Radioimmunoassay of fibrinogen-fibrin degradation products: Assay for fragment E-related neoantigen -methodological aspects

THROMBOSIS Pergamon

c

RESEARCH 16; 601-615 Press Ltd.1979. Printed

in Great Brirain OOirs-3943 /79~1201-~~601 508 . c_7i3 ~‘0

RADIOIMMUNOASSAY OF FIBRINOGEN-FIBRIN DEGRADATION PRODUCTS: ASSAY FOR FRAGMENT E-RELATED NEOANTIGEN -METHODOLOGICAL ASPECTS* James P. Chen and Richard S. Schulof** Department of Medical Biology/Memorial Research Center University of Tennessee Center for the ?iealth Sciences Knoxville, Tennessee 37920 U.S.A.

(Received 8.1.1979; in revised form 26.7.1973. Accepted by Editor H.L. Nossel)

ABSTRACT E fragment and its antigenically-related degradation products of human fibrinogen (Fg) and/or fibrin (Fb) have been measured in both normal and pathological plasmas by a specific radioimmunoassay (RIA) for the neoantigenic expression of E(Eneo) which is engendered after theplasmic cleavage of Fg or Fb. High titer antisera were produced in rabbits against Fg-E (E derived fromFg). Theanti-Fg-E sera harvested were absorbed with a cyanogen bromideactivated immunoadsorbent column coupled with intact Fg. The Fg-absorbed antisera were employed to develop a double antibody RIA which detected < 1 ng/ml of Fg-E and exhibited little cross-reactivity of Fg at 1 mg/ml. This Eneo RIA did not discriminate between Fg-E and Fb-E (E derived from Fb) in the competitive inhibition profile, and hence The displacement curves it measured total Eneo antigens. of normal plasmas generally showed a parallel relationship to the standard which permitted the calculation of normal Considerably plasma Eneo immunoreactivity (ca. 15 ng/ml). higher plasma Eneo immunoreactivities have been observed in patients with consumption coagulopathy. Modifications of the Eneo RIA which permit the attainment of this sensitivity are described.

*A part of this work was done at the Biomedical Research 77058 while Division, NASA Johnson Space Center, Houston, TX J. P. Chen was under the tenure of Senior Research Associateship awarded by the National Research Council. Memorial **Present address: 10021, U.S.A. New York, New York

Sloan-Kettering

Cancer

Center,

IYi'RODL'CTI91; Plasmin digestion of fibrinogen (Fg) or fibrin (Fb) exposes new antigenic sites on the fibrinogen-fibrin degradation products (FDP) which are thought to be sterically hidden on the native Fg or Fb molecule. These neoantigens, -which have been extensively characterized by Plow and Edgington (l-3) and aiso by Gordon et aA. (4,5), are useful markers of circulating FDP. Since antiserum specific for the neoantigens will not react with native Fg or Fb, plasma levels of FDP can be determined. Due to immunological cross-reaction between Fg and FDP, the currently available immunoassay of FDP, e.g., the hemagglutination inhibition immunoassay (6), utilizes the serum instead of the plasma of patients. In the absence of proteolytic inhibitors, considerable generation of FDP. takes place in vitro during the process of clotting fresh blood. Various fibrin-c inhibitors greatly reduce the release of FDP but do not completely eliminate the in vitro fibrinolysis which occurs during or immediately foll%inge,normal clotting of blood (7). Hence the testing of serum may, in many cases, indicate a spurious concentration of FDP in blood. By determining FDP in plasma, a better estimate of its effective concentration should result. Because of the cross-reactivity of the anti-FD? with Fg in plasma, an assay for a noncross-reacting FDP neoantigen is therefore necessary. In a plasma assay, however, it is difficult to develop a RIA sensitive enough to detect normal plasma FDP levels. The avidity of the antibody is one of the dominant factors determining RIA sensitivity, and we have found that the avidity of the antibodies generated against the neoantigen of FDP is not exceptionally high. the sensitivity of neoantigen RIA is freConsequently, quently insufficient to detect the minute quantity of FDP present in the blood of a normal individual. In this paper we describe the development of a RIA for determining the neoantigen associated with fragment E and E-related FDP which is sensitive enough to measure Eneo immunoreactivity in normal and pathological plasmas. Since both Fg-E and Fb-E fragments inhibited the reaction between anti-Fg-Eneo serum and Fg-E tracer in a nearly identical manner, the Eneo RIA measured total Eneo antigens.

