Clinica Chimica Acta, 202 (1991) 179-190 0 1991 Elsevier Science Publishers B.V. All rights reserved 0009~8981/91/$03.50


CCA 05103

Sandwich enzyme immunoassay for serum integrins using monoclonal antibodies Masahiko


Tohru Kurome, Kazuki Yamamoto, Fumitsugu Hino and Ikunoshin Kato



Biotechnology Research Laboratory, Takara Shuzo Co., Ltd., Otsu, Shiga (Japan)

(Received 10 October 1990; revision received 19 July 1991; accepted 22 July 1991) Key words: Integrins; Monoclonal antibodies; Enzyme immunoassay; Liver disease

Summary We produced monoclonal antibodies (mABs) against human integrins. Competitive enzyme-linked immunosorbent assay (ELISA) revealed that each mAB bound to different antigenic determinants. We then developed sandwich-type enzyme immunoassays (EIAs) to measure the concentration of fibronectin receptor (FNR) and vitronectin receptor (VNR). Serum immunoreactive integrin levels were measured using these EIAs in various liver and malignant diseases. In almost all cases of liver cirrhosis (LC) and hepatocellular carcinoma (HCC), serum integrin levels were significantly elevated, but were in the normal range in gastric, colon, lung cancer, and acute hepatitis (AH). The correlation between serum FNR and VNR levels was statistically significant in all cases of liver disease, and no correlation was observed between these integrin levels and conventional biochemical markers such as AST, ALT, and GGT. The serum integrin levels were demonstrated to be a potential diagnostic marker for hepatic fibrogenesis and carcinogenesis, and these sandwich EIAs could be useful for determination of these integrins in clinical laboratory tests.

Introduction Specific adhesive interaction range of biological processes,

between cells and substrata are crucial to a wide including organ development, phagocytosis, cell

Correspondence to: M. Katayama, Biotechnology Research Laboratory, Takara Shuzo Co., Ltd., Otsu, Shiga 520-21, Japan.


motility, and immune response. These interactions often involve the recognition of adhesion proteins by specific cell surface receptors. One family of receptors that appears to provide the interactions between cells and extracellular matrix is known as the integrin superfamily [l]. These receptors, consisting in mammals of an ((Yand P-subunit which are noncovalently associated, recognize the Arg-Gly-Asp (RGD) sequence in fibronectin and vitronectin [2]. Recently, it has been reported that transforming growth factor P-stimulated fibronectin receptor synthesis and increased receptor expression may help drive fibronectin deposition into matrix [3,4]. Furthermore, tumor cell invasion and metastasis are complex processes that probably involve interactions of extracellular matrix molecules and their receptors, and the increases in integrins might contribute to the invasive phenotype, and enhanced integrins expression were observed in Rous sarcoma virus (RSV)-induced tumor in vivo [5]. We describe here the preparation of monoclonal antibodies (mABs) against human integrins by immunizing affinity purified placental fibronectin receptor (FNR) and vitronectin receptor (VNR), and the application of mABs to sandwich enzyme immunoassays (EIAs) for the simple and sensitive determination of human integrins. With these assays we demonstrated elevated immunoreactive integrins in the sera of patients with liver disease, and serum integrin levels reflected selectively the hepatic fibrogenesis and carcinogenesis. Materials and methods Purification of integrins

Human FNR and VNR were purified from placenta essentially as described by Pytela et al. [6], with the following modification. CBP-Agarose (Takara Shuzo, Japan), which was prepared by coupling the 30 kDa recombinant DNA derived cell-binding peptide (CBP) of human plasma fibronectin to Sepharose 4B, was used instead of the 110 kDa fibronectin fragment-coupled Sepharose 4B described previously [6]. Human term placenta was homogenized in a blender in phosphatebuffered saline containing 1.0 mmol/l CaCl,, 1.0 mmol/l MgCl, (PBS2+), and 3 mmol/l phenylmethylsulfonyl fluoride (PMSF) and 50 mmol/l octyl P-Dthioglucopyranoside (Sigma). The homogenate was stored for 30 min at 4°C and then the supernatant was loaded on the Sepharose 4B precolumn, and the pass through fraction was applied to the Gly-Arg-Gly-Asp-Ser-Pro (GRGDSP)-Sepharose 4B column. The pass through fraction from the GRGDSP-Sepharose 4B column was further applied on the CBP-Agarose column. These columns were washed with PBS2+ with 25 mmol/l octyl P-D-thioglucopyranoside and 1 mmol/l PMSF, and the receptors were eluted with 10 mmol/l EDTA in the same buffer. The FNR and VNR were eluted from the CBP-Agarose and GRGDSP-Sepharose 4B, respectively. The proteins in eluted fractions were analyzed by SDSPAGE. The further purification, using immobilized wheat germ agglutinin column chromatography reported elsewhere [6], was not necessary in this study, because the affinity chromatography gives nearly homogeneous receptor. The CBP-Agarose


and GRGDSP-Sepharose PBS.

