METHOD FOR THE IDENTIFICATION AND PURIFICATION OF HUMAN LUNG TUMOR-ASSOCIATED ANTIGENS (hLTAA) AND CLINICAL DETECTION AND DETERMINATION OF THESE ANTIGENS

Abstract: Method for the purification of a human lung tumor-associated antigen (hLTAA) specific to human lung tumors of diverse histological characteristics; serum levels of hLTAA correlate with lung tumor incidence, and appear to usefully discriminate between various stages of the malignancies. The invention further comprises an immunoassay predicated on purified hLTAA for the detection and quantitative determination of hLTAA in biological fluids, particularly blood serum, and diagnostic systems for clinical immunoassay procedures.

Description: METHOD FOR THE IDENTIFICATION AND PURIFICATION OF HUMAN LUNG TUMOR-ASSOCIATED ANTIGENS (hLTAA) AND CLINICAL DETECTION AND DETERMINATION OF THESE ANTIGENS BACKGROUND OF THE INVENTION Immunologic approaches to cancer management have focussed in recent years on attempts to identify and isolate human tumor-associated antigens which will prove clinically useful in the immunodiagnosis of cancer. While it is well known in theory that malig¬ nant tumors can be detected by radioimmunoassay (RIA) techniques for associated cell-surface antigens, the theory is limited in its practical clinical application to malignant tumors for which specific cell-surface antigens are available in highly purified form; also, these antigens must be released into the bloodstream. and the determinants must retain antigenicity when radiolabelled. Further, clinical utility of the RIA in cancer management is dependent upon biological fluid levels of the selected antigen which substantially correlate with tumor burden; utility is further enhanced if the selected antigen is site-specific, i.e., associated with tumors of specific organs. As a result of the difficulties encountered in attempting to isolate and study a protein for which no function or activity is known, and for which the method of isolation relies on the presumption that it is more prevalent in tumor tissues than in normal tissues, satisfactory clinical antigenic markers have not been heretofore characterized for many malignant tumors, in particular, human lung tumors. SUMMARY OF THE INVENTION The invention comprises a method for the purification of a human lung tumor-associated antigen (hLTAA) specific to human lung tumors of diverse histological characteristics; serum levels of hLTAA correlate with lung tumor incidence, and appear to usefully discriminate between various stages of the malignancies. The invention further comprises an immunoassay predicated on purified hLTAA for the detection and quantitative determination of hLTAA in biological fluids, particularly blood serum, and dia¬ gnostic systems for clinical immunoassay procedures. BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 is graphic representation of radioiodi- nated antigen (pool 1 of Fig. ) incubated 16 h at h°C in the presence of 51-1 normal rabbit serum (NRS) (panel A) or 5 μl R-201 (panel B) and then electro- phoresed on 7% polyacrylamide gels. The recovery of radioactivity was 98$ in A and Q2% in B. TD = bromphenol blue tracking dye. Fig. 2 is a graphic representation of the binding of individual components, hLTAA-I and hLTAA-II to immune serum. Shown are 12S I-hLTAA-I binding to - R-201 (φ φ ), and antiserum to a-,-antichymostryp- sin ( ___ ) , • and 125 I-hLTAA-II binding to R-201 (+ +) and antiserum to α-,-antichymotrypsin (_____ ) . Free and antibody bound antigen were separated using Panεorbin. Binding the presence of normal serum was subtracted. Fig. 3 is graphic representation of a radio- immunodi fusion assay for hLTAA. Glass slides were coated with 1% agarose containing a fifty-fold dilution of R-152 IgG. Dilutions of a standard lung tumor extract, LT-37, were applied to wells in the plate and allowed to diffuse for at least 42 h. An example of the plate appearance is shown in the photograph on the right. Dilutions of LT-37, at 1.0, 3-3, 5-0, 10.0, 14.3, 20.0 and 28.6 units/ml are shown in ascending order in the photograph. Area is expressed as mm 2 . Fig. 4 is a graphic representation of DEAE- cellulose chromatography of crude LT-120 extract. Tumor antigen activity was measured by radial diffusion assay. Antichymotrypsin activity was also determined with a radial diffusion assay. Absorbance at 280 nm and the conductance of the effluent were also deter¬ mined. The NaCl gradient (0-0.2 M) ran from fraction number 355 to 635 and was linear as determined by conductivity measurements. One molar NaCl was applied at the end of the gradient. Pools of fractions were collected as follows: pool I, 11-175; pool II, 375- 469; pool III, 471-575; pool IV, 651-669. Fig. 5 is a photographic representation of the results of Ouchterlony double diffusion analysis of DEAE-purified antigens, of Fig. 4. Center well, ad¬ sorbed immune serum R-152; 1, pool I; 2, pool II; 3 and 6, LT-37; 4, pool III; 5, pool IV. The minor reactivities seen with wells 1, 4 and 5 are residual normal reactivities due to incomplete adsorption and are