MATERIALS Fibrinogen

and Fg-E and Fb-E

AND METHODS

fragments

Human Fg (grade L) was purchased from KABI, Stockholm, Sweden. Four ml of KABI Fg (5 mg/ml) were clotted at pH 7.4 in the presence of 0.02 M EDTA by adding 10 NIH units of bovine thrombin (Parke, Davis and Co., Detroit, Michigan) for each mg The clots were soluble of Fg, then incubated for 3 hr at 4'C. (within 15 min) in 5 M urea at room temperature indicating the The fibrin clot was washed with absence of cross-linked fibrin. several changes of isotonic saline per day for 2-3 days at 4". Both Fg and Fb were digested by urokinase-activated plasminogen.

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FUGNEST

E-RELATED

SEOXNTIGES

6!,2

FOT

each mg of Fg or Fb 0.063 caseinolytic Iunits of plasminogen The plasminogen was then activated by adding (KABI) was added. 0.564 Plough units of urokinase (Leo Pharmaceutical Products, Digestion at 37" was terCopenhagen, Denmark) per mg Fg or Fb. acid (EACA) at a minated after 5-6 hrs by adding z-aminocaproic final concentration of 0.2 M. The digest was frozen immediately and stored at -50" for later use. Fragment E was isolated by QAE-Sephadex ion-exchange chromatography as previously described (8). To ensure purity, the isolated Fg-E and Fb-E fragments were checked by immunoelectrophoresis using antisera R80 and R41 directed against Fg and normal human serum respectively and by 7.5% polyacrylamide gel electrophoresis in 0.1% sodium No contaminating proteins were detecdodecyl sulfate (SDS). table on immunoelectrophoresis or SDS gel electrophoresis of concentrated solutions (5 mg/ml) of the Fg-E or Fb-E preparation. Antisera Three mg of antigen was dissolved in 1 ml of 0.15 M saline and thoroughly mixed with an equal volume of Freund's adjuvant. Each rabbit was administered weekly with the adjuvant mixture (subcutaneously at the neck and intramuscularly in the hind legs) for a three-week period. For the initial challenge, the antigen was emulsified in complete adjuvant, and for the second and third injections, the antigen was emulsified in a 1:8 mixture of complete and incomplete adjuvants. The first blood sample was taken 4 weeks after the initial injection (this was the time when most rabbits began to produce anti-E antibodies) and the rabbits were bled from the ear vein every 10 days thereafter. Antisera R80 and R41 were produced against Fg and normal human serum respectively. Antisera R45 and R77, used throughout these studies, were prepared by immunization with fragment Fg-E. On immunoelectrophoresis with normal human plasma, antiserum R45 showed strong precipitin lines against Fg and weak lines against a:-macroglobulin. However, antiserum R77 was monospecific against Fg. Of the eight rabbits immunized with either Fg-E or Fb-E, antisera R45 and R77 were found to be most suitable for the RIA of fragment E. (See RESULTS section for the criteria of selecting anti-E sera for RIA). Immunoadsorbent Before coupling the Fg to cyanogen bromide (CNBr)-activated Sepharose 4B, steps were taken to remove traces of plasminogen from the commercial preparation of KABI Fg. Plasminogen was removed essentially by the method of Mosesson (9). When the deplasminogenated Fg was incubated with urokinase at 37", no fibrinogenolysis could be seen within a 24-hr period by immunoelectrophoresis against R80 anti-Fg serum. The activation of Sepharose 4B with CNBr and subsequent protein conjugation were carried out according to a modification of the method of Axen et al . (10) as suggested by Eveleigh (11). Briefly, 2 g of CNBr was used to activate 10 ml of packed Sepharose 4B at pH 11.0 in an ice bath under the hood. The pH of the