4B were regenerated

by washing with 8 mol/l

urea in

Production of mABs

Male BALB/c mice were immunized intraperitoneally with 50 pg of human placental FNR or VNR emulsified with Freund’s complete adjuvant. The antigens were injected 3 wk later with Freund’s incomplete adjuvant and again after another 2 wk. Two weeks after the final injection, the mice received an iv. booster dose of 2.5 pg of antigens. Three days later, the spleen was excised. Spleen cells were fused with mouse myeloma P3Ul in the presence of polyethylene glycol 1500 (Wake Pure Chemical, Japan), according to the previous method described by Oi and Herzenberg [7]. The cells were inoculated in microtiter plates. Hybridomas were screened for the secretion of specific antibodies by enzyme-linked immunosorbent assay (ELISA) using microtiter plates coated with antigens and peroxidase (POD)-conjugated goat anti-mouse IgG (Cappel, USA). Positive wells were cloned twice by limiting dilution. Ascites fluids were prepared by intraperitoneal injection of hybridoma cells. To purify the mABs, they were first precipitated with 50% saturated ammonium sulfate, then dialysed against 50 mmol/l Tris-HCI buffer (pH 7.5) and purified by Mono Q-FPLC (Pharmacia, Sweden). Specificity of mABs

Sodium dodecyl-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of integrins was performed according to Laemmli [S]. Gels were stained with Coomassie blue or electroblotted to nitrocellulose membrane (Schleicher and Schuell) as described by Towbin et al. [9]. Strips of the membrane were incubated with mABs. Bound mABs were visualized using POD-conjugated goat antimouse IgG, 4-chloro-l-naphthol, and H,O,, as described by Kittler et al. [lo]. Preparation of POD-conjugated mAB

Ten mg of each purified mAB to integrins were conjugated to 5 mg of POD (Boehringer Mannheim, FRG), as described by Nakane and Kawaoi [ll]. Competitive binding ELISA

Competitive binding to integrins of the mABs was studied by ELISA [121. Serial dilutions of unlabeled mAB in PBS containing 1% bovine serum albumin (BSA) were incubated for 1 h in microtiter plates coated with integrins, after which the suboptimal diluted POD-conjugated mAI was added. After 3 h the plates were washed, and then the POD activity bound to the plate was measured for 20 min at room temperature with a solution (100 PI/well) of 0.1 mol/l citrate-O.2 mol/l phosphate buffer, pH 5.0, containing 1 g/l o-phenylenediamine (OPD) and 0.01% (v/v> H,O,. The reaction was stopped by the addition of 100 ~1 of 1 N sulfuric


acid. Absorbance value at 492 nm was measured by microtiter plate reader (Flow Laboratories, USA). EL4 for integrins

Polystyrene beads (Immunochemicals, Japan) were coated with mAB solution (10 pg/ml in PBS) and incubated overnight at 4°C. The beads were washed three times with PBS and blocked with 1% BSA in PBS containing 0.01% thimerosal overnight at 4°C. The mAB coated beads could be stored for several months without significant loss of activity at 4°C. Twenty ~1 of standard or lo-fold diluted serum sample was added to each tube, 200 ~1 of POD-conjugated mAB, diluted in PBS containing 1% BSA and 0.05% Tween 20 was added to each tube, and then a mAB coated polystyrene bead was added. The tubes were left for 90 min at room temperature without shaking. Following incubation, the bead was washed three times with PBS. The POD activity bound to the beads was measured for 20 min at room temperature with a solution (300 pi/tube) of 0.1 mol/l citrate- 0.2 mol/l phosphate buffer, pH 5.0, containing 1 g/l o-phenylenediamine and 0.01% (v/v) hydrogen peroxide. The reaction was stopped by the addition of 1 ml of 1 N sulfuric acid. The absorbance value at 492 nm was measured by Quantum II (Abbot Laboratories, USA). The molecular size of integrin antigens in 1.5 ml serum samples was determined by EIAs after molecular-sieve chromatography on a 1.5 X 130 cm Sephadex G-200 (Pharmacia, Sweden) column, equilibrated with PBS. Serum samples

