Monoclonal antibodies from rats immunized with fragment D of human fibrinogen

THROMBOSIS RESEARCH 22; 309-320, 1981 0049-3848/81/090309-12$02.00/O Printed in the USA. Copyright (c) 1981 Pergamon Press Ltd. All rights reserved.

MONOCLONAL ANTIBODIES FROM RATS IMMUNIZED WITH FRAGMENT D OF HUMAN FIBRINOGEN*

Stephen J. Kennel ‘, James . P. Chen2, Patricia K. Lankford' and Linda J. Foote' %iology Division, Oak Ridge National Laboratory, P. 0. Box Y, Oak Ridge, Tennessee 37830, and %niversity of Tennessee, Memorial Research Center, Knoxville, Tennessee 37920.

(Received 21.12.1980; in revised form 4.5.1981 Accepted by Editor T.S. Edgington) ABSTRACT Fischer rats were immunized with fragment D (Fg-D) of hunan fibrinogen (Fg) to obtain antibody specific for neoantigens unique to this molecule. Absorption of serum with whole Fg indicated that some of the antibody produced reacted preferentially with Fg-D. Hybridoma cultures were prepared by fusion of immune rat spleen cells with mouse myeloma P3-X63-Ag8. Monoclonal antibodies obtained from these cultures fell into two classes: (a) Those reacting equally well with Fg and Fg-D. (b) Those reacting preferentially but not absolutely with Fg-D. Antibody from hybridoma 104-14, a member of the first group had an affinity for Fg-D of 1.5 x log M- 1 while antibodies from 106-59 and 106-71 (jro_"p1 2) demonstrated much lower affinities of 1.0 x lo7 and 4.7 x 10 M respectively. The cross reactivity of antibodies in the second group indicated that they react with protein conformations that are altered during production of Fg-D from Fg.

INTRODUCTION Enzymatic degradation of fibrinogen (Fg) by plasmin and leukocyte proteases results in the production of specific protein fragments (see reviews I, 2). These fragments can be distinguished from the parent molecule by antibodies which recognize neoantigens (3). Quantitative assay of fibrinogen-fibrin degradation products (FDP) should be useful in assessing the clotting status of patients as well as assaying for proteases that produce these fragments (i.e. plasmin) (4, 5, 6). Competition radioimmunoassays using heterologous antisera are currently available (5, 7, 8) which distinguish FDP, viz. fragment E (Fg-E) and fragment D (Fg-D) from the parent molecule. Key words: Monoclonal antibody, fibrinogen, fragment D, neoantigen, conformation antigen, affinity constants.