reaction mixture was maintained Seti;2en l.c.5 3x3 11.5 for 1 ?.r 5~ the addition with vigorous stirring of 10% XaOZ at 4". The activated agarose gel was washed with 1 L of cold water, and with 100 ml of cold 0.1 M sodium bicarbonate-O.5 M XaCl buffer, pH 8.2 (hereafter called bicarbonate-buffered saline). The Fg was coupled to activated Sepharose 4B in the ratio of 2.5 mg per ml gel, and the reaction mixture was rocked seesaw-like in a screwcapped tube in the cold room overnight. The agarose gel was washed successively three times with 100 ml each of 0.1 1~1sodium acetate0.5 M NaCl buffer (pH 4.0) and bicarbonate-buffered saline. The gel was then treated with 100 ml of 0.1 M ethanolamine in bicarbonate-buffered saline for 1 hr to block remaining active groups. The gel was packed into a 20 ml column, and the immunoadsorbent column was washed extensively with 0.1 M sodiuim borate-C.25 $1 NaCl buffer, pH 8.2 (hereafter called borate-buffered saline) until the eluates had an ODZBQ of less than 0.001 and pE of 6.2. Two ml of Fg-E antiserum were applied to the Fg-immunoadsorbent column, followed by washing with approximately 50 ml of borate-buffered saline. The absorbed antiserum was concentrated to its original volume by negative pressure dialysis. The column was regenerated by washing with 0.1 M glycine HCl buffer (pH 2.4) and 0.1 M glycine HCl buffer plus 0.5% Tween 80 (polyoxyethylene of less than sorbitan monooleate) until the eluate had an ODZB~ 0.005. Radioimmunoassay RIA buffers The buffers for radioimmunological studies consisted of four kinds of phosphate-buffered saline (PBS) containing 0.01 $1 sodium phosphate buffer (pH 7.41, 0.15 M sodium chloride and 0.1% sodium aside. Diluent

PBS:

Fibrinogen

Collecting

Carrier

PBS:

PBS:

PBS:

PBS, pH 7.4 0.1% bovine serum albumin PBS, pH 7.4 0.1% BSA 0.25 M sodium 0.2 !I EACA

(BSA)'

citrate

PBS, pH 7.4 2% BSA PBS, pH 7.4 4% (v/v) normal

'RIA grade BSA was purchased St. Louis, Missouri.

rabbit

serum'

from Sigma Chemical

Co.,

'Four parts of normal rabbit serum were treated with one part of barium sulfate slurry in order to remove prothrombin. This step was introduced to help prevent fibrin clots in assay