Serum samples were obtained from 19 cases of hepatocellular carcinoma (HCC), 12 of liver cirrhosis (LC), 12 of acute hepatitis (AH), 6 of stomach cancer, 4 of colon cancer, 3 of lung cancer, and 18 healthy individuals. The serum samples were stored at -40°C until assayed. The activity of aspartate transaminase CAST), alanine transaminase (ALT), and y-glutamyl transpeptidase (GGT) in serum were determined with the specific reagents purchased from Wako Pure Chemical (Japan). The d a t a are expressed as the mean f SD. Student’s two-tailed t test was used to determine statistical significance among goups and P < 0.05 was construed as significant. Results mABs production

Four clones and 8 clones were obtained that secreted anti-FNR and anti-VNR, respectively, and two anti-FNR mABs (FNRS and FNR31) and two anti-VNR mABs (VNR3 and VNRS) were selected based on their high titer. The subclass of these all mABs was IgG,,. Western blotting of human placental FNR and VNR clearly showed that all mABs recognized p-subunits of FNR and VNR, respec-


Fig. 1. Immunoblot analysis of the specificity of mABs. The 2 pg proteins of FNR (A) and VNR (B) were separated on 7.5% SDS-PAGE under nonreducing conditions respectively. The proteins transferred electrically on nitrocellulose membranes were then stained with Coomassie blue (lanes 1) or immunostained with POD-labeled mABs of FNR31 (lanes 2, 3) and VNR3 (lanes 4, 5).

Anti-FNR mAJ3s did not react with VNR and anti-VNR mABs did not react with FNR (Fig. 1). The N-terminal amino acid sequences of purified FNR and VNR, used for immunagen and antigen in imm~ological analysis, were analyzed in the PSQ-1 automated protein sequencer system (Shimadzu Corporation, Japan) using Edman degradation. Some integrin p subunits were previously demonstrated to have blocked N-terminal residues [13]. The p chains of FNR and VNR, recognized specifically by mABs used in this study, were also found to have the blocked N-terminal residues. However, we observed that co-isolated (Ychains of FNR and VNR evidently have the N-terminal residues (Phe-Asn-Leu-Asp-Ala-Glu-Ala-ProAla-Val-Leu-Ser-Gly, and Phe-Asn-Leu-Asp-Val-Xaa-Ser-Pro-Ala-Glu; Xaa means indeterminant residue) which are identical to the N-terminal sequences of (Y chain of FNR and VNR, respectively, reported in the previous results [14,15]. Additionally, we performed the immunopurification of integrin p chains from 200 tively.


ml of normal human serum using immobilized mABs on agarose column, separated the proteins eluted from column on SDS-PAGE, and immunostained the target antigens with rnABs. Serum FNR antigen isolated using immobilized FNR31 mAB showed the apparent molecular mass of 120 kDa, and serum VNR antigen isolated using immobilized VNR3 mAB showed the apparent molecular mass of 100 kDa, on SDS-PAGE under nonreducing condition. Several reports have already suggested that FNR p subunit has a slightly higher molecular mass than VNR p subunit apparently [6]. The molecular masses of serum FNR and serum VNR were completely distinguishable on electrophoresis. These results substantiate that the two kinds of receptor antigens purified from placenta are truly FNR and VNR respectively, and that the serum substances reactive with anti-FNR mABs are distinct from those reactive with anti-VNR mABs. A solid phase competitive ELISA was used to determined whether FNRS and FNR31 recognize the different epitopes on FNR. A 100-fold excess of unlabeled FNRS did not compete with the binding of POD-conjugated FNR31 to FNR. A lOO-fold excess of unlabeled VNR5 did not compete with the binding of POD-conjugated VNR3 to VNR. These results indicate that these mABs against FNR and VNR recognize distinct epitopes on each antigen, respectively. EL4 for integrins