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Monoclonal antibody specific for these fragments would circunvent the need for immunoadsorption to remove antibodies against common determinants shared between Fg and FDP, and therefore may be useful for developing more direct assay procedures for FDP in plasma. To this end, we have undertaken the production of monoclonal antibody to neoantigens of hlananFg-D. We report here characterization of rat-mouse hybridoma (9) antibodies reacting with Fg-D. MATERIALS and METHODS Protein preparations Human Fg (grade L purchased from AB Kabi, Stockholm, Sweden) was proteolyzed by urokinase-activated plasminogen as described by Chen and Schulof (10) except that the plasmin digestion of Fg was performed in the presence of 2 rrMcalcium chloride as suggested by Haverkate and Timan (11). Fg-D was isolated from the reaction mixture by QAE-Sephadex chromatography according to the method of Chen et al. (12). Fg and Fg-D were each radioiodinated using chloramine T (13) and 1251 NaI (Amersham, Arlington Heights, IL) to specific activities approaching 100,000 cpm/ng. The iodinated proteins were purified by gel filtration through an AcA 34 (LKB) column equilibrated with 0.01 M sodium phosphate (pH 7.4) and 0.15 M NaCl (PBS) containing 5 mg/ml of Pentex bovine serum albumin (BSA) (Miles Laboratories, Elkhart, IN) and 0.02M e-aminocaproic acid. The eluant fractions were pooled and stored at a concentration of 100 ng/ml at -2O'C for no more than I month. Proteins were analyzed for purity by sodiun dodecyl sulfatepolyacrylamide gel electrophoresis using a 4-1096gradient of acrylamide (14). Molecular weights (MU) of Fg and Fg-D were determined to be 340,000-350,000 and 98,000, respectively, compared to MW of marker proteins used (i.e. ovalbumin, 43,000; bovine serum albunin, 68,000; phosphorylase-B 94,000; B-galactosidase, 130,000; myosin, 200,000). Freshly iodinated samples demonstrated similar MW's, however, upon storage for more than 1 mo., breakdown to MWs of 240,000 for Fg and 70,000 for Fg-D was noted. Immunization and antibody assay Specific pathogen free Fischer 344 rats (Oak Ridge National Laboratory colony) were immunized by intraperitoneal injection of 200 ug Fg-D emulsified in complete Freund's adjuvant followed by 3 weekly injections of Fg-D (200 ug) in incomplete Freund's adjuvant. Three days prior to sacrifice, animals were boosted with 370 pg Fg-D in saline. Immune serum was collected by cardiac puncture just prior to sacrifice. Absorption of serum to remove antibodies reacting with the native Fg molecul_ewas done by incubating 1.0 ml of Fg (20 mg/ml) in PBS containing 0.02 M caminocaproic acid mixed with 1 ml of rat antiserum to Fg-D for 2 hrs at 4'C. The mixture was centrifuged at 20,000 Xg for 10 min. to remove large immune complexes. Titration curves for binding of radiolabeled antigens (antigen binding capacity - 50%) (ABC-SO) as well as antigen binding data for determination of affinity constants were done in a final reaction volune of 85 ~1 as described previously (15) except for antibodies from culture 104-14 which were tested in 1.0 ml volune so that lower antigen concentrations could be used.

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Hybridoma

MONOCLONAL ANTIBODY TO FG-D

311

production

Cells were cultured in Dulbecco's modified Eagles medium (catalogue no. 430-2100, GIBCO, Grand Island, NY) supplemented with 2 nF1 glutamine, 100 ug of streptomycin per ml and 100 U of penicillin/ml and 20% fetal calf serum (Microbiological Associates, Walkersville, MD) (DME-20). Medium without calf serum is designated DME. Hybridoma cultures (16) were produced by fusion of lo* viable lymphocytes, prepared from the spleen of an immune rat, with lo7 P3-X63-AgB cells (provided by Dr. Frank Fitch, University of Chicago, with permission from C. Milstein). Fusion was accomplished in 1 ml of DME containing 45% (w/v) polyethyleneglycol 1540 (Koch-Light Laboratories Ltd., Colnbrook Bucks, England) for 1 min at 25°C. Cells were slowly diluted into DME-20 (8 mls), washed once, incubated in 10 ml DME-20 overnight at 37°C in 7.5% COz. The following day, cells were diluted with 30 ml DME-20 mediun containing hypoxanthine, aminopterin, and thymidine (DME-HAT) (17) to select against unfused parent myeloma cells and plated into five 96 well plates (Falcon 3042, Becton Dickinson and Co., Oxnard, CA). Cells were fed with DME-HAT on days 1 and 4 and with mediun lacking aminopterin on days 7 and 11. Hybridoma cultures were screened for antibody production to Fg-D between days 12 and 14 and cultures positive for antibody production were subcultured into 24 well plates and then to 25 ml flasks (Corning Glass Works, Corning, NY). Screening Supernatant fluids from hybridoma cultures were tested for antibody production to Fg-D. Antigen was bound to plastic plates by incubation of 50 ul of 20 ug/ml Fg-D in PBS, in each well for 18 hr at 37°C. Wells were saturated by adding 120 ul of 50 mg/ml BSA in PBS for 2 hrs and then each well was washed with PBS with an automatic cell harvestor (Otto Hiller Co., Madison, WI)* Hybridoma culture fluid (50 Ill/well) was added for 2 hrs at 37'C and the plates were washed, and finally incubated with affinity purified (15) 1251 rabbit antibody to rat IgG (50 ~1 of 10 ug/ml at lOO,OOO-200,000 cpn/pg protein). The plates were washed and placed in film cassettes with RP-5 X-ray film (Eastman Kodak, Rochester, NY) on Cronex intensifier screens (Dupont, Wilmington, DE) for 18 hrs at -70°C. Rat immunoglobulin (Ig) was classified by double diffusion analysis in 1% agarose in PBS using class and subclass specific antisera to rat Ig (Miles Antibodies from supernatants of hybridoma cultures in log Laboratories). phase (5 x lo5 to 1 x lo6 cells/ml) were concentrated by precipitation with Precipitates were dissolved in l/50 the anonium sulfate (50% saturation). original volume of PBS and dialyzed against PBS before storage at -2O'C. Cloni