not related to the major tumor antigenic compon¬ ent. Fig. 6 is a graphic representation of G-200 chromatography of DEAE pool III, of Fig. 4. Fractions were tested for absorbance at 280 nm ( ____, - _____ ), hLTAA (+ - +) and antichymotrypsin ( ___ - [~~J) activity by radial immunodif usion. Two pools of activity were collected, pool C, 549-905 ml and pool D, 913- 1171 ml. Standards are as indicated in Fig. 7. Fig. 6A is a tabular representation of the quantitative aspects of the purification steps of Figs. 4, 6, 7 and affinity immunoadscrption. Fig. 7 is a graphic representation of G-200 chromatography of DEAE pool II of Fig. 4. Fractions were tested for absorbance at 280 nm ( _____ - ____ ) and hLTAA content by the radial diffusion assay (+ - +). Standards indicated are: BD, blue dextran; BSA DIM, dimer of bovine serum albumin; BSA, bovine serum albumin; C G, cymotrypsinogen; N-,-, sodium azide. Two pools of activity were collected as follows: pool A, 654-900 ml and pool B, 913-1095 ml. Fig. 8 is a photographic representation of the results of Ouchterlony double diffusion analysis of G-200 purified antigens of Figs. 6 and 7. Center well, adsorbed immune serum R-201; 2, 4 and 6, LT-37, pool A (from Fig. 7); 3, pool B (from Fig. 7); 5, pool C (from Fig. 6). Fig. 9 is a photographic representation of the results of polyacrylamide gel electrophoresis of affinity-purified hLTAA and detection of antigenic activity. Two % polyacrylamide gels, each containing 27 u g protein were electrophoresed, one was sliced in 2.5 mm sections and the other stained with Coomassie Blue. Each slice was placed in an appropriate size well and the protein allowed to diffuse against R-201 (center well). Upper and lower wells (unlabelled) in each pattern contained LT-37 for comparison. Slices 9-24 were all negative for antigen. Fig. 10 is a photographic representation of the results of Ouchterlony double diffusion analysis of affinity-purified hLTAA. Center well, adsorbed immune serum R-201; 1, crude extract; 2, affinity pool 1; 3 and 6, LT-27; 4, affinity pool 2; 5, affinity pool 3. Fig. 11 is a standard binding inhibition curve for the hLTAA radioimmunoassay . Radiolabelled hLTAA-I was incubated 16 h at 4°C with immune serum in the presence of varying amounts of unlabelled antigen. Antigen bound to antibody was separated using Pansorbin and quantitated as described in Methods. Fig. 12 is a graph of the quantitatIon of hLTAA in the serum of lung cancer patients versus normal individuals. Inhibition of binding of radiolabelled hLTAA to immune serum in the presence of test sera was converted to antigen concentration in the sera by interpolation from the standard curve (Fig. 11). Fig. 13 is a graphic representation of the gel electrophoresis of radioiodinated antigen (of Fig. 1) for 1 h after the tracking dye ran off the bottom. This resulted in separation of the major peak into two components (I and II) and better separation of these from the unreactive protein (peak III). Fig. 14 is a graphic representation of the polyacrylamide gel electrophoresis of isolated peaks I and II from Fig. 13. The R f values were calculated relative to the dye front. . ' Fig. 15 is a graphic representation of the Sephadex G-200 chromatography of } 25 I-hLTAA-I (panel A) and 12S I-hL AA-II (panel B). The smaller peak at fraction number 104 in panel A represents free 125 I. Standards are: blue dextran, V ; apoferritin, M = 480,000; BSA dimer, M r = 134,000; BSA monomer, M r = 67,000; chymotrypsinogen-A, M = 25,000; and cytochrome c, M r = 12,500. Fig. 16 is a graphic representation of the high performance liquid chromatography of unlabelled hLTAA-I (panel A) and hLTAA-II (panel B) . A Spherogel TSK 3000SW size exclusion column was used at a flow rate of 0.7 ml/min with 0.05 M sodium phosphate, pH 7.0, containing 0.3 M NaCl as the mobile phase. HPLC profiles of the pool 1 material from which hLTAA-I and hLTAA-II were isolated indicated the peak eluting at 20 min to be the major component, with minor components eluting at 14.5, 22 and 25 min (panel C). These contaminants were effectively removed by prepara- tive gel electrophoresis since they are not detected in A and 3. Fig. 17 is a graphic representation of the sucrose gradient sedimentation analysis of hLTAA-I (panel A) and hLTAA-II (panel B). Radiolabelled antigen ( _____ - ZΛ ) was compared to E. coli alkaline phos- phatase (+ +) and α-chymotrypsin ( _~\ J~~l ) as standards. Centrifugation was for 22 h at 5°C at 149,632 x g_ (average). Direction of sedimentation was from right to left. Fig. 18 is a graphic representation of the isoelectric focusing of hLTAA-I (panel A) and hLTAA-II (panel B). Samples were focused for 27 h at 4°C in a 110 ml gradient of 5-50% glycerol. Each fraction was counted for 125 l content and the pH was determined. Fig. 19 is a graphic representation of the sodium dodecyl sulfate gel electrophoresis of hLTAA-I (panel A) and hLTAA-II (panel B). Shown are results obtained with 1.5% acrylamide gels. Gels containing ' 10 and 12.5% acrylamide were also used and the standard curves obtained in