD-labeled monoclonal antibody (Fab’) of clone HL-4H3 as a conjugate (Fig. 1). The reaction time of the assay was 60 min for immunoreaction and 15 min for the color reaction. The assay coefficient of variation (CV SE;)of the standards was examined at 10 different laminin levels from 0.039-20 ng/well (n = 10 each}. CV values obtained were 1.9-9.3%.
TABLE I I~ogIobuIin type of monoclonal antibodies and their i~unor~c~~ty fragment haying rel mol mass of 200 kDa
with purified Iaminin Pl
Immunoreactivity with purified laminin a
HL-2D12 HL-4H3 HL-5AlO 22-1El 22-2Al 22-3BlO 22-5C9 22-6B4 22-7C5 22-8E9 22-967 22-10HI 22-24D5 22-25B7 22-26E10 22-27E7
+ f + + -t + + + + + + + + +
kWK IgGZa/K IgGl/K IgGl,‘K IgGl/K IgGl/K igGl/K IgGl/x IgGl,‘K IgGl/K IgGl,‘K IgGl,‘K 1gGl/~ 1gGl/~
a After SDS-PAGE of-O.2 pg purified laminin Pl fragment, the protein was transferred onto a nitroce&Iose fiiter. The filter was then treated with each monoclonal antibody and POD-labeled goat anti-mouse i~unoglob~ins as described in ‘Materials and Methods’.
Fig. 1. Standard laminin curve by one-step sandwich EIA system. The purified laminin Pl fragment was determined with a combination of a mouse monoclonal antibody of clone 22-3BlO as a solid phase and a mouse monoclonal antibody (Fab’f of clone HL-4H3-POD conjugate using a MicroWell Module in frame as described in ‘Materials and Methods’. Values in the parentheses are expressed as con~ntration of laminin Pl as /.lg/l.
Precision The assay CV values of intra-assay in the determination of normal and pathological sera laminin having 100,173,440 and 531 pg/l were 4.4~6.3% (n = 10). On the other hand, CV values of inter-assay were 3.0-6.2% (n = 10) at serum laminin levels of 117, 259, 375 and 464 pg/l (Table II).
TABLE II Intra-assay and inter-assay variations of one-step sandwich EIA method
Normal serum Pathological sera
lOO& 4.98 173* 9.10 440 & 27.9 531 f 23.3
(5.0) (5.3) (6.31 (4.4)
Normal serum Pathological sera
117* 259* 375 f 464+
(6.21 (3.0) (5.61 (5.91
7.20 7.84 21.1 27.6
Intra- and inter-assay variations for one normal and 3 pathological samples were expressed as mean + SD and CV value.
Recoveries of standard laminin added to human sera into laminin concentrations of 94 or 250 pg/l over a range of 31-500 pg/l were 105.6 + 1.6% and 102.6 k 5.2% (mean k SD), respectively (Figs. 2A and B). These results indicate that apparently no serum components interact with laminin to interfere with monoclonal antibody binding. Immunoreaction temperature
Immunoreaction temperature variations of the assay were examined at 10 different serum laminin levels from 80-770 pg/l. CV values of immunoreactions temperature between lo-20°C and 20-30°C were from 5.0-5.4s. These data suggest that the assay method was not affected by ambient temperature. Determination of laminin in human sera
The newly developed one-step sandwich EIA method was applied to detect immunoreactive laminin levels in 15 sera from healthy individuals (N) and patients with liver cirrhosis (LC), hepatocellular carcinoma (HCC) and primary biliary cirrhosis (PBC). Immunoreactive laminin concentration in the sera from the N group was 103 f 15 pg/l (mean * SD) against 228 + 70 pg/l, 341 f 163 pg/l and 232 + 93 pg/l in patients with LC, HCC and PBC, respectively (Fig. 3). The upper reference value (mean f SD) was exceeded by 14/15,15/15 and 11/15 of the cases in the LC HCC and PBC groups, respectively. Specificity
Cross-reactivity of the two monoclonal antibodies used in the assay system was examined in pepsin-solubilized type IV collagen, 7-S domain of type IV collagen and other types of collagens, including types I, III, V and VI. No or negligible crossreaction was observed with these collagens (Table III). Only the laminin Pl fragment contained one immunoreactive collagenous band with rel mol mass of 200
Fig. 2. Recoveries
of the laminin added to human serum. Laminin was determined EIA method as described in ‘Materials and Methods’.