nq

Cells from actively growing hybridoma cultures were cloned by limited At 2-3 weeks of growth, wells containing dilution in prewarmed DME-20. colonies were screened for antibody and several cultures were expanded and frozen. Two dimensional

gel analyses

Rat antibodies were purified from concentrated, cloned hybridoma culture fluids (18) and iodinated using chloramine T (13). After gel filtration on AcA 34 resin in 5 mg BSA/ml PBS, aliquots from pooled preparations were analyzed by two dimensional gel electrophoresis. The method of O'Farrell

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(19) was modified as suggested by Dr. Russell Tracy, Mayo Clinic (personal communication) to extend the pH range of isoelectric focusing gels and 10% acrylamide slab gels were used for the electrophoresis dimension. Labeled spots were detected by autoradiography. RESULTS To demonstrate that rat sera can preferentially recognize antigens on Fg-D, sera from animals immunized with hunan Fg-D were tested for antibody binding to radiolabeled Fg and Fg-D. Freshly iodinated preparations were incubated with rat antiserun and the resulting immune complexes precipitated with secondary rabbit antibody to rat IgG (15). Data from antibody binding assays (Fig. 1) show that the rat antisera is very high titer, demonstrating ABC-50's (15) of 0.14 and 0.83 mg/ml for Fg-D and Fg, respectively. The higher ABC-50 for Fg than Fg-D can be partially explained by differences in their molecular weights since ABC-50 values are calculated on a weight rather than molar ratio. Absorption of rat antiserum to Fg-D with whole Fg resulted in a 10,000 fold decrease in ABC-50 for Fg but only about a 35-fold decrease in ABC-50 for Fg-D (Fig. 1). Thus at least some antibody in the rat antiserum reacts preferentially with Fg-D. To produce monoclonal antibody to Fg-D, spleen cells from immune rats were fused with mouse myeloma cells (P3-X63-Ag8). Hybrid cells growing in 96 well microtiter plates were tested for antibody production using antigen coated Of 517 cultures tested, 17 scored as positive at the first screening plates. (Table 1). Cells in these wells were transferred to 24-well plates and supernates retested for antibody after 3-5 days of growth. Eleven of the 15 cultures which were transferred (2 of the original 17 failed to grow) were selected for expansion and frozen storage.

1

10

8

I

I

100

1,000

10,000

ioo,ooo

ANTISERA (raciprocol dilution)

FIG.

1

Antigen binding titer of rat antiserun for Fg-D open symbols) or for Fg (closed symbols). Serun was tested before (d ,m) or after (0, ) absorption with Fg. One ng of 125I antigen was incubated with 5 ~1 ot test serum in a final volume of 85 ~1 for 18 hrs at 4OC before precipitation of Results are normalized to 100% for immune complexes using secondary antibody. maximum antigen bound, background levels of binding not subtracted.

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MONOCLONAL ANTIBODY TO FG-D

1

TABLE Screening

of hybridoma

313

fluids for antibody to Fg-D

Fusion Number

First Screen*

Second Screen*

Binding if 1 ng of antigen**

104 105 106

151350 O/72 2195

9/13

7113

212

2/2

*Positive/nunber tested. Culture fluids were scored for antibody by visual analysis of autoradiograms of 96 well screening plates. **Culture fluids binding at least 10% of the 1 ng antigen sample were considered positive.