each were subjected to linear regression analysis prior to estimation of the M of the unknowns. The average value obtained is independent of acrylamide concentration. The standards used and their molecular weights were: phoεphorylase b, 94,000; bovine serum albumin, 67,000; ovalbumin, 43,000; carbon anydrase, 30,000; soybean trypsin inhibitor, 20,000; and α-lactalbumin, 14,400. DETAILED DESCRIPTION OF THE INVENTION The clinical marker antigen of the invention (hLTAA) comprising two (2) components designated human lung tumor-associated antigen I and II (hLTAA-I and hLTAA-II) is purified according to the method of the invention by affinity immunoadsorption from a crude extract of lung tumor source material, prefer¬ ably after resolution of the crude extract into frac¬ tions of high antigenic activity by conventional non-specific procedures such as ion-exchange chro a- tography and/or gel filtration. The affinity matrix comprises the IgG fraction of an adsorbed xenoantiserum raised against a pool of crude lung tumor extract. Preferably, at all stages of purification, the product is assayed for hLTAA, as by radiali munodiffusion (RID) against purified antibody. The product antigen, purified hLTAA, is useful in clinical immunoassay procedures according to the present invention for de¬ tecting and quantitating serum levels of hLTAA, partic- - - ularly concentrations below about 1 ug/ml which are typically encountered in clinical applications. Useful lung tumor extract source material for hLTAA comprises human lung tumor extract of the four major histologic types: small cell and squamous carcinoma, large cell un ifferentiated carcinoma, and adenocarcinoma. The crude tumor extract is puri¬ fied by affinity immunoadsorption against an affinity matrix of purified antibody, derived from an isera raised In heterologouε species animals such as rabbits or goats against these carcinoma extracts. These antiεera develop hLTAA.-speci ic antibodies which are purifiable by conventional adsorption techniques against extracts of normal adult and fetal lung tissue, other normal tissue, and malignant tumors of organs other than lung. Various Immunoadsorption techniques are suitable for purification of hLTAA from crude tumor extract. In an exemplary immunoadsorption procedure, hLTAA antibody is covalently coupled to a solid support such as agarose gels, especially SEPHAROSE gels, or porous glass or polyacrylamide beads by known methods, and antigen is removed from solution by complexing it with the immunoadsorbent. hLTAA is thereafter disso- ciated from anti-hLTAA by customary dissociation agents such as ammonium or potassium thiocyanate, potassium idodide, urea, or propanoic acid. Preferably, the crude tumor extract is treated prior to affinity immunoadsorption to reduce the protein complexity of the extracted material. Suitable pretreatment proced¬ ures include ion-exchange chromatography, which separates proteins on the basis of electrostatic binding capacity, and gel filtration, which separates proteins on the basis of size. Detailed descriptions of these and other non-specific protein separation procedures useful for resolution of protein mixtures are found in many texts, for example, Garvey, et al. , Method in Immunology, 3rd ed. , published by W. A. Benjamin, Inc., Reading, Mass. (1977)- After each purification step, the product is monitored for the presence of hLTAA. Standard RID techniques are useful, employing as standard serial dilutions of the source material or purified antigen. In the purification process exemplified, purification is accomplished by sequential DEAE cellu¬ lose chromatography, gel filtration and affinity immunoadsorption. The proteins obtained by icn- exchange chromatography appear heterogenouε, with immunological reactivity detected in three different peaks; however, all the active components were immuno- logically identical, producing lines of identity in Ouchterlony analysis. Gel filtration of the major antigenic component from DEAE-cellulose similarly demonstrated a further fractionation into several active, immunologlcally identical forms. These results suggest a charge/size isomeric relationεhip among the various forms, all of which possess a common and identical antigenic site. The ma or component was isolated throughout the purification scheme. The final product representing 9 % of the input activity, produced a single, though broad, protein-staining region on 7% polyacrylamide gels, which was coincident with the antigenin the crude extract as well as with that in an extract from another lung tumor. The rela¬ tively low recovery is partially attributable to the subdivision of activity at various steps of purifica¬ tion. The total activity recovered during the affinity immunoadsorption step was 20% of that in the crude extract. Recoveries of 75, 50 and 68% were obtained from DEAE, gel filtration, and immunoadsorption, respectively. The antigen was undetectable by the Ouchterlony technique in the sera of lung cancer patients, indicating a circulating level of <1 μg/ml. Two immunore active forms of hLTAA. were