-(continued) tubes. The BaSO1, suspension, -was prepared as follows: A solution of 152.5g BaClz. 2H-0 in 500 ml of water was mixed with a solution of 92.5g Na2SOI, in 500 ml of water. After precipitation of the BaSO,, the supernatazt fluid was poured off and 0.9% :;aCl was ad-ded to nearly the original volume. The settled suspension was stirred thoroughly and again left to settle. This procedure was repeated 5-7 times till the supernatant fluid gave no further re>ction on sulfate ion wit:h the addition of 0.2 t! BaClr. After desantation, approximately 330 ml of a thick BaSOk suspension of Even after 6 -38% concentration was kept in a closed flask. months the adsorptiT;e capacity was undiminishLed. Radioiodination Fg-E fragment r&as labeled with ("'I) :;aT by a modification (l_'I) !YaI of the chloramine-T method of Greenwood et al. (12). (dissolved in dilute NaOH solution, pH 8-11) was purchased from Union Carbide Radiochemicals, Tuxedo, New York. Typically, one mCi of carrier free ('*'I) >:a1 was pipeted into a disposable glass culture tube (12 x 75 mm) and the pB adjusted to 7.4 by adding 25 ~1 of 0.4 X sodium phosphate buffer. Wenty ~1 of a 1 mg/ml solution of Fg-E was added and the iodination was started by the addition of 10 ul of a freshly prepared 2.5 mg/ml solution of chloramine-T (obtained from Eastman Organic Chemicals Div., Rochester, 5!!y) in 0.01 M phosphate buffer, pH 7.4. After 40 5213, the reaction was stopped by tte rapid simultaneous addition of 100 1-l of a 2.5 ng/ml solution of sodium neta-bisulfite (NazSiO?, Eastman) in 0.01 ?l phosphate buffer and 50 ;,l of normal rabbit The two reagents were not added sequentially but together. serum. The reaction mixture was immediately transferred to a Sephadex G-25 column (1 x 20 cm) previously equilibrated with diluent PBS. The column was eluted with diluent PBS in order to remove unreacted iodide from iodinated Fg-E. Fractions (0.5 ml) were collected in tubes containing 0.5 ml of collecting PBS to prevent ncnspecific adsorption of radiolabeled Fg-E to the test tubes. By this technique Fg-E was routinely iodineted to specific activities of approximately 25 ,+Ci/;g. Double

antibody

RIA

Assays were performed in disposable glass tubes. For inhibition studies: (a) 0.7 ml of a RIA buffer mixture which consisted of 5 parts carrier PBS, one part diluent PBS and one part fibrinogen PES, E whici? had been diluted (b) 0.1 ml of "'I-labeled with diluent PBS to give ca. 20,000 cpm, (c) 0.1 ml of Fg-E antiseru? suitably diluted in diluent PBS and id) 0.1 ml of standard solutions of nonradioactive Fg-E or Fb-E. When titering antise rum, step (d) was omitted and 0.8 ml of another RIA buffer mixi_ ture (5 parts carrier PBS, 2 parts diluent PBS and one part t=;brinlogeriPBS) was substituted in step (a). All assays were performed in duplicate and included contrcl tubes with labeled E fragment but without anti-E serum (copre*Sf to of r.:aTents, =-i’Zes 5:erz ;tirrp.d 5;. clpitation). GILr .~
vortexing, incubated for 2 days at 4', ckn the antibody-b.3JT.d fraction was precipitated by adding 0.1 ml each of high titer goat anti-rabbit gamma globulin (GARGG, purchased from Antibodies, Inc., Davis, California) and incubating for an additional 24 hrs. The tubes were centrifuged for 4 hr at 2500 rpm, the supernatants removed by gentle aspiration, and the precipitates counted for 2 min in a Packard model 5110 autogamma counter. Counting efficiency of this instrument was 53.7% for iodine-125. An appropriate amount of GARGG to be added in the second reaction was established by using lz51-Fg-E and Fg-absorbed R45 antiserum. 0.05 ml, 0.1 ml and 0.2 ml of GARGG was added to assay tubes containing identical amounts of "'I-Fg-E, R45 anti-Fg-Eneo (suitably diluted) and carrier normal rabbit serum (2% final concentration). Maximum binding was observed using 0.1 ml of GARGG. Initially the double antibody RIA was performed as described above. Subsequently, the plasma assays were carried out by preincubating the antibody with test plasma before the radiolabeled antigen was added; this procedure led to a higher sensitivity than did the simultaneous incubation of the three components (labeled tracer, nonradioactive antigen and antibody) (13). To obtain standard curves for the plasma assay, nonradioactive Fg-E or Fb-E standards were also treated in the same manner. Normal plasma samples were assayed at four concentrations 1400 Ul, 200 lJ1, 100 ~1 and 50 ~1) and the volume of RIA buffer mixture was adjusted to make up a total of 0.9 ml volume for For assaying plasma, Fg-E antiserum and RIA buffer combined. pathological plasmas, generally smaller plasma volumes were reTest samples were first inquired, e.g., 12.5 ~1, 6.25 ~1, etc. cubated with anti-Eneo serum at 37" for 4 hr and 4" for an addiThe preincubation mixture was mixed with 0.1 ml tional 17% hr. of '251-Fg-E in diluent PBS, and incubated at 4" for two days. Then the antibody-bound fraction was precipitated by GARGG in a similar manner as above. Mean values from duplicate determinations were used for all For graphic display either (Bi/Bo) x 100 vs. anticalculations. gen concentration or logit (Bi/Bo) vs. antigen concentration was plotted (14); Bi/Bo x 100 represents normalized % binding relative to maximum binding in the absence of nonlabeled ligand, whereas Bi is % binding in the presence of inhibitor and BO is Plasma values were % of cpm bound in the absence of inhibitor. determined by comparing with the values (Bi/Bo) of Fg-E standard Plasma values were assayed at 4 dilutions dose response curve. and mean values are reported.

Immunochemical

analyses

Immunoelectrophoresis was performed in 2% Noble agar (Difco Laboratories, Detroit, Michigan) in 0.05 M sodium barbital buffer, (25 x 75 mm) for 30 min. pH 3.2, at 30 volts per microslide

Double immunodiffusion was carried NaCl and 0.13 sodium azide.

out in 1% ?;oble agar in 0.15 :!

-Analytical procedures Protein concentrations were estimated by measuring the absorbancy at 280 run with a Zeiss PMQ II spectrophotometer. For human Fg, an extincti n coefficient of 15.1 was used for a 1% B protein solution. E!cinat 280 rrmof D ard E fragmentsw-e assumed to be 20.8 and 10.2 respectively (15). The molecular weights of Fg-E and Fb-C fragments were determined from their mobilities and the mobilities of proteins of known; molecular weight by SDS gel electrophoresis (16). Reference proteins used were chymotrypsinogen A, NW 25,000; ovalbumin, ."IW45,000; human u-chain, Wd 75,000 and rabbit phosphorylase-a, YW 92 , 5 00 . Plasma

samples

colle'ction

Nine ml of venous blood were collected into a polypropylene tube together with 1 ml of an anticoagulant solution containing 0.1 M sodium citrate and 0.1 N EACA. Immediately after the venipuncture, blood and anticoagulant solution were thoroughly mixed and then centrifuged at 3,500 rpm at 4" for 20 min. Plasma samples were stored at -50" within 2 hr after the venipuncture.

RESULTS Radiciodination Using unabsorbed R45 anti-Fg-E serum, it was found that at specific activities greater than 25 pCi/pg, the immunological reactivity of Fg-E was increasingly destroyed. Figure 1 shows that the corrected percentage of Fg-E tracer bound to the antibody in R45 antiserum decreased progressively as the specific activity for Fg-E was increased from 25 pCi/ug to 100 and 250 uCi/iJg respectively. Figure 1 also shows that R45 antiserum contained high titer antibodies since at a 1:300,000 dilution of this antiserum, 50% of 1251-labeled Fg-E remained bound to the antibody. Antiserum

specificity

(before absorption

with Fg)

At a final working dilution of l/300,000 unabsorbed R45 antiserum was utilized to develop a double antibody RIA with "'1-Fg-E. The graphic display in Figure 2 illustrates that the unabsorbed antiserum, as expected, had specificities against both native Fg and Fg-E fragment. As shown in Figure 2, Fg-E fragment appears to be more efficient than the whole Fg in the competitive inhibition with Fg-I: tracer, as indicated by the different slopes for their inhibition curves. This may be partly because Fg-E with molecular weight 50,000 is nearly seven times smaller than native Fg (340,000) and therefore more efficient in displacing labeled Fq-E from the antigen-antibody complex. It

-SPEC!FiC ACTIVITY =25pci/pg ------'SPECIFIC ACTIVITY~IOO~~~/JJ~ z SPECIFiC ACTlVITY~250~ci/~g

goi 80-l

CORRECTED 70% BOUND 6050403020 IO] I

FIG.