by one step sandwich
Fig. 3. Determination of immunoreactive laminin in the N group and patients with LC, HCC and PBC. Laminin was determined by one-step sandwich EIA method as described in ‘Materials and Methods’. Vertical bars show mean + SD. A horizontal dotted line indicates the upper reference value (mean of the N group + 2 SD).
TABLE III Cross-reactions of basement membrane collagen and interstitial collagens Cone
1000 fig/l (20 ng/well)
Collagen types I
a Pepsin-solubilized tw IV collagen. Values in the table are expressed as cross-reactivity as a percentage of the arithmetic mean on the basis of laminin Pl fragment value.
Fig. 4. I~~oblot~g of purified laminin Pl fragment and laminin in sera. After SDS-PAGE of 0.2 pg purified laminin Pl fragment (lane A) and 10 ~1 each of concentrated eluates from LC patient serum (lane B) or normal serum (lane C), the proteins were stained as described in ‘Materials and Methods’.
kDa by i~unoblotting after SDS-PAGE in serum from a normal indi~du~l or a patient with LC (Fig. 4). Discussion Several competitive radioimmunoassays have been developed for human, mouse, rat laminin Pl fragment, using rabbit antisera [3,7,9]. No immunoassay system has, however been established utilizing monoclonal antibody against laminin Pl fragment. We developed a reliable one-step sandwich EIA system with microplate as a solid support using a combination of two monoclonal antibodies specific to the laminin Pl fragment. The principle of our present one-step sandwich EIA system was based on simultaneous i~unoreaction for only 60 min of antigen, monoclonal ~tibody-moated microplate as solid phase, and POD-labeled mon~lonal antibody (Fab’), and the color reaction for 15 min after washing the solid phase. The sensitivity of our present one-step sandwich EIA system with a short assay time is 2.580-fold higher than those of a radioimmunoinhibition assay system with a longer assay time (30-36 h) reported by Niemelii et al.  and Brock et al. . The one step sandwich EIA system is only able to detect antigens having two different epitopes, each of which is recognized by its corresponding monoclonal antibody. Thus this assay is much more specific than the radioimmunoinhibition assay, as shown in Figs. 2 and 4. This difference between two assays might be reflected in the difference of laminin levels in sera. As shown in Fig. 3, the concentration of immunoreactive laminin (103 + 15 pg/l) in sera of normal subjects by the immuno~say is higher than that of laminin PI (26.0 k 5.5 pg/l) estimated by the ra~oi~unoi~bition assay using rabbit antisera . The mon~lon~ antibody (clone HL-4H3) utilized as the conjugate in our present one step sandwich EIA system was found to be specific for laminin and showed little or no cross-reactions with the basement membrane type IV collagen and 7-S domain of type IV collagen or the interstitial collagens, types I, III, V, VI (Table III and Fig. 4). The elevated concentration of immunoreactive laminin in sera of patients with LC, HCC and PBC was observed in our preliminary examination (Fig. 3). This suggests that the assay may be useful for disease involving basement-membrane metabolism. In this study, the numbers of sera from patients with LC, HCC or PBC were not large enough to allow a definite conclusion. Studies are now under way, in our laboratory to assay laminin in additional serum samples from normal subjects, as well as patients with liver diseases, and tumor growth and metastasis . Acknowledgements We would like to express our gratitude to Karim T. A. Malik and Tomoe Sakai for their excellent help in preparation of the manuscript. References 1 Martinez-Hemandez 677.