Calibration curves were prepared with a standard assay system by using purified FNR (Fig. 2A) or VNR (Fig. 2B). In EIA for FNR, the minimum detectable concentration calculated from the mean + 2 SD of 10 control samples without FNR was 10 ng/ml, and the linearity was obtained to 1,280 ng/ml by combination of mAB of FNRS as a solid phase and a POD-conjugated mAI of FNR31 (Fig. 2A). In EIA for VNR, the minimum detectable concentration calculated from the mean + 2 SD of 10 control samples without VNR was 30 ng/ml, and the linearity was obtained to 3,840 ng/ml by combination of mAB of VNR5 as a solid phase and a POD-conjugated mAB of VNR3 (Fig. 2B). The total assay time was 2 h, for each. The dilution curves for 3 serum samples are shown in Fig. 2. The curves appear to be parallel, indicating that the same immunoreactive substance is measured in the different dilution series. These two assays were specific for FNR and VNR, respectively, because collagen type I, II, III, IV, fibronectin, vitronectin, and laminin showed no reactivity (data not shown). Assay validity

The precision of the assays was tested by assaying 4 serum samples 10 times (intra-assay) and in 10 consecutive assays (interassay). Table I shows the results of this testing, and the coefficient of variation (CV) values were < 10%. Analytical recoveries of standard added to human serum were 90.2-113.0% and 88.6-111.8% for FNR and VNR, respectively. This indicates that apparently no serum component interacts with FNR and VNR to interfere with mAI binding.

185 Dilution .16







Dilufon -8



2.0 A



0.05 20












Fig. 2. EIA for FNR and VNR. Standard curves (0) for FNR (A) and VNR (B) and dilution curves of serum samples from a healthy subject t A ) and patients with HCC (0) and LC (0 1 are shown. Serum samples were originally diluted IO-fold with PBS containing 1% BSA. Serial dilutions of pre-diluted sera are denoted above the curves.

TABLE I Intra-assay and interassay precisions of EIAs for FNR and VNR Serum sample



mean f SD kg/ml)

cv (o/o)

mean f SD (/*g/m0

Intra-assay (n = 10)


1.2OkO.05 2.71 f 0.10 6.51+ 0.27 10.81 kO.70

4.2 3.7 4.1 6.5

3.66+0.10 14.82 f 0.64 16.41 f 0.63 28.29k 1.63

2.8 4.3 3.8 5.8

Interassay (n = 10)


1.50f0.12 3.48 & 0.20 8.47+0.41 9.66 f 0.89

8.0 5.8 4.9 9.2

2.96+0.20 10.01 f 0.65 16.54+ 1.36 22.03 f 1.66

6.8 6.5 8.2 7.5

The precision of the EIAs was evaluated by assaying four samples of serum 10 times each in a continuous series (intra-assay) or twice each in 10 consecutive assay (interassay).


-. r*-%-l#r-,







: “c

= .



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is a



a 2 LA




? . .



2 >

. . .





J . .

. l

. b .. + . l>* .I






0; &



01 AH







Fig. 3. Serum FNR levels (A) and serum VNR levels (B) in liver diseases. The bars represent the SD of FNR and VNR levels. Student’s two-tailed t test was used to compare the results between two groups: significant differences were assessed at the level of P < 0.05 (*), P < 0.01 (* *), and P < 0.001 (* * *). N, normal; AH, acute hepatitis; LC, liver cirrhosis; HCC, hepatocellular carcinoma; OC. cancer other than HCC.

Determination of integrins in human sera

The serum integrin levels of patients and healthy subjects are shown in Fig. 3. The FNR concentration (mean + SD> was 6.30 f 3.20 pg/ml in HCC, 6.31 k 2.76 in LC, 1.84 * 0.67 in cancer except for HCC, 2.57 + 1.11 in AH, and 1.82 + 0.63 in healthy subjects. The serum FNR levels were significantly higher in HCC and LC than in cancer other than HCC, AH, and healthy subjects, as shown in Fig. 3A. The VNR concentration (mean ~frSD) was 22.7 rt 11.9 kg/ml in HCC, 27.8 + 21.9 in LC, 7.23 & 2.85 in cancer other than HCC, 10.3 k 4.66 in AH, and 5.45 + 2.16 in healthy subjects. The serum VNR levels were significantly higher in HCC and LC than in cancer other than HCC, AH, and healthy subjects, as well as FNR (Fig. 3B). If it is assumed that the cut-off value for normal FNR and VNR concentration in serum is 3.08 and 9.76 pg/ml (these values correspond to the mean values + 2 SD for 18 healthy control subjects), respectively, high serum FNR levels were found in 18 of the 19 (95%) patients with HCC and in 10 of the 12 (83%) with LC, and high serum VNR levels were found in 16 of the 19 (84%) with HCC and in 10 of the 12 033%) with LC.