1

Decrease in the irnTunoreactivity of "'I-labeled Fg-E against unabsorbed antiserum R45 with increase of its specific activities. Corrected % bound represents 3 of radiolabel bound to the antibody after the correction has been made for background counts.

‘e\ \\ \ \ \ \\ i\\

Bi/BoBo Xl00 7o

'.,Fg '.-.

50 40

: 30

Fg-E \

20

\

IO1 ‘ng

'.

'.

,/!, \ t

1

'3.

_.

._ I-----~

1/16 i/Bl/4i/2 I IOOng

r long AMOUNT

TION PLASMA/iOO+ I kg

COMPETING

FIG.

‘O!Jg

/ 1@Jpg

fmg

ANTIGEN/TUBE

2

Competitive inhibition profile of Fg and Fg-E in the assay system involving '251-F~-E and unabsorbed R45 anti-Fg-E serum. Serial dilutions of 3 nonzal plasmas show Bo=51.6% (corrected). a parallelism to a Fg standard.

must be pointed out that Fg, which was present in much greater concentration-mean value 2.9 mg/ml (17)-than fragment E, in plasma reacted primarily with unabsorbed anti-Fg-E serum (Figure It can be seen that the inhibition slopes of three normal 2). plasmas paralleled that of the Fg standard, thereby suggesting that Fg immunological activity was being assayed in plasma. Antiserum

specificity

(after absorption

with Fg)

Antisera R45 and R77 were each absorbed with Fg coupled to a CNBr-activated immunoadsorbent column as described in the Materials and Methods section. This procedure reduced the titer of unabsorbed R45 anti-Fg-E serum from one particular bleeding (separate from that shown in Figure 1) from l/700,000 to l/2,500 (the titer of anti-Fg-Eneo serum). However, antibodies to Fg-Eneo still showed the binding characteristics typical for The titer is defined as the radiolabeled antigen and antibody. dilution of antiserum that will bind 50% of added radioligand. The avidity of the antibody is generally the important denomThe Ka for Fg-absorbed R45 and R77 inator of assay sensitivity. antisera were calculated; they were 2.1 x lo9 and 4.1 x 10' il/mol Although varying with different batches of bleedrespectively. ing, the -best titers of R45 and R77 anti-Fg-Eneo sera were deterAntisera R45 and mined to be l/2,500 and l/2,000 respectively. R77 were judged more suitable than other anti-Fg-E and anti-Fb-E sera based on their Ka values and titers. Double

antibody

RIA

The double antibody Eneo RIA employing "'1-Fg-E as a labeled tracer demonstrated, as shown by slight inhibition by Fg at 1 mg/ml (Figure 3), that the cross-reactivity of anti-Fg-Eneo with Fg had been effectively removed. The working range for Fg-E Since Fg is nearly fragment was between 1 ng/ml and 1 pg/ml. seven times larger than Fg-E or Fb-E, % Bi/Bo was plotted against Almolar concentration of antigen rather than antigen weight. though not shown in Figure 3, both Fg-D fragment and az-macroglobulin (remotely possible contaminants in Fg-E tracer preparation) showed little cross-reactivity with R45 and R77 anti-Fg-Eneo sera at 1 ug/ml. It is noteworthy that R45 anti-Fg-Eneo did not distinguish between Fg-E and Fb-E as can be seen by the similarity in the two inhibition curves. The sigmoid inhibition curves could be transformed into a straight line by logit transformation, by applying the formula

logit

(Y) = In [ &]

where

Y is Bi/Bo x 100

(14).

The results again revealed the superimposition of the inhibition curves of Fg-E and Fb-E so that the two curves could be considered identical (Figure 4). The combined slope and intercept calculated considering all of the points that made up both Fg-E and Fb-E curves corresponded closely with the slopes and intercepts calculated for Fg-E and Fb-E individually and had a correlation coefficient of -0.9931. The method of least squares was used for the

100 8

80

*

60

g

40 20

I O-l4

IO’” COMPETlN6

IO”0 ANTIGEN

iMJ

10-8

CONCENTRATION

/TUBE

FIG. 3 Inhibition pattern of Fg, Fg-E and Fb-E in the RIA involving "'1-Fg-E and absorbed R45 anti-Fg-Eneo serum. Bo=14.58 (corrected). *

lo-’

IO"' COMPETING

[M] ANTIOEN CONCENTRATKIN / TUBE FIG.

4

Logit plot of the inhibition pattern same assay system as in Figure 3.

for Fg-E and Fb-E in the

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calculations of the slopes and intercepts (19). The slope and intercept of combined Fg-E and Fb-E points were -2.160 and 2.779 respectively; whereas the slope and intercept of Fg-E were -2.299 and 3.163, and those of Fb-E were -2.099 and 2.547. The probability of obtaining the above sample correlation coefficient b:; chance alone is less than 0.001. Since the two standard curves for Fg-E and Fb-E were indistinguishable, in our hands the Eneo RIA measured total Eneo antigens. Logit transformation of the displacement curves of normal and pathological plasmas produced a straight line which exhibited a parallel relationship to a Fg-E standard cilrve. This procedure permitted the calculation of the total Eneo in both normal and pathological plasmas. A test for parallelism between the displacement curves of test plasma and Fg-E standard was ascertained The test involved a ratio comparison by a two-stage procedure. between the standard deviation of the concentrations to the standard deviation of the logit (Bi/Bo x 100) values for test plasma The second step was a test for differing and Fg-E standard. standardized slopes using Fisher's Z transformation on correlation coefficients for test plasma and Fg-E standard (18). A linear least squares analysis was performed on all the Eneo data It was determined that the probabiliobtained from plasma RIA. ties of obtaining any of the correlation coefficients for the samples analyzed by chance alone are less than 0.05. The RIA data on normal and pathological plasmas are tabulated in Table 1. Normal plasma Eneo immunoreactivities were approximately 15 ng/ml and presumably reflected normal Fg turnover. Considerably higher Eneo plasma immunoreactivities have been obIn acute disserved in patients with consumption coagulopathy. seminated intravascular coagulation (DIC) syndrome, such as in patients with abruptio placentae and Hodgkin's disease followed by sepsis, a plasma Eneo inununoreactivity of nearly 1.0 ug/ml and 2.9 ug/ml respectively was observed. In chronic DIG represented by patients with multiple forms of neoplasms, plasma Eneo immunoreactivities ranged from 43 to 270 ng/ml.

DISCUSSION A new antigenic determinant can be exposed in a protein A newly exposed antimacromolecule upon proteolytic cleavage. genie determinant on an altered Fg molecule is termed neoantigenie expression (1). Plow and Edgington (2,3,19) subsequently reported that the D or E neoantigens are exposed early in plasmin digestion of Fg and are present on fragments X, Y and D or E. These investigators laid down considerable ground work which established the steric basis of D and E neoantigen (2,3,19). They also found in the Eneo RIA that early E fragments, i.e., X and Y, possessed quantitatively less neoantigen than terminal E fragments (19). Nevertheless, Fg-Eneo can be used as a specific immunochemical marker of the structural changes associated In addition, with the plasmic cleavage of Fg and/or Fb molecules. &_I@ to the high specificity and sensitivity exhibited by RIA, the Eneo RIA could presumably provide a suitable RIA for E-relhted

Table 1 Eneo Immunoreactivityin Plasma Compared with Other Coagulation Data

Patient

Diagnosis

A.B D.S.

Abruptio placentae Hodgkin's disease, sepsis

E.D. I.D. N.G. T.G. D.H. T.M.

Breast carcinoma Breast carcinoma Ovarian carcinoma Pancreatic carcinoma Multicentric hepatoma Acute promyelocytic leukemia Lung carcinoma Breast carcinoma Malignant melanoma

R.M. M.P. M.R.