ob¬ served and isolated after electrophoresis of labelled antigen in 7% polyacrylamide gels. These components, referred to as hLTAA-I and hLTAA-II, in order of mobility, were both judged homogenous and characterized by gel electrophoresis, Sephadex gel filtration, size exclusion high performance liquid chromatography (HPLC) and by sedimentation velocity analysis. The latter three techniques produce identical profiles for both forms of the hLTAA. Sephadex chromatography and HPLC analyses indicated the maεε of the antigen, aεεuming a globular configuration, to be approximately 140,000-150,000 daltons with a D 2Q w of 4.2-4.3 x 10 "7 cm 2 s -1 . The ε o values for both were 4.5-4.6S. Sodium dodecyl sulfate (SDS) gel electrophoresis of hLTAA-II gave a single component of M = 81,700 while hLTAA-I had a major component Identical in size to hLTAA-II, and two minor components of M = " 50,000 and 27,700. The observed M was independent of the concentration of the acrylamide, indicating that very little, if any, carbohydrate is associated with the antigen; both forms failed to bind to a series of insolubilized lectin columns. The SDS gel electrophoresis indicated a high degree of purity for hLTAA-II. The sum of the sizes of the two minor peaks from hLTAA-I very closely approximates the size of the major component, εuggeε- ting the occurrence of proteolytic cleavage at one site, producing two fragments. Peptide mapping of hLTAA-I and hLTAA-II show a high degree of overlap between the patterns , indicating that the se two forms are st ructurally very s imilar . The is oelectric point of hLTAA-II (peaks at pH 2. 6 and 3 - 2 ) generally was more acidic than hLTAA-I (maj or component centered at pH 4 . 7, minor component at pH 3. 1 ) . Becaus e of the diffi culties in c onverting result s from gel filtrat ion analysis of unknown pro¬ teins int o reliable M„r estimates , 3 the diffusion coefficient of hLTAA was also calculated, which permitted a calculation of the Stokes radius, which is more reliably related to the behavior of proteins o on gel filtration. Thus, the value of 51 A obtained by this calculation can be considered a more accurate reflection of the size of the antigen, whereaε the value of 140,000-150,000 daltons, although less reliable, is useful in initial comparisons which can be made between laboratories involved in similar studies. Purified hLTAA is generally useful in a known radioimmunoassay. Immunoassays according to the Invention are particularly useful for detecting and quantitating levels of hLTAA below about 1 ug/ml, which are undetectable by methods such as Ouchterlony analysis. Since serum levels below 1 yg/ml have been found by the process of the present invention to be those most commonly associated with the presence of lung tumors, Ouchterlony and related procedures of similar sensitivity are not clinically useful. In a particular embodiment, an immunoassay of the invention broadly comprises a radioimmunoassay employing radio- labelled purified hLTAA and anti-hLTAA in known amounts as an assay mixture for serum analysis for hLTAA. Bound labelled hLTAA is recovered from the test serum, and the concentration compared to a standard curve correlating the concentration of bound radio¬ active antigen. Serum concentrations of hLTAA is then employed as an aid in the diagnosis of tumor burden. In an alternate embodiment, the immunoasεay of the invention comprises a solid-phase radioimmuno- aεεay technique of the type based on the SANDWICH principle. In this assay, patient serum or plasma is incubated with a solid support such as a disc con- taining coupled anti-hLTAA (e.g., goat). Subsequently, radiolabelled anti-hLTAA (e.g., equine) Is added, which binds to any hLTAA which has reacted with anti-hLTAA coupled to the support. The complex gives a radioactivity measurement which is compared to a standard curve to give the level of hLTAA in the specimen. Radio immune as says with a useful detection range from 1-100 ng/ l, were developed with hLTAA-I for clinical use. Evaluation of binding of radio- iodinated antigen to antibody showed that 20% of the radioiodinated antigen was bound at an antiserum dilu¬ tion of 1:3500 (Fig. 2). Quantitative binding occurred at higher antibody concentrations, as shown in Fig. 1, which graphically represents a study wherein the major component (which contains both hLTAA-I and hLTAA-II) was completely excluded in the presence of immune serum. In another experiment (Fig. 2), using iodinated hLTAA-I, R-201 at a final dilution of 1:2000, and a second antibody (goat anti-rabbit IgG) in place of Pansorbin to separate free from bound antigen, 9 % of the radiolabelled antigen was found to be reactive with the antibody. Many additional immunoasεay procedures well knovm to those skilled in the art may be based on the hLTAA and anti-hLTAA of the present invention, such as enzymatic radioisotope assays (ERIA), ultrasensitive enzymatic radioimmunoassays (USERIA), and enzyme potentiated radioimmunoassay (EPRIA). Broadly, various heterogenous enzyme immunoassay systems such as EIA, ELIA, ELISA, CELIA and IEIA are contemplated, as well as assays employing fluorescents as markers. Various radioactive isotopes such as . 