Antibody from supernatant fluids of these cultures was further characterized after ammonium sulfate precipitation from culture medium. Immunoglobulins were tested for class and subclass by double diffusion analysis (Table 2). Approximately 10 pg of antibody from each concentrate was tested for binding of 1 ng of 12% Fg or 1251 Fg-D in a double antibody precipitation test (15). Two groups of antibodies were recognized (Table 2); (a) antibodies from cultures 104-1, 104-13, 104-6, 104-11, and 104-14 reacting with Fg and Fg-0 equally, and (b) antibodies from cultures 106-59, 106-71, 104-2 and 104-5 reacting with Fg-D preferentially but not absolutely. Cultures 104-7 and 104-B yielded no significant precipitation of 1251-Fg-D.

TABLE 2 Characteristics

of Hybridoma

Antibody ----

Binding Antibody from culture

of 1 ng of antigen*

Fg-D

Fg

Antibody Class and Subclass

104-14 104-13 104-6 104-11

98 55 17 21

97 61 17 15

91

IgG2b IgG2a IgG2a _ *** _ ***

106-59 106-71 104-2 104-5

72 76 53 43

27 40 28 12

IgGl IgGl IgG2a IgG**

104-l

93

104-7 104-8

63

Normal rat IqG

2

*% of total antigen bound. **Sticlass could not be identified. ***Class could not be identified.

L 10

I gG2b _ ***

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Antigen binding curves were constructed for three of these antibodies to comoare their titers and bindinq soecificities (Fig. 2). Antibody from culture 104-14 has very high ABC-50's o? 0:22 and 0.63 mg/ml for Fg-D and Fg, respectively. Antibodies from 106-59 and 106-71 have much lower ABC-50's of about 65 ug/ml for Fg-D, but demonstrated a 30-50 fold preference for Fg-D over Fg. Cloning of cultured lines Fusions 104 and 106 from which these hybridoma lines were-developed, demonstrated relatively low fusion frequencies (i.e.
O0

t 10

I 100 ANTIBOOY

f

I 1,000 (reciprocal

to,000

100,000

dilution)

FIG. 2 Antigen binding titer of hybridoma antibodies for Fg-D (open symbols) or Fg (closed s bols) as in Fig. 1. 104-14 (0 e, 106-59 (&A) 106-71 (0.5,. 10 ~1 of 0.5 mg/ml monoclonil aniibodies were use: for the first dilution. Background levels shtracted.

MONOCLONAL ANTIBODY TO FG-D

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315

we demonstrated that less than 10% of the isotope was present in mouse P3 IgG and that >90% of the isotope was bound to rat IgG. Some representative two dimensional gel profiles of the radioiodinated antibodies are shown in Fig. 3. The profiles indicate that the purified antibodies are much more homogeneous c, d) than normal rat IgG (Fig. III, pool a). Patterns from clones (Fig. IIIb, 106-71A and 106-71B derived from the same parent culture, 106-71, are nearly identical.

106-59 B

* 106-718

106-71 A

f

0 56 -

.r)

??

22 510

5:5

Q.0

615

;.O

7:5 PH

FIG.

3

Autoradiograms of two dimensional gel electrophoresis of purified, Normal rat IgG, panel A; 106-59, panel B; radioiodinated antibodies. 106-71A, panel C; and 106-71B, panel 0.

Concentrated supernate fluids from cloned cultures 106-71A and 106-718 were tested for binding of 12? FgD and Fg (Fig. 4). These antibodies show similar binding profiles for the two target antigens and also have similar affinity constants for FgD (Table 3). Antibodies were further characterized by constructing curves for the amount of antigen bound at various concentrations by a constant amount of antibody (Fig. 5). A double reciprocal plot of this data (Fig. 6) allows graphic determination of affinity constants and maximum antibody levels (20, 21). Data for monoclonal antibody-antigen interactions result in a straight line relationship while data using antisera give non-linear graphs The intercepts on the abcissa (-l/Ka) and reflecting their heterogeneity. ordinate (l/Ah max) were determined graphically and used to calculate the affinity constant (l/Ka) and the maximum antigen binding at infinite antigen

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60 0,. 0,m E .-[r z 040 I- 50 i

71A 718

0 \

0

16

ANTIBODY

33

(r&iproc~l

64

dilution)

-

{?A ’

‘--

FIG. 4 Antigen binding titer of cloned hybridoma antibodies for FgD (open s

Fg (closed

symbols)

as in Fig.

1; 106-71A (a,

0)

and 106-71B (m,

bols)

and

fl).

TABLE 3 Affinity

constants

and amounts of antibody l/Ka

106-59 106-71 106-71A 106-718 104-14*** 104-14B*** 104-14A*** Rat anti FgD sewn.**

1.0 4.7 1.9 2.1 1.5

x x x x x

l/h 107 lo& lo& 106 109

4.0 6.0 x 109 log

*

0.3-10.5

x 10s

1.2 0.7 0.7 0.7 0.3

binding max

x x x x x

to FgD Pb max ( ug antigen/ml

107 107* 107* 106 10’0

72 130 138 123 57

0.2 1.2 x 10’0

;:

-

-

*Average of duplicate determinations. **Undiluted antibody preparations are 0.5-1.0 mg/ml. ***Antibody tested at l/2000 dilution in a 1 ml reaction

volume.

/Ab ) **

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MONOCLONAL ANTIBODY TO FG-D

I/" /

0 A/

2 5:

/ /

l#!f 0

I 10

I 20

1 30

1251 Fg-D PRESENT

FIG. Antigen binding by hybridoma antigen concentrations.

antibodies

I 40

8 50

(Mxt0-8)

5 106-59

&

and 106-71

(0)

at different

2.4-

I

I 0.4

I

I

I

I 1.2

0.8

I 1.6

1

I 2.0

l/free (l/mole x108)

FIG.

6

Double reciprocal plot of data from Fig. 5. ordinate intercepts, l//-b max.

Abcissa intercepts,

-l/Ka and

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MONOCLONAL ANTIBODY TO FG-D

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concentrations (Ab max). Data in Table 3 show that 104-14 antibody and its clones have very high affinities (1.5-6.0 X log M-l) while antibodies from 106-59 and 106-71 have much lower values, 1.0 x lo7 and 4.7 x lo6 M-l, respectively. Similar data for whole antiserun gave a non-linear plot which could be represented by two components of l/Ka = 0.3 x log and 10.5 x log. DISCUSSION Serological analysis of Fg degradation products has been useful in determining the structure of the Fg molecule as well as in quantitation of its breakdown (5, 6). Monoclonal antibody reagents have the advantage that they bind to only a single antigenic determinant and thus have absolute specificity. We have started to develop a battery of these reagents from rats immunized with Fg-D. The Fg-E derived from hunan Fg is poorly immunogenic in rats (S. J. Kennel, unpublished observations, 1980). We demonstrated that serun from immune rats contained antibody that react preferentially with Fg-D. Quantitation of specific serum antibody indicated that it should be possible to isolate monoclonal antibody which was specific for Fg-D. Analysis of antibody from our first panel of hybridoma cultures showed two groups of antibodies produced: 1) those reacting with Fg and Fg-D equally and 2) those reacting preferentially but not absolutely with Fg-D. No antibodies with absolute specificity for Fg-D were detected. Hybridoma antibody 104-14 has a very high affinity for Fg-D and Fg, while antibodies from 106-59 and 106-71 have much lower affinities but demonstrated a 30-50 fold preference for Fg-D over Fg. Plow and Edgington (5) also reported that significant loss of native Fg antigenic expression was observed with the progressive cleavage of Fg. Hence, antibodies 106-59 and 106-71 are probably directed against the surface determinant of Fg-D which represents conformational shifts from the parent Fg molecule. Although antibodies directed against neoantigens of Fg-D can be isolated by immunoadsorbent methods from heterologous antisera, their isolation from supernatants of cultured hybridoma cells is certainly much more convenient. Unfortunately, 106-59 and 106-71 antibodies show preferential, though not absolutely specific, binding to Fg-D. These antibodies will not be useful for clinical assays of FDP in plasma, but they may be of sufficient sensitivity to detect FDP in serun. We are currently screening more hybridoma cultures to probe Fg-D structure and develop specific D-neoantigen assays. REFERENCES 1.

DOOLITTLE, R. F. Fibrinogen and fibrin. Bloom and Thomas, in press, 1981.

In:

Haemostasis._andThrombosis.

2.

DOOLITTLE, R. F. Structural aspects of the fibrinogen-fibrin conversion. Advances in Protein Chemistry 27, l-109, 1973.

3.

PLOW, E. F., HOUGIE, C. and EDGINGTON, T. S. Neoantigenic expressions engendered by plasmin cleavage of fibrinogen. J. Immunol. 107, 1496-1500, 1971.

4.

SACK, E. S. and BURASCHI, J. J. Med. 284, 1441, 1971.

Fibrinogen degradation products.

N. Eng,

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5.

PLOW, E. and EDGINGTON, T. S. Immunobiology of fibrinogen: Emergence of neoantigenic expressions during physiologic cleavage -in vitro and -in vivo. J. Clinical Invest. 52, 273-282, 1973.

6.

GROPP, C., EGBRING, R. and HAVEMAN, K. Fibrinogen split products, antiproteases and granulocytic elastase in patients with lung cancer. Eur. J. Cancer 16, 679-684, 1980.

7.

CHEN, J. P. and SHURLEY, H. M. A simple efficient production of neoantigenic antisera against fibrinolytic degradation products: Radioimmunoassay of fragment E. Thrombosis Res. l, 425-434, 1975.

8.

PLOW, E. F. and EDGINGTON, T. S. A cleavage-associated neoantigenic marker for a Y chain site in the NHZ-terminal aspect of the fibrinogen molecule. J. Biol. Chem. 250, 3386-3392, 1975.

9.

GALFRE, G., HOWE, S. C. MILSTEIN, C., BUTCHER, G. W. and HOWARD, J. C. Continuous cultures of fused cells secreting antibody of predefined specificity. Nature 266, 550-552, 1977.

10.

CHEN, J. P. and SCHULOF, R. S. Radioimmunoassay of fibrinogen-fibrin degradation products: Assay for fragment E - related neoantigen methodological aspects. Thromb. Res. I6, 601-615, 1979.

11.

HAVERKATE, F. and TIMAN, G. Protective effect of calcium in the plasmin degradation of fibrinogen and fibrin fragments D. Thromb. Res. IO, 803-812, 1977.

12.

CHEN, J. P., SHURLEY, H. M. and VICKORY, M. F. A facile separation of fragments D and E from the fibrinogen/fibrin degradation products of three mammalian species. Biochem. Biophys. Res. Commun. 6l, 66-71, 1974.

13.

A method of trace iodination of McCONAHEY, P. J. and DIXON, F. J. proteins for immunologic studies. Int. Arch. Allergy 29, 185-189, 1966.

14.

LAEMMLI, U. K. Cleavage of structural proteins during assembly of the head of bacteriophage T4. Nature (London) 227, 680-684, 1970.

15.

KENNEL, S. J. Purification of a glycoprotein from mouse ascites fluid by immunoaffinity chromatography which is related to the major glycoprotein of murine leukemia viruses. J. Biol. Chem. 251, 6197-6204, 1976.

16.

KOHLER, G. and MILSTEIN, C. Antibodies to major histocompatibility antigens produced by hybrid cell lines. Nature 256, 495-497, 1975.

17.

LITTLEFIELD, J. W. Selection of hybrids from matings of fibroblasts in vitro and their presumed recombinants. Science 145, 709-710, 1964. --

18.

KENNEL, S. J., FOOTE, L. J., and LANKFORD, P. K. Analysis of surface proteins of mouse lung carcinomas using monoclonal antibodies, submitted to Can. Research.

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O'FARRELL, P. H. High resolution two-dimensional electrophoresis of proteins. J. Biol. Chem. 250, 4007-4021, 1975.

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20.

ACCOLLA, R. S., CARREL, S. and MACH, J. P. Monoclonal antibodies specific for carcinoembrionic antigen and produced by two hybrid cell lines. PE. Natl. Acad. Sci. 77, 563-566, 1980.

21.

CELADA, F., MACARIO, A. J. L. and deMACARI0, E. C. Enzyme activation by antibodies: A method to determine the binding constant of the activating antibody towards one determinant of E. coli B-D-galactosidase. Immunochemistry l0, 797-804, 1973.