187 TABLE II Comparison between serum levels of integrins and serum activities of other biochemical markers in liver disease Correlation coefficient a






0.629 h

0.153 0.187

-0.101 - 0.096

0.037 0.088

a Serum AST, ALT, and GGT activities were measured simultaneously with determination of serum levels of integrins in 33 patients with liver diseases. Correlation coefficients were calculated between the levels of integrins and the activities of other 3 markers, and between the levels of two integrins, FNR and VNR. ’ A significant correlation was observed on statistical regression analysis only between the levels of two integrins (P < 0.01).

We established the molecular size of immunoreactive integrins in serum by molecular-sieve chromatography (Fig. 41, and they appear to be relatively uniform in size with a molecular weight between y-globulin and albumin. Standard of









0’ 5 : B


g 8 y




200 -


o_L J







Fig. 4. Characterization of the size of FNR (A) and VNR (B) antigens in normal (0) human serum and in serum of HCC patients (01 by molecular-sieve chromatography on a column of Sephadex G-200. Elution position of globular proteins of known molecular mass, which were used for calibration, are indicated by arrows.


integrins also eluted as one major peak at the position with the same molecular weight. No difference in the molecular weight was observed between in HCC serum and in normal serum (Fig. 4), as seen in the analysis of sera of two different patients with HCC and two different patients with LC (data not shown). Comparison of integrins and other biochemical markers in patients with liver diseases

Table II shows the correlation between serum integrin concentration and AST, ALT, and GGT levels in all cases of liver disease. A correlation between levels of FNR and VNR was observed (P < 0.011, but no correlation between the levels of these integrins and conventional biochemical markers of liver disease was observed. Discussion

In experiments reported here we have developed EIAs that use mABs direct epitopes on human integrins to measure the soluble form of these receptor molecules in human sera. Although mABs against integrins were prepared by many investigators [16-181, no immunoassay system has been established using mABs. This is the first report that immunoreactive integrins circulate in soluble forms and their levels in serum are elevated in the patients with LC and HCC. An association between immunoreactive integrins in serum and purified standard of integrin was obtained from gel fractionation in which both eluted from the column with major peaks in the same fraction at approximately loo-120 kDa (Fig. 4). These immunoreactive integrins probably are intact p-subunit of integrins, when considered with the specificity of these mABs, as shown in Fig. 1. It has been known for a long time that neoplastic cells present several adhesive abnormalities that may contribute to their invasive ability [19-211. In particular, an enhanced integrin expression was observed in transformed cells [5]. Tumor cells may use their adhesion receptors to facilitate migration through tissue. The involvement of integrins in tumor cell invasiveness is also suggested by the observation that RGD peptides inhibit the migration of tumor cells through tissues and restrict metastatic dissemination of tumor cells [22-241. Tumor cell attachment and migration is thought to play a crucial role in the complex multistep process of tumor invasion and metastasis. Moreover, it has been suggested that elevated expression of integrins and extracellular matrix proteins play a pivotal role in matrix synthesis characterizing fibroproliferative responses to tissue injury such as liver fibrosis [3]. In this study, we examined the integrin levels in sera of patients with various liver diseases and cancers. In HCC, the serum integrin levels were significantly higher than those in cancer other than HCC and healthy subjects. In LC, the serum integrin levels were also higher than those in AH and healthy subjects. It is well known that patients with LC are a high-risk group for the development of primary hepatomas. The low level of serum integrins in AH suggests that serum integrins reflect selectively the fibrosis of liver. Moreover, there is no correlation


between the level of serum integrins and that of serum AST, ALT, and GGT. From the results, the generation of serum integrins in soluble form does not appear to be the result of cell necrosis with subsequent integrin release. We are at present trying to study the elevation of integrins expression in hepatic tissues by immunohistochemical method. Present EIA systems for soluble forms of integrins in sera, however, could be established as a tool with a possible diagnostic value in certain clinical situations of fibrotic liver disease and HCC. References 1 Hynes RO. Integrins: a family of cell surface receptors. Cell 1987;48:549-554. 2 Ruoslahti E, Pierschbacher MD. New perspectives in cell adhesion: RGD and integrins. Science 1987;238:491-497. 3 Roberts CJ, Birkenmeier TM, McQuillan JJ, et al. Transforming growth factor p stimulates the expression of fibronectin and of both subunits of the human fibronectin receptor by cultured human lung fibroblasts. J Biol Chem 1988;263:4586-4592. 4 Heino J, Ignotz RA, Hemler ME, Grouse C, Massague J. Regulation of cell adhesion receptors by transforming growth factor-p. J Biol Chem 1989;264:380-388. 5 Saga S, Chen W, Yamada KM. Enhanced fibronectin receptors expression in Rous sarcoma virus-induced tumors. Cancer Res 1988;48:5510-5513. 6 Pytela R, Piershbacher MD, Argraves WS, Suzuki S, Rouslahti E. Arginine-glycine-aspartic acid adhesion receptors. Methods Enzymol 1987;144:475-489. 7 Oi VT, Herzenberg LA. Immunogloblin-producing hybridoma cell line. In selected methods in cellular immunology. Mishell BB, Shiigi SM, eds. San Francisco: W.H. Freeman & Co., 1980;351372. 8 Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970;227:680-685. 9 Towbin H, Staehelin T, Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: Procedure and some applications. Proc Nat Acad Sci USA 1979;76:4350-4354. 10 Kittler JM, Meisler NT, Viceps-Madore D, Cidlowski JA, Thanassi JW. A general immunochemical method for detecting proteins on blots. Anal Biochem 1984;137:210-216. 11 Nakane PK, Kawaoi A. Peroxidase-labelled antibody. A new method of conjugation. J Histchem Cytochem 1974;22:1084-1088. 12 Stahli CV, Miggiano J, Stocher T, et al. Distribution of epitopes by monoclonal antibodies. Methods Enzymol 1983;92:242-253. 13 Cheresh DA, Smith JW, Cooper HM, Quaranta V. A novel vitronectin receptor integrin (a,,PX) is responsible for distinct adhesive properties of carcinoma cells. Cell 1989;57:59-69. 14 Argraves WS, Pytela R, Suzuki S, Mill&r JL, Pierschbacher MD, Ruoslahti E. cDNA sequences from the (I subunit of the fibronectin receptor predict a transmembrane domain and a short cytoplasmic peptide. J Biol Chem 1986;261:12922-12924. 15 Suzuki S, Argraves WS, Pytela R, Arai H, Krusius T, Pierschbacher MD, Ruoslahti E. cDNA and amino acid sequences of the cell adhesion protein receptor recognizing vitronectin reveal a transmembrane domain and homologies with other adhesive protein receptors. Proc Natl Acad Sci USA 1986;83:8614-8618. 16 Hemler ME, Huang C, Schwartz L. The VLA protein family. J Biol Chem 1987;262:3300-3309. 17 Cheresh DA, Harper JR. Arg-Gly-Asp recognition by a cell adhesion receptor requires its 130.kDa alpha subunit. J Biol Chem 1987;262:1434-1437. 18 Gresham HD, Goodwin JL, Allen PM et al. A novel member of the integrin receptor family mediates Arg-Gly-Asp-stimulated neutrophil phagocytosis. J Cell Biol 1989;108:1935-1943, 19 Poste G, Fidler IJ. The pathologenesis of cancer metastasis. Nature 1980;283:139-146. 20 Liotta LA, Rao CN, Wewer UM. Biochemical interactions of tumor cells with the basement membrane. Annu Rev Biochem 1986;55:1037-1057.


21 Juliano RL. Membrane receptors for extracellular matrix macromolecules: relationship to cell adhesion and tumor metastasis. Biochim Biophys Acta 1987$07:261-278. 22 Humphries MJ, Olden K, Yamada KM. A synthetic peptide from fibronectin inhibits experimental metastasis of murine melanoma cells. Science 1986;44:517-518. 23 Humphries MJ, Yamada KM, Olden K. Investigation of the biological effects of anti-adhesive synthetic peptides which inhibit experimental metastasis of B16-FlO murine melanoma cells. J Clin Invest 1988;81:782-790. 24 Ugen KE, Mahalingam M, Klein PA, Kao K. Inhibition of tumor cell-induced platelet aggregation and experimental tumor metastasis by the synthetic Gly-Arg-Gly-Asp-Ser peptide. J N C I 1988;80:1461-1466.

Sandwich enzyme immunoassay for serum integrins using monoclonal antibodies.

We produced monoclonal antibodies (mABs) against human integrins. Competitive enzyme-linked immunosorbent assay (ELISA) revealed that each mAB bound t...
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