A, Amenta PS. The basement membrane in pathology. Lab Invest 1983;48:656-
2 Timpl R, Engel J, Martin GR. Laminin-a multifunctional protein of basement membranes. Trends Biochem Sci 1983;8:207-209. antigen in 3 Niemell 0, Risteli L, Sotaniemi EA, Risteli J. Type IV collagen and laminin-related human serum in alcoholic liver disease. Em J Clin Invest 1985;15:132-137. 4 Nouchi T, Womer TM, Sato S, Lieber CS. Serum procollagen type III N-terminal peptides and laminin Pl peptide in alcoholic liver disease. Alcohol Clin Exp 1987;11:287-291. 5 van Zanten RAA, van Leeuwen RE, Wilson JHP. Serum procollagen III N-terminal peptide and laminin Pl fragment concentrations in alcoholic liver disease and primary biliary cirrhosis. Clin Chim Acta 1988;177:141-146. 6 Hbgemann B, Voss B, Altenwerth FJ, Schneider M, Rauterberg J, Gerlach U. Concentration of 7S collagen and laminin Pl in sera of patients with diabetes mellitus. Klin Wochnschr 1986;64:382-385. I Brocks DG, Strecker H, Neubauer HP, Timpl R. Radioimmunoassay of laminin in serum and its application to cancer patients. Clin Chem 1986;32:787-791. 8 Rao CN, Margulies IMK, Tralka TS, Terranova VP, Madri JA, Liotta LA. Isolation of a subunit of laminin and its role in molecular structure and tumor cell attachment. J Biol Chem 1982;257:97409744. 9 Risteli J, Rohde H, Timpl R. Sensitive radioimmunoassays for 7-S collagen and laminin: application to serum and tissue studies of basement membranes. Anal B&hem 1981;113:372-378. 10 Obata K, Iwata K, Ichida T, et al. One step sandwich enzyme immunoassay for human type IV collagen using monoclonal antibodies. Clin Chim Acta 1989;181:293-304. 11 Risteli L, Timpl R. Isolation and characterization of pepsin-fragment of laminin from human placental and renal basement membranes. Biochem J 1981;193:749-755. 12 Mayne R, Zettergren JG, Mayne PM, Bedwell NW. Isolation and partial characterization of basement membrane-like collagens from bovine thoracic aorta. Artery 1980;7:262-280. 13 Matsumoto E, Muragaki Y, Ooshima A. Increased amount of serum type IV collagen peptides in human liver fibrosis as determined by enzyme-immunoassay with monoclonal antibodies. Acta Path01 Jpn 1989;39:217-223. 14 Risteli J, B&inger HP, Engel J, Furthmayr H, Tip1 R. 7-S collagen: characterization of an unusual basement membrane structure. Eur J Biochem 1980;108:239-250. 15 Oi VT, Herzenberg LA. Immunoglobulin-producing hybrid cell lines. 1n:Mishell BB, Shiigi SM, eds. Selected methods in celhilar immunology. San Francisco: W.H. Freeman and Company 1980;351-372. immunoassay (ELISA) for 16 Remmrd SI, Berg R, Martin GR, Foidart JM, Robey PG. Enzyme-linked connective tissue components. Anal Biochem 1980;104:205-214. 17 Ey PL, Prowse SJ, Jenkin CR. Isolation of pure IgG,, IgG,, and IgG,, immunoglobulins from mouse serum using protein A-Sepharose. Immunochemistry 1978;15:429-436. Y, Yoshitake S, Ishikawa E, Endo Y, Ohtaki S. Improved procedure for the conjugation 18 Hamaguchi of rabbit IgG and Fab’ antibodies with B-D-galactosidase from Eschen’chia coli using N,N’ -o-phenylenedimaleimide. J Biochem 1979;85:1289-1300. for the 19 Hashida S, Imagawa M, Inoue S, Ruan K-H, Ishikawa E. More useful maleimide compounds conjugation of Fab’ to horseradish peroxidase through thiol group in the hinge. J Appl Biochem 1984;6:56-63. of the head of bacteriophage T4. 1. DNA packaging events. J 20 Laemmli UK, Favre M. Maturation Mol Biol 1973;80:597-599. 21 Tanabe K. Western blotting (in Japanese). Cell Technol 1983;l & 2:1061-1068. VP, Williams JE, Liotta LA, Martin GR. Modulation of the metastatic activity of 22 Terranova melanoma cells by laminin and fibronectin. Science 1984;226:982-985.