125 I are employed as the radiolabel. Conventional antisera are a cuεtomary source of antibodies for bioaεsay procedures according to the present invention; goat antiserum containing anti- boides to hLTAA is on deposit at the American Type Culture Collection, Rockville, Maryland, identified by ATCC accession number 40057- However, monoclonal antibodies having the desired specificity to the anti¬ gen are the reagents of choice in many immunological applications, particularly sensitive radioimmunoaεsayε. Monoclonal antibodieε (McAb) uεeful in the detection and evaluation of hLTAA are obtained from εelected hybrid myelomaε produced according to well known protocolε. Broadly, hybrid cell cultureε pro¬ duced by fuεion of myeloma cellε with εpleen cells producing the desired antibody are screened to isolate hybrids which produce McAB of the desired specificity, and the εelected hybrldε cloned. In contraεt to conventional antiεera which generally contain a mixture of antibodieε of varying avidity and specificity, monoclonal antibodies secreted by a single hybrid clone are homogenous, thus providing accurate and reliable aεsay reεultε. Further, they are relatively inexpen¬ sive to produce once a clone has been identified, generally being propagated in tissue culture or mice. The need for large animals or humans as a source of conventional antisera is thus obviated. The preεence of hLTAA in most lung tumor extracts of all histologic types and its absence at significant levels from all normal tissues tested (as measured by double diffusion analysis), qualifies it as a useful lung tumor marker. Since there is no known satsifactory marker for the screening or diag¬ nosis of lung cancer, the hLTAA described here has particular relevance. The development of radloimmuno- aεsayε with a sensitivity of 100-1000 times greater than double diffusion assays permits the asεay of εera to determine the preεence of hLTAA. Further, the quantitation of hLTAA preεent in εera permitε at least a preliminary evaluation of the degree of tumor burden, and may prove to reliabily permit discrimination between Stage I and Stage III tumors as well as to permit a reliable prognosis following medical Intervention by surgery or medical therapy. Practial applications include the screening of sera of populations at high risk of lung cancer, such as smokerε and aεbestos workers. EXAMPLES I. Identification and Purification of hLTAA from a Primary Lung Tumor A. Materials Cyanogen bromide-activated Sepharoεe 4B, Protein A-Sepharose and Sephadex G-200 were purchased from Pharmacia Fine Chemicals, Piscataway, N.J., and DEAΞ-cellulose was obtained from Whatman, Inc. , Clifton, N.J. Agarose was obtained from L 1 Industrie Biologique Francaise, S. A., Gennevillierε, France. All chemicals were the best grades commercially available. OMFI B. Methods 1- Lung Tumor Extraction — Primary tumor and metastaεis to the liver, from a patient with small cell carcinoma of the lung, were combined, giving a total weight of 1657 g. This pool of tissue is referred to herein as LT-120. The tissue was cut into 0.5 inch cubes, divided into eight (8) portions, and 275 ml PBS was added to each. The tissue was diεrupted by two 30 ε full-power bursts in a Brinkman Polytron homogenizer, using a PT-35 generator. The homogenate waε centrifuged for 1 h at 16,300 x g_ in a Sorvall GSA rotor. The supernatantε were poured through a gauze filter, combined and εtored at -20°C. A second pool of tiεεue, deεignated LT-37 was obtained in a similar manner, and found to have immunologic identity with pool LT-120 within the scope of the invention. 2. Antisera — Rabbit antisera, designed R-152 and R-201, were obtained according to Braatz, et al., Purification and Characterization of a Human Lung Tumor-Asεociated Antigen, J. Natl. Cancer Inεt . , 61:1035-1046, 1978. The R-152 and R-201 antiεera were raised against a pool of lung tumor extract having immunological identity with the LT-120 and LT-37 pools, as confirmed by double diffusion in gel resolutions showing detection of the same antigenic determinants by these antisera. These adsorbed antisera detected a protein in 84 of 98 lung tumor extracts and 1 of 13 extracts of tumors from organs other than lung, but were negative, at the level of sensitivity of the double immunodif fusion test, with, normal lung extracts, normal tissueε, fetal tissues and normal serum. - - 3- hLTAA Assay — A radial immunodif usion (RID) test was used to quantitate hLTAA after each purifica¬ tion step. The test waε similar to that deεcribed by Mancini (Mancini, et al., Immunological Quantitiation of Antigens by Single Radial Immunodif fusion, Immuno- chem, 2:235-254, 1965) and Fahey and McElvey (Fahey, et al. , Quantitative Determination of Serum Immono- globulins in Antibody-Agar Plates, J. Immunol. 