THE dOURNAL OF BIOLOGICAL CHEMISTRY L 'd 255. No. l i . Iswe of September 10, pp. 8334-839, 19H(1 Prrnted m I'.S A.

A Human Lung Tumor-Associated Antigen Cross-reactive with a,-Antichymotrypsin"

(Received for publication, February 29, 1980, and in revised form, May 14, 1980)

Shaik A. Gaffar,+ Gerald L. F'rincler, K. Robert McIntire, and James A. Braatz8 From the Laboratory oflmmunodiagnosis, National Cancer Institute, National Institutes of Health, Bethesda, Maryland . . - 20205

A protein constituent of normal human plasma which causes inhibition in a double-antibody single-label ra- dioimmunoassay for a human lung tumor-associated antigen has been identified as the antiprotease al-an- tichymotrypsin. The inhibitor was isolated by Sepha- dex G-200 chromatography, DEAE-cellulose ion ex- change chromatography, and polyacrylamide gel elec- trophoresis. After each step, the peak of radioimmu- noassay-inhibitory activity was tested in Ouchterlony double diffusion analysis for precipitating reactivity with 46 commercial antisera to individual plasma pro- teins, After gel filtration, ion exchange chromatogra- phy, and acrylamide gel electrophoresis, the pools of fractions containing peak inhibitory activity reacted with 25, 3, and 1 antisera, respectively. Only antisera directed against al-antichymotrypsin reacted with all three pools. Antiserum against al-antichymotrypsin, but not antisera against 10 other plasma proteins, was capable of removing all radioimmunoassay-inhibitory activity from plasma. Purified al-antichymotrypsin ex- hibited an inhibitory dose response curve in the ra- dioimmunoassay parallel to that obtained with plasma, lung tumor extract, and purified lung tumor antigen. Despite the immunologic relationship between al-anti- chymotrypsin and the lung tumor antigen, the two proteins are physically distinct since they can be sep- arated by acrylamide gel electrophoresis and gel filtra- tion. In addition, the a,-antichymotrypsin inactivated a-chymotrypsin on a 1:l molar basis, but the tumor antigen was totally without effect on chymotrypsin esterase activity. Insolubilized chymotrypsin was shown to bind only al-antichymotrypsin, leaving the tumor antigen free in solution. T h i s observation pro- vides a practical method for separation of al-antichy- motrypsin from the tumor antigen. These findings will have an immediate application in a lung tumor antigen radioimmunoassay, the usefulness of which had been limited because of the cross-reactive plasma compo- nent identified here as al-antichymotrypsin.

In previous studies, by serologic analysis we have described the presence of a common tumor-associated antigen in a large percentage of lung cancers (1). The physicochemical features of two antigens purified from different tumor extracts were found to be very similar (2 , 3). Making use of purified and

* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "aduertisernent" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. + Present address, Department of ExperimentaI Pathology, Uni- versity of Kentucky Medical Center, Lexington, Kentucky 40536.

3 To whom inquiries regarding this paper should be addressed.

labeled lung tumor antigen and a highly adsorbed rabbit immune serum, we developed a radioimmunoassay which was subject to competition by a cross-reacting protein in normal human plasma, but not sera from other species (4). The immune serum employed in our studies was adsorbed exten- sively with insolubilized normal serum and with insolubilized normal lung extracts until no further precipitation was ob- served by Ouchterlony double diffusion analysis. Neverthe- less, inhibition was still observed with normal human plasma in the RIA.' Therefore, studies were undertaken to identify the inhibitor and study its molecular relationship to the lung tumor antigen. In this paper, we describe the results of iden- tification and isolation of the inhibitor molecule from normal human plasma.

EXPERIMENTAL PROCEDURES

Materials Outdated normal human plasma was obtained from the National

Institutes of Health Blood Bank. Sephadex G-200 was from Phar- macia Fine Chemicals, Inc., and DEAE-ceUulose was purchased from Whatman. Acrylamide and N,h'"methylenebisacrylamide were pur- chased from Aldrich Chemical Co. Antisera to various plasma proteins were obtained from Dako (distributed by Accurate Chemical and Scientific Corp., Hicksville, NY), Calbiochem-Behring Corp., La JoLla, CA, and Miles Laboratories, Inc., Elkhart, IN.

Methods Gel filtration-Normal human plasma was centrifuged for 30 min

at 27,000 X g and the supernatant filtered through glass wool. The resuking clear plasma was adjusted to a final concentration of 0.1 M with solid NaCl and applied to a column of Sephadex G-200 (4 X 124 cm; void volume, 565 ml). This column was equilibrated and the sample was eluted with 50 m~ Tris, pH 8.1, containing 100 mM NaCI. Fractions of 5.0 ml were collected. Portions of each fraction were analyzed for optical density at 280 nm and assayed for inhibitory capacity in the lung tumor antigen RIA.

Ion Exchange-DE-52 microgranular ion exchanger was equilii- brated with 50 n m acetate buffer, pH 5.0. A portion of the pool of inhibitory activity from Sephadex G-200 chromatography (approxi- mately 1.0 mg of inhibitor) was dialyzed 12 h at 4°C against 10 mM acetate buffer, pH 5.0, and then centrifuged 30 min at 27,000 x g. The clear supernatant was loaded onto the DEAE column (20-ml bed volume), and fractions of approximately 1.1 ml were collected. The unadsorbed proteins were eluted by washing with the initial buffer, and the bound proteins were eluted with a linear concentration gradient of sodium acetate (0.02 to 0.1 M, 60 ml total), pH 5.0. Finally, the column was washed with 0.2 M sodium acetate, pH 5.0.

Polyacrylamrde gel electrophoresis-Six per cent acrylamide gels were employed using a Tris-borate buffer (gel system 1 ) at pH 9.0 (5). At the end of electrophoresis, the gels were sliced into 2-mm slices

"_" ' The abbreviations used are: RIA, radioimmunoassay; hLTAA-2b.

human lung tumor-associated antigen purified from LT-109; LT-109, saline extract of a human lung adenocarcinoma; LT-37, saline extract of a human lung squamous cell carcinoma; a,ACT. a,-antichymotryp- sin; LTAA, lung tumor-associated antigen.

8334

Tumor Antigen Cross-reactivity 8335

with an electric gel slicer (Bio-Rad). The slices were incubated in 1 ml of 10 mM sodium phosphate buffer, pH 7.0, for 16 h at 4°C to extract the protein. Staining for protein was performed using Coo- massie blue as previously described (2).

antigen hLTAA-2b was performed as previously described (4). Com- Immunoassays-The radioimmunoassay for lung tumor-associated

petitive inhibitors were tested in duplicate at each dilution, and the data were subjected to linear regression analysis by the method of least squares.

Radial immunodiffusion analysis for a,-antichymotrypsin was per- formed as previously described (6) , using purified a,-antichymotrypsin (the generous gift of Behringwerke, Marburg, Germany) as the stan- dard. In our hands, this assay was linear over a range of 5 to 200 pg/ ml. Double diffusion analysis was performed in 1% agarose gels as previously described (2). Protein was determined using the Bio-Rad protein assay reagent with bovine serum albumin as standard (7). Chymotrypsin activity was measured using N-acetyl-L-tyrosine ethyl ester as substrate (8).

Binding Studies with Znsolubilized Chymotrypsin-Enzite-aga- rose chymotrypsin (Miles Laboratories, Inc.) was washed and sus- pended in an equal volume of 50 m~ Tris-HC1, pH 8.1, containing 0.1 M NaCl. Binding was performed at room temperature for 10 min in 0.4-ml polystyrene Microfuge tubes in a final volume of 0.35 ml. Final concentrations of the following components were: Tris-HC1, 20 mM, pH 8.1; NaC1, 0.1 M; bovine serum albumin, 1 mg/ml; radiolabeled protein, 6000 cpm (approximately 50 pCi/pg). The mixture was in- verted several times during the incubation, then centrifuged 1 min at 12,000 X g, and washed three times with 50 mM Tris-HC1, pH 8.1, containing 0.1 M NaCl and 1 mg/ml of bovine serum albumin. After the last wash, the supernatant was aspirated, and the tip of the tube containing the washed gel was excised and counted for radioactivity.

RESULTS

Our previous studies with a double antibody radioimmu- noassay have shown that lung tumor antigen levels in plasma from lung cancer patients could be distinguished from those of normal individuals (4). However, the high levels of reactiv- ity found in normal plasma suggested the presence of a similar or cross-reactive substance. That the inhibitory substance in normal plasma was not identical with the lung tumor antigen was suggested by their slightly different elution characteristics on Sephadex G-200 (Fig. 1). Similar gel filtration experiments have confirmed that hLTAA-2b reproducibly elutes after this inhibitory component.

In order to identify this cross-reactive plasma component, its isolation was attempted, and its inhibitory activity in the RIA was followed throughout the isolation procedure.

As an initial step, Sephadex G-200 gel filtration of pooled normal human plasma was performed, with a typical result shown in Fig. 2. The plasma proteins eluted characteristically as three major protein peaks with the inhibitory activity eluting essentially as a single peak between the second and third protein peaks. Peak fractions showing inhibition in the RIA were pooled and further purified by DEAE-cellulose chromatography at pH 5.0 (Fig. 3). Greater than 90% of the protein did not adhere, and no inhibitory activity could be detected in the unbound fractions. The RIA-inhibitory activ- ity was eluted with 0.2 M sodium acetate, and the active fractions (120 to 140) were pooled and concentrated for further analysis.

In order to identify the plasma proteins present in the G- 200 and DEAE pools, Ouchterlony double diffusion analysis was performed using a variety of commercial antisera to human plasma proteins. Table I indicates the reactivities observed. As expected, the G-200 pool contained many reac- tivities. Of the 46 antisera tested, 25 gave precipitin lines in agarose gel. However, most of these reactions were eliminated by the relatively efficient DEAE-cellulose step. In the DEAE pool, reactivity was observed only with antisera to hemopexin, P-lipoprotein, and a,-antichymotrypsin.

In order to decide which of these proteins was responsible

1.2 -

1.0 -

5 0.8

1 -

u" 0.6 - z m e 2 0.4 m

-

0.2 -

0

FRACTION NUMBER

FIG. 1. G-200 chromatography of normal human plasma mixed with L261-labeled lung tumor antigen. One milliliter of plasma was mixed with 1 ml(6600 cpm) of labeled antigen and applied to a column (2 X 92 cm) equilibrated with 50 mM Tris-HC1, pH 8.1, containing 100 m~ NaCl. Fractions (2.15 ml) were collected at 4°C at a flow rate of 6.6 ml/h and tested for absorbance at 280 nm (W), radioactivity (13 - - - a), and for inhibitory activity in the RIA for hLTAA (A-A). The void volume (VO) was 112 ml.

FRACTION NUMBER

FIG. 2. Fractionation of pooled normal human plasma by G- 200 chromatography. Plasma (100 ml) was processed and applied to the column at 4°C as described under "Methods." Fractions were tested for protein content (M) and for inhibitory activity in the RIA for hLTAA (o"--o).

FRACTION NUMBER

FIG. 3. DEAE-cellulose chromatography of the inhibitor re- gion from G-200. Arrows indicate the points of application of the gradient and the final wash buffer.

8336 Tumor Antigen Cross-reactivity

TABLE I Reactivity ofpartially purifiedplasma inhibitor fractions urith

commercial antisera Fractions from G-200 which contained inhibit0r.v activity in the

RIA were tested in double diffusion against 46 monospecific antisera to known plasma proteins. Twenty-one antisera to the following proteins were negative with the G-200 fractions and were not tested further: C1 esterase inhibitor, Clq, C3, C4, C5, C reactive protein, calcitonin, ferritin, haptoglobin, hemoglobin. lactoferrin, lysozyme. cr.-macroglohulin, myoglobin, /{.-microglobulin. pregnancy-associated o.-glycoprotein, pregnancy-specific PI-glycoprotein, secretory com- ponent. cx,Al’ glycoprotein. factor XI11 (subunit A). factor XI11 (sub- unit SI.

” ~- Antisera [ ( I

”

( ; - 3 N I DEAE PACE”

Albumin /{.-Glycoprotein I + /lL-Glycoprotein 111 + P I - H glycoprotein C I Activator + - n.d.“ Ceruloplasmin + - ol-Antichvmotrvpsin + + + Fibrinogen + - n.d. GI. Globulin + - nl-Glycoprotein - n.d. Hemopexin + + n2-HS Glvcoprotein Immunoglobulin (polvvalent) o-Lipoprotein + P-Lipoprotein Prothrombin + Plasminogen + - n.d. I’realbumin + - n.d. Retinol-binding protein + ol-Antitrypsin + Inter-tr trypsin inhibitor + Transferrin Antithrombin 111 + - (11-T glvcoprotein + - - Zn-cr&lycoprotein

- ~ ~~

+ I’ - - - - - -

+ - -

-

-

+ -

+ +

- - - n.d. -

+ -

+ - - -

- - - - - -

+ - n.d. -

- . .~ . ~

+ - -

” PAGE, polyacrylamide gel electrophoresis.

‘ Indicates no reactivity. ‘I Not determined.

Indicates precipitating reactivity.

for the inhibition in the radioimmunoassay, portions of pooled human plasma were reacted with a select group of commercial monospecific antisera against individual plasma proteins. After removal of the precipitate, the immunodepleted super- natants were tested for inhibitory capacity in the lung tumor RIA. Table I1 indicates that neither hemopexin nor P-lipopro- tein depletion had an effect on the RIA. Only the antiserum to al-antichymotrypsin produced a significant effect on inhib- itory activity, and this completely eliminated reactivity.

In confirmation of this finding, a preparation of RIA-inhib- itory material from DEAE chromatography was subjected to polyacrylamide gel electrophoresis, and the extract from each gel slice was assayed for RIA inhibition and for alACT by radial immunodiffusion. Fig. 4 shows the profile obtained, which clearly relates both activities to the same electropho- retic position in the gel, corresponding to the major protein component.

The eluate from the slice containing the peak of activity (gel slice 34) was tested in double diffusion for reactivity with the group of antisera as shown in Table I and analyzed for contaminants by polyacrylamide gel electrophoresis. Only antisera to alACT produced a precipitin line (Fig. 5A). The homogeneity of this fraction is demonstrated by the single protein band in a polyacrylamide gel (Fig. 5B).

Additional evidence implicating alACT as the plasma pro- tein responsible for inhibition in the RIA is shown in Fig. 6. Purified alACT from this study (Curve A ) and from Behring- werke, (Curve B ) were tested at different concentrations for

TAHLE I1 Immunoprecipitation of normal human plasma by antisera to

normal plasma proteins ~-

Incubation with antisera to” 1,T-109 RIA

# / m l -~ ~~

Antithrombin 111 43R tr-Lipoprotein 414 /j-Lipoprotein 34 2 Ceruloplasmin .3w Hemopexin 450 n,-Antitrypsin 3 1 2 nl-Antichymotrypsin t l nl-Glycoprotein 396 ltl-T glycoprotein 702 tr,-HS glycoprotein 378 Zn-n?-glycoprotein 666 PBS 336 NUS 4 14

“ One milliliter of a 1:50 dilution of plasma was incubated with 200 pI of antisera. The precipitate was removed by centrifugation. and the amount of inhibitor remaining in the supernatant was measured in the RIA.

GEL SLICE FIG. 4. Polyacrylamide gel electrophoresis of partially pu-

rified inhibitor. Electrophoresis was carried out at 4°C on a 6 8 gel (1 X 11 cm) at 8 mA/tube until the tracking dye was about 1 cm from the bottom of the gel. The gel was sliced longitudinally; one half was stained for protein, and the other half was sliced laterally and the protein extracted from each 2-mm slice. The eluates were assayed for inhibition in the LTAA-RIA and for a lACT using the radial immu- nodiffusion procedure as described under “Methods.”

inhibitory activity in the RIA. It is evident that these proteins not only produced strong inhibition but also yielded dose response curves parallel to those obtained with a lung tumor extract (Curve C) and purified hLTAA (Curve D), indicating recognition of similar sites by the antibody. Unfractionated normal human plasma also produced a parallel dose response (Curve E).

The slope of each line generated was calculated by linear regression analysis using the method of least squares. The slopes obtained for each curve were A , -1.20; B, -1.18; C, -1.20; D, -1.08; E , -1.18. The result of seven separate deter- minations of the slope of the line using LT-37 extract as displacer was -1.12 f 0.09. Thus the slopes of each of the five displacers can be considered parallel within experimental error.

Sera from four animal species were tested as displacers in the RIA. Whereas normal human plasma produced significant inhibition a t dilutions greater than 1:1OO,OOO indicating inhib-

Tumor Antigen Cross-reacticlity 8337

phoretic mobility of the hLTAA (3). In addition, this differ- ence in electrophoretic mobility can also be demonstrated using purified radiolabeled alACT and hLTAA (data not shown). Also, as shown in Fig. 1, these two proteins could be separated by Sephadex G-200 chromatography.

Additional evidence supporting a structural divergence be- tween these two proteins was obtained by examining their respective abilities to inhibit chymotrypsin activity. As ex- pected, the alACT purified from plasma was capable of com- plete inhibition of enzymatic activity (Fig. 8). In addition, the pattern of inhibition was stoichiometric since 1 mol of chy- motrypsin was inhibited by 1.02 mol of alACT. However, hLTAA-2b. when tested in equivalent proportions. failed to have any measurable effect on chymotrypsin activity. The inhibition by alACT occurred rapidly since it required no preincubation for a maximal effect, whereas preincubating

FIG. 5. A. I)ouble diffusion testing o f the eluate from gel slice 34, Fig. 4 (center wd0, against antisera to I , (rlACT; 2. antithrombin 111; 3 , retinol-binding protein: 4. prothrombin; 5. &-glycoprotein I; and 6, /&glycoprotein 11. R , 7 5 polyacrylamide gel electrophoresis of same sample applied to center well in A; 13 pg of protein applied. Anode is at the top.

+ 5 t + 4 -

+ 3 -

+ 2 -

- + 1 - m ,

0 - t U 3 - 1 .

- 2 "

- 3 -

- &

" " - 1:ld 1305 1:lo.

.I I

SERUM DILUTION

0.5 1.0 5.0 10 50 loo INHIBITOR, ng/rnl

FIG. 6. LTAA-RIA displacement curves. A. alACT, slice 34 from Fig. 4. M, E , alACT from Behringwerke, W C, LT- 37, A-A; D. LT-109, same material used for trace label, A-A; and E , unfractionated normal human plasma, W. Each point is the average of duplicates.

itor concentrations in the range of 200 to 500 pg/ml, fetal calf, chicken, horse, and goat sera were only weakly inhibitory a t a final dilution of 1:lO. Inhibitor concentrations in the xenosera were all less than 25 ng/ml.

The actual relationship between alACT and the hLTAA can only be determined when larger quantities of tumor antigen become available. However, it is apparent that while the two are immunochemically related, physicochemically they can be shown to be distinct. Fig. 7 demonstrates separa- tion of hLTAA from plasma inhibitory activity after co-elec- trophoresis on a 6% polyacrylamide gel. The process of iodi- nation was previously shown to have no effect on the electro-

J

0 10 20 30 40 50 GEL SLICES

FIG. 7. Polyacrylamide gel electrophoresis of a mixture of partially purified inhibitor (from G-200) plus 12sI-labeled pu- rified LT-109 antigen. Electrophoresis was performed at 4OC in 6'T gels (1 X 12 cm). Each gel slice was counted for radioactivity and eluted; the eluate was assayed for inhibition in the RIA. The arrou' indicates the positon of the tracking dye as well as the [""I]iodide.

1.01

0

I 1 1 I I

" 0.2 0.4 0.6 0.8 1.0

\

INHIBITOR, c(g

FIG. 8. Effect of plasma inhibitor and tumor antigen on chy- motrypsin esterase activity. Additions of alACT (-1 or hLTAA (M) were made a t room temperature to the substrate prior to addition of enzyme (0.3 pg per assay). Activity was determined as soon as all components were mixed.

8338 Tumor Antigen Cross-reactivity

CHYMOTRYPSIN/AGAROSE. PI

FIG. 9. Binding of radiolabeled plasma inhibitor and tumor antigen to insolubilized chymotrypsin. Binding of "'I-alACT (t-3) and '"I-hLTAA-2b ( D " - f 3 ) to chymotrypsin/agarose gel was corrected for nonspecific binding to an equivalent amount of Sepharose 4B gel. Inset, binding of "'I-anACT in the presence of varying concentrations of unlabeled a1 ACT using 50 p1 of chymotryp- sin/agarose suspension.

chymotrypsin with hLTAA-2b resulted in no detectable loss of enzymatic activity.

Since the RIA for hLTAA in its present form is hampered by interference with alACT, a simple means of removing &,ACT from clinical samples could render the assay more clinically useful. With the finding that the plasma ~ I A C T , but not the tumor antigen, inhibited chymotrypsin activity, it seemed likely that this could provide the basis for separating the two proteins. When comparable amounts of equivalently labeled alACT and hLTAA were mixed with insolubilized chymotrypsin, only the former bound to the enzyme gel (Fig. 9). The binding which took place using "'I-alACT was rapid (complete in less than 1 min), stable (was not displaced by three buffer washes), and was inhibited by excess unlabeled aIACT (inset). Radiolabeled tumor antigen, on the other hand, failed to exhibit any significant binding to the insolu- bilized enzyme.

DISCUSSION

We previously developed a radioimmunoassay for a purified human lung tumor-associated antigen which we are currently studying in an attempt to develop a useful immunodiagnostic test for lung cancer (4, 9). Although the RIA is capable of discriminating normal from lung cancer sera, high levels of reactivity, between 200 to 500 pg/ml, are found in the sera of healthy individuals. Initial experiments indicated the inhibi- tory substance in plasma to be distinct from the LTAA since separation could be achieved by gel fitration. The work reported here identifies the plasma inhibitor as the antipro- tease, al-antichymotrypsin, and describes a method for elim- inating this interference.

Sequential chromatography on Sephadex G-200 and DEAE-cellulose followed by electrophoresis in polyacryl- amide gels was performed on normal human plasma, and the fractions were tested for inhibitory activity in the RIA for tumor antigen. In each profile, only one region of inhibitory activity was identified and subsequently subjected to the next separation procedure. Each region, after pooling and concen- trating, was qualitatively tested for the presence of various plasma proteins by double diffusion in agarose using a battery of commercial antisera to individual proteins. The pool of inhibitory activity obtained after G-200 chromatography, d- though well separated from the bulk of the protein, was reactive with 25 of the 46 monospecific antisera. Elimination of all but three of these reactivities was effected by DEAE- cellulose chromatography. The efficiency of the ion exchange step is also apparent in the profile (Fig. 3). At pH 5.0, virtually

all of the protein and little if any inhibitory activity eluted with the solvent, whereas all the activity and little protein which bound to the column eluted with 0.2 M acetate. Poly- acrylamide gel electrophoresis of the post-DEAE pool indi- cated one major protein component with a lesser staining region of slower mobility. Analysis of the eluates from individ- ual gel slices indicated that the major staining component coincided with both RIA-inhibitory activity and precipitating reactivity with antisera to a,ACT. Quantitative analysis across the gel demonstrated that both activities were in fact associ- ated with the same component.

Additional evidence implicating alACT as the plasma pro- tein responsible for inhibition in the RIA was obtained by immunodepletion of whole plasma with various commercial antisera and then testing the resulting supernatant for inhib- itory capacity in the RIA. Only plasma depleted of culACT was significantly altered in its ability to interfere in the RIA. In fact, the inhibitory activity was completely removed by this treatment.

Finally, we have observed that purified aIACT obtained from a commercial source produced a dose response curve in the radioimmunoassay which was parallel to that obtained with a crude lung tumor extract as well as with purified lung tumor antigen, purified alACT obtained in this study, and unfractionated normal human plasma. These parallel dose response curves indicate that lung tumor antigen and alACT have similar antigenic sites, as recognized by the antibody to hLTAA. Taken together, these results demonstrate that a I - antichymotrypsin is the component, and the only component, of human plasma which inhibits our radioimmunoassay for a human lung tumor-associated antigen.

The demonstration of a normal plasma protein cross-reac- tive with a human tumor-associated antigen has also been reported by Chawla et al. (10). These workers have identified a protein fragment, EDC1, which is present in the urine of patients with different types of cancer and is immunologically related to the normal plasma protein inter-a trypsin inhibitor. EDCl is smaller (M, 27,000) than inter-a trypsin inhibitor ( M , 170,000) and inhibits trypsin and chymotrypsin. The authors suggest that EDCl is not produced directly by the tumor cells but instead is a result of a tumor protease acting on inter-a trypsin inhibitor.

The relationship between alACT and the tumor antigen discussed in this report i s not clear. Although highly cross- reactive immunologically, the two species appear to be dis- tinct, as evidenced by their separation by polyacrylamide gel electrophoresis and gel fdtration, as well as by their inhibition and binding patterns with chymotrypsin. Previous character- ization of the LTAA indicates the best estimate for its molec- ular weight to be between 73,000 to 77,000, whereas atACT has a molecular weight of 68,000 (11). Thus, in contrast with the results of Chawla et al., the lung tumor antigen is larger than the plasma protein to which it is related. One possible explanation for this type of relationship is that the tumor expresses information not generally expressed by lung tissue and does so incorrectly by producing a precursor form of the molecule. When made elsewhere under appropriate conditions the precursor would be converted to that form found in plasma. The actual relationship between these two protein species is currently under investigation, and attempts are being made to convert one form to the other.

One highly significant product of the work reported here is the finding that the lung tumor antigen and the plasma inhibitor can be separated on the basis of a simple binding procedure. This observation followed from the demonstration of the inability of tumor antigen to inhibit chymotryptic activity, even when added in amounts which for alACT pro-

Tumor Antigen Cross-reactivity 8339

duced complete inhibition. Along this line, our results indicate that purified aIACT inactivates chymotrypsin on an equimo- lar basis which is consistent with the report of Travis et al. (1 1) who demonstrated enzyme - inhibitor complexes, stable in sodium dodecyl sulfate, with molecular weights of 90,OOO which would suggest a 1:l molar ratio.

These findings suggest that immobilized chymotrypsin can be used to deplete the alACT from the sera of lung cancer patients; the LTAA could then be quantitated in the absence of interfering substances using the radio-immunoassay. All pathologic sera which have been tested in the LTAA-RIA to date have yielded results which had to be interpreted in the presence of a high basal level caused by the plasma inhibitor, identified here as alACT. Although discrimination between normal and lung cancer sera has been possible (4), it is suspected that removal of the high background caused by alACT will permit the specific quantitation of LTAA in the circulation with the implication that a more quantitative assessment of disease status may become available. A specific and quantitative assay for a marker of this type may be applicable to early diagnosis of lung cancer or for providing an indication of the patient’s response to therapy.

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Kortright, K. H., and Princler, G. L. (1979) in Lung Cancer: Progress in Therapeutic Research (Muggia, F., and Ro- sencweig, M., eds), pp. 183-189, Raven Press, New York

2. Braatz, J. A., McIntire, K. R., Princler, G . L., Kortright, K. H., and Herberman, R. B. (1978) J. Natl. Cancer Znst. 61, 1035- 1046

3. Gaffar, S. A,, Braatz, J. A,, Kortright, K. H., Princler, G. L., and McIntire, K. R. (1979) J. Biol. Chem. 254, 2097-2102

4. McIntire, K. R., Braatz, J. A,, Gaffar, S . A., Princler, G. L., and Kortright, K. H. (1979) in Carcino-Embryonic Proteins (Leh- mann, F. G., ed) Vol. 2, pp. 533-540, Elsevier/North Holland Biomedical Press, New York

5. Maurer, H. R. (1971) Disc Electrophoresis and Related Tech- niques of Polyacrylamide Gel Electrophoresis, pp. 50-51, Wal- ter de Gruyter, New York

6. Princler, G. L., and McIntire, K. R. (1980) in Conference on Serologic Analysis of Solid Tumor Antigens (Rosenherg, S., ed) Academic Press, New York, in press

7. Bradford, M. M. (1976) Anal. Biochem. 72, 248-254 8. Braatz, J. A,, and Heath. E. C. (1974) J. Biol. Chem. 249, 2536-

2547 9. Braatz, J. A,, McIntire, K. R., Gaffar, S. A,, and Princler, G. L.

(1980) in Conference on Serologic Analysis of Solid Tumor Antigens (Rosenherg, S., ed) Academic Press, New York, in

10. Chawla, R. K., Wadsworth, A. D., and Rudman, D. (1978) J . press

Zmmunol. 121, 1636-1639 11. Travis, J., Garner, D., and Bowen, J. (1978) Biochemtstry 17,

5647-5651