Q1 mg % 73 126

Thrombin Time set 17/19*

Platelets ml-l

high

62,000

975.7 2,875.O

205,000 17,000 7,000 36,000 334,000

123.0 42.8 270.3 92.0 47.9 160.4

250,000 25,000 250,000

68.0 266.1 202.3

155 210 215 255

32.9/19 >120/19

90 >40 358 16

70

19/19

16

18-20

(2

200-400

20/19

Eneo ng/ml

-FDP' ug/ml

Pool (6) J.C. P.F. V.H. T.W.

150,000400,000 14.5 8.0 9.5 11.5 13.5

'Determined by Clauss procedure (Acta Haematol. l7_, 237, 1957). *Exnressed as time required to clot for test plasma/control plasma. 'Measured bv stanhvlococcal clumcinq test (,l.Lab. Clin. lied.'5, 93, 1971).

FDP, i.e., Fg.

X,

Y and F, in plasma

in the presence

of circulating

The presence in blood of immuno-detectable FDP may have considerable diagnostic significance since excessive amounts commonly occur in patients with defibrination syndrome or a fibrinolevels of FDP Clinically, normal and pathological lytic state. are usually measured in serum instead of plasma because of the Quantitation immunological cross-reactivity between Fg and FDP. of FDP in serum, however, can have only relative values as it Some of the FDP such as fragment X depends on many variables. are 85% thrombin-clottable and almost completely removed from plasma in the clotting process (15). Although fragments Y, D, and E are present in serum, some of them might have been generated by the action of thrombin in vitro (7). The availability of reliable data on the plasma level ofP iS therefore important if hemostatic dysfunctions are to be properly evaluated by this criterion.

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t;l?

As suggested by the findings of Plow and Edgington :19), Eneo immunoreactivity in normal and pathological plasmas could reflect cross-reactivity by X or Y fragment as well as reactivity Studies to characterize the molecular weight of of E fragment. the Eneo activity of plasma in normals and patients are in progress and should resolve this question. Recently Edgington and Plow (20) observed immunochemical differences in Fg-Eneo expressions exhibited by Fg-E and Fb-E They reported that a parallelism was observed between fragments. the inhibition curves of Fg-E and Fb-E but a 3.5 molar excess of Our Fb-E was required to achieve the same inhibition as Fg-E. Eneo RIA, on the other hand, has shown that Fg-E and Fb-E are antigenically indistinguishable. Divergent findings by Edgington and Plow (20) and by us on Eneo expressions by Fg-E and Fb-E should probably be attributed to antigenic specificities of our respective Eneo antisera. I"_ is desirable to develop a neoantigen RIA which can differentiate between the lytic products of Fg and Fb in normal and pathological plasmas, and hence define primary versus secondary fibrinolysis. Originally our aim was to prepare Fg-Eneo or Fb-Eneo antiserum which could discriminate subtle antigenic differences between Fg-E and Fb-E, and to develop a RIA (based on such an antiserum) which can serve as a descriptor of primary versus secondary fibrinolysis. Although the Eneo RIA described by Edgington and Plow (20) noted antigenic differences between Fg-Eneo and Fb-Eneo, their assay system recognized primarily the cross-reactivity between the two molecular species, as evidenced Our Eneo RIA by #a parallelism between the two inhibition curves. Therefore, did not discriminate between Fg-Eneo and Fb-Eneo. neither RIA systems can be used to distinguish between the plasmic degradation products of Fg and Fb. It is nevertheless important to establish a neoantigen RIA which can directly identify FDP in normal and pathological plasmas regardless of the source of the degradation products. Work is presently underway to develop the Eneo RIA system described here into a solid-phase RIA which is more applicable in clinical situations.

ACKNOWLEDGMENT The authors are indebted to Vicki Huff and Helen Shurley for excellent technical assistance, and to Dr. William Chua for suoplying plasmas of patients with defibrination syndrome. This work was supported by grants from the U.S. National Institutes of Health (HL 20014) and American Cancer Society (IN 89J).

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