94:84- 90, 1965). The IgG fraction from rabbit antiserum R-152 was isolated by precipitation with 45% (NH_ j ) 2 « - SOi, followed by adsorption to Protein A-Sepharose and elution with 0.1 M glycine-HCl, pH 3-0. After adjus¬ ting the pH to 7.0 with 0.5 M Na-,C0-, the volume of the solution waε brought to the original volume of the antiserum used for the Isolation. A 1:25 dilution of this purified IgG in 0.05 M Veronal buffer, pH 8.5 at 35-4θ°C waε mixed with an equal volume of 2% agaroεe in the same buffer at 75° C. Of this mixture, 8.5 ml were applied to a 5 x 7-5 cm glasε slide (previously coated with 0.3% agarose and dried), kept in a moist chamber at 4°C and allowed to gel. Wells (1.3 nm) were punched In the agarose and sampleε of product from each purification step (approximately 3 μl) were added in duplicate. Serial dilutions of a lung tumor extract, LT-37, were used as standardε and a unit was arbitrarily defined as the amount of antigen required to produce a circular precipitin ring of 2.7 mm in diameter. The plateε were incubated under light parafin oil for 42 h at 37° C, rinsed and washed overnight in PBS. The gel surface waε covered with with filter paper, dried at 37°C, stained 10-15 min with 0.1% Ami do Black and then destained with. 5% acetic acid until a clear background waε obtained. The diameter ε of the precipitin rings were measured and the calculated areas were plotted against the concentration of standard as shown in Fig. 3. The assay waε linear over a range of 1-30 units. \Vl 4. Antichymotrypsin Assay — A radial immunodif- fusion asεay, similar to the RID assay for hLTAA, was performed according to Princler, et al., Quantitative Assay for α-1-antichymotrypsin by Radial - Diffusion, in Conference on Serologic Analysis of Solid Tumor Antigens, pp. 709-710, (S. Rosenberg, ed.), NY Academic Press, 1980. This asεay has a range of 5-200 μg of ACT. The assay was included owing to observed competition between a previously reported antigen, h.LTAA-2B, and serum α--antichymotrypεin in double antibody radioimmunoasεayε . 5. Double Diffusion Analysis in Agarose — Ouchterlony double diffusion analysis was performed in 1% agarose gel for 24-48 h at 20°C as previously described by Braatz, et al., J. Natl. Cancer Inst . , 61:1035, supra. 6. DEAE-Cellulose Chromatography — A column of DE-52, 350 ml bed volume, was equilibrated with 5 m Tris-HCl, pH 8.1 (buffer A) at 4°C until the effluent had the same conductance as the starting buffer. The crude extract was dialyzed against buffer A containing 0.5 mM phenylmethylsulfonly fluoride and 0.02% NaN_, and then loaded onto the column which was washed with buffer A until the absorbance of the effluent at 2δ0 nm was less than 0.07. A linear NaCl gradient from 0 to 0.2 M, with a total volume of 4 liters, was applied to the column, followed by a 1 M NaCl wash. All NaCl solutionε were prepared in buffer A. Portionε of fractions were concentrated using a Minicon Type B15 concentrator (Amicon Corp., Lexington, Masε. ) and teεted for LTAA or ACT content. Fractions eluting with the NaCl gradient that contained antigenic activity were pooled as indicated and then concentrated by ultra- filtration with an Amicon concentrator and a UM-20 membrane (Amicon Corp., Lexington, Masε.) to a final volume of 35 ml. 7- Gel Filtration — A column of Sephadex G-200, 3-5 x 143 cm, was equilibrated with 50 mM Triε-HCl, pH 8.1, at a flow rate of 15 ml/h. The column waε calibrated with blue dextran and chymotrypsinogen-A (Slgnma Chem. Co., St. Louis, Mo.), bovine serum albumin (Miles Laboratories Inc., Elkhart, Ind. ) , and NaN-,. 8. Affinity Chromatography — The IgG fraction from rabbit antiεerum R-152 (25.8 mg, prepared as described under hLTAA assay, B-3, supra) was coupled to CNBr-Sepharose (35 cc gel) using the manufacturer's recommended procedure. Pooled fractions of antigenic activity from the G-200 profile were pasεed through the affinity column at 4°C. The column waε waεhed with the equilibrating buffer, 0.01 M Triε-HCl, pH 8.1, containing 0.5 M NaCl and then eluted with 2.5 M NH_,SCN. pH 8.0. Fractions which eluted with thiocyanate were immediately pooled, dialyzed against the equilibrating buffer and stored at 20°C. 9- Gel Electrophoresiε — Analytical polyacryl- amide gel electrophoresis was performed according to Davis (Diεcelectrophoreεiε , II. Method and Application to Human Serum Proteinε, Ann. NY Acad. Scie., 121:404- 427, 1964), without stacking gelε . Staining for pro¬ tein waε performed using Coomassie Brillant Blue G-250 as detailed in Braatz, et al. , J. Natl. Cancer Inst., 6l :1035 s supra. For the determination of antigenic activity in the gel after electrophoresis , 2. 5 mm s lices were placed in agarose wells of the same dia¬ meter and double diffusion analysis against rabbit antiεerum R-201 was performed. 10. Analytical Procedureε — Protein was deter¬ mined with the Bio-Rad protein assay reagent , using bovine serum albumin as standard, according to Bradford, M.M. , A Rapid and Sensi-Method for the Quantitation of Mlcrogram Quantities of Protein Utilizing the Principle of Protein Dye-Binding, Anal . Biochem. , 72 : 248-254 , 1976. C . Discussion of Results The crude extract from tumor tissue (ca. 2 liters ) was subj ected to DEAE-cellulose chromatography. The elution profile indicated multiple peaks of ant i¬ genic reactivity (Fig. 4 ) . Three maj or regions con¬ taining antigenic activity are referred to as peaks I, II and III in their order of elution. A negligible amount of activity , peak IV, eluted with 1 M NaCl . The predominant component , peak II, eluted soon after the application of the gradient , at about 0.045 M NaCl, and contained more than four (4 ) times as much antigen as peak III which eluted at about 0. 08 M NaCl . Peak I contained almost one-fourth as much activity as peak II but did not result from overloading the column: when It waε reapplied to a fresh column of DEAE-cellulose three-fourths of the antigenic activity was again recovered in the unbound fractions . Depsite the fractionation of antigenic activity into multiple components , the antigens in peaks I , II and III were observed to give precipitin lines of identity by double - - diffusion analysiε (Fig. 5)- In addition, each of the three (3) componentε produced lineε of identity with the εtandard lung tumor extract, LT-37, which itself is representative of most lung tumor extracts which have been treated in this faεhion. Therefore, these components appeared to have identical antigenic εiteε, which were also common to human lung cancers in general. The quantitative aεpectε of this and the εub- sequent purification stepε are preεented in Table I (Fig. 6A) . The sum of the activity present in the four (4) peaks represents 75% of the activity applied to the column, while peak II alone accounted for 9 of the activity. Fractions were alεo aεεayed for α,-antichymo- trypεin (ACT) for compariεon with a previously isolated lung tumor antigen, hLTAA-2b, which immunochemically related to ACT. As shown in Fig. 6, peak II was clearly separated from ACT and thus by this analysiε waε diεtinct from lung antigen hLTAA-2b. Peak III εhowed some overlap with ACT, however, the two (2) peaks appeared distinct. Baεed on additional evidence to be preεented below, it waε concluded that they are unrelated. Concentration of peak II by ultrafiltration followed by Sephadex G-200 chromatography resolved the antigenic activity into two (2) additional components (Fig. 7). The major peak eluated at a v " e /V 0 of 1.4, corresponding to globular proteinε of M = 135,000 - 150,000. Approximately 41% of the applied activity was contained in this pool of fractions, whereas the minor peak eluted at V /V of 1.75 and contained about 10% of the applied activity. The major peak may represent a more polymerized form of the minor peak, εince the latter eluted in a region containing globular proteins of approximately 80,000 daltonε, roughly one-half the size of the major component. The two (2) forms were Immunochemically related, giving a precipitin line of identity in Ouchterlony double diffusion (Fig. 8). Each peak was further purified by iffinity immunoadsorption chromatography. The IgG fraction iεolated from the rabbit immune εerum (R-152) waε coupled to Sepharose 4B and used as the affinity matrix. Each one of the two G-200 pools containing antigenic activity was applied to the column and εeparated into two (2) fractions: the unbound fraction which was eluted with Tris buffer (0.01 M, pH 8.1, containing 0.5 M NaCl) and the bound fraction which eluted with 2.5 M NH^SCN, pH 8.0. The latter fraction waε immediately dialyzed vs Triε buffer to remove NH^SCN and εtored at -20°C. The unbound fraction waε recycled through the column twice in order to remove all antigenic activity. The total antigenic activity recovered after the three (3) cycles of binding and elution represented about 67% of the activity first applied to the column (Table I, Fig. 6A). Polyacrylamide gel electrophoretic analysis of the fraction which bound to and was eluted from the antibody column demonstrated the degree of purity of this material. Figure 9 showε a repreεentative pattern obtained when the affinity-purified preparation waε electrophoreεed on a 7% acrylamide gel. One major protein staining component waε evidenced, with some diffuse anodal staining. For the purpose of locating the antigenic reactivity, an identical gel with the same protein load (27 μg) was electrophoresed at the same time. The tracking dye migrated to the same poεition in both gels, 50 mm. Sliceε (2.5 mm) of the unεtained gel were teεted for immunoprecipitable activity by Ouchterlony analysis using the rabbit immune serum R-201. The antigenic activity corres¬ ponded closely to the major staining component, with R f ranges of 0.09-0.22 and 0.11-0.17, respectively. Electrophoresiε in 5% acrylamide gels gave a similar result, with the protein and activity showing increased, but identical mobilities. The primary antigen described here is the affinity-purified, first G-200 peak (peak A) which in turn was obtained from DEAE-peak II. In addition to this, peak III from DEAE was also chromatographed on Sephadex G-200. The antigenic profile obtained (Fig. 6) was similar to the profile obtained with peak II (Fig. 7), again showing two (2) peaks of antigenic activity (peaks C and D). It appears that these various forms are immuno chemically related size and/or charge is omers . Affinity immunoadεorption was also performed on peak B (Fig. 7) and peak C (Fig. 6). When these pools of affinity-purified antigens (affinity pools 2 and 3, reεpectively ) along with the first affinity- isolated antigen (affinity pool 1) were tested in double diffusion, lines of identity resulted not only among each other, but also with the antigen present in a standard extract, LT-37 (Fig. 10), indicating the common nature of these antigens. Lines of identity between the purified and crude forms of the antigens were alεo demonstrated (wells 1 and 2), suggesting that the antigen was Isolated intact. II. Characterization of hLTAA and Develoυ ent of Radioimmunoassay A. Materials Proteins used to standardize size exclusion columns were: aldolase, bovine serum albumin, chymo- trypsin-A and cytochrome c, from Pierce Chemical Co., and apoferritin and IgG from Schwarz, Mann. Esherichia coll alkaline phosphatase and bovine pan¬ creatic α-chymotrypsin were from the Sigma Chemical Co. Antisera to α-,-antichymotrypsin waε obtained from Dako, distributed by Accurate Chemical and Scientific Corp., Hicksville, NY. Pansorbin and Sansorbin (Protein A-containing and deficient forms of inactiva¬ ted Staphylococcus aureus, respectively) were pur- chased from Calibiochem Corp., Bolton-Hunter reagent, 4000 Ci/mmol, was obtained as soon aε possible after preparation from New England Nuclear. Lung cancer sera (kindly supplied by Dr. J. Concannon, Allegheny General Hospital, Pittsburgh, PA) were pretreatment specimens from newly diagnosed patients with lung cancer of various histologic typeε. Normal εera were obtained from the National Inεtitutes of Health Clinical Center Blood Bank. B. Methods 1. Radlolodlnatlon — Partially purified lung tumor-asεociated antigen (pool 1 from the affinity immunoadεorption εeparation described in I-B-8, supra) was radioiodinated by the procedure of Bolton and Hunter (The Labelling of Proteins to High Specific Activities by Conjunction to an 25 I-containing Acylating Agent, Biochem. J., 133:529-538, 1973). Ten microgramε of protein in 100 1 of 0.1 M εodium borate, pH 8.6 waε added to 1 mCi N-εuccinimidyl-3- (4-hydroxy-5C 125 I]-iodophenyl) propionate (Bolton- 5 Hunter reagent) after careful removal of benzene from the latter with a gentle εtream of N-, . After 20 min at 0°C, 0.5 ml of 0.5 M glycine waε added for an additional 5 min and the mixture waε chromato raphed over a PD-10 column (Pharmacia), previously equili- 10 brated with PBS 2 containing 30% glycerol. Routinely, the void volume of the column was collected and further purified by preparative gel electrophoresis as described below. 2. Gel Electrophoreslε — Analytical polyacryla- 15 mlde gel electrophoreεiε waε performed according to Davlε and εtained with Coomaεsie Blue G-250 as pre¬ viously described in Braatz, et al. , J. Natl. Cancer Inst., 61:1035, supra. SDS gel electrophoresiε was conducted according to Weber and Osborn, The 20 Reliability of Molecular Weight Determinationε by Dodecyl Sulfate-Polyacryla lde Gel Electrophoreεis, J. Biol. Che ., 244:4406-4412, 1969, and stained as in Braatz, et al. , J. Natcl. Cancer Inst., 61:1035, supra. / 2 For the preparative isolation of radioiodinated antigen, 1 ml of the void volume from the PD-10 column was loaded onto standard 7% gels (1 x 11 cm) and electrophoreεed at 4°C for an additional 2 h after the tracking dye eluted from the gel. This 30 allowed better resolution of the two (2) slower moving components that were of interest. The gels were then sliced into 5 mm sectionε and the slices containing radlolablled antigen were re-electrophoresed In order to extract the labelled proteins, according to Braatz, et al . , A Rapid and Efficient Method for the Iεolation of Proteins from Polyacrylamide Gels, Prep. Biochem. , 7:495-509, 1977. Recoveries of iodinated antigen were generally in the 50-70% range. 3- Gel Filtration — Analytical Sephadex G-200 chroma ography was performed at 4°C on a 1.2 x 94 cm column equilibrated with 0.05 M Tris HC1, pH 8.1 containing 0.1 M NaCl at a flow rate of 4.1 ml cm "2 h "1 . Fractions (0.86 ml) were collected and protein waε determined by absorbance at 280 nm, or 410 nm for cytochrome c. Radioactivity was determined with a BioGamma counter (Beckman Instruments, Inc.). The standards used and their elution volumes were: blue dextran, 32.7 ml; apoferritin, 33 • 5 ml; aldolase, 44.7 ml; BSA dimer, 43.0 ml; BSA monomer, 53-8 ml; chymotrypsinogen-A, 67.1 ml; and cytochrome c, 73-5 ml. The D- n __ for the antigen was calculated using these data and the known diffusion coefficients for each standard by constructing a plot of 1 D Q W versus the elution volume/void vol e ratio. (Ackers, Molecular Exclusion and Restricted Diffusion Processes in Molecular Sieve Chromatography, Biochemistry, 3: 723-730, 1964; Andrews, The Gel Filtration Behavior of Proteins Related to their Molecular Weights Over a Wide Range, Biochem. J. , 96:595-605, 1965; DeVincenzi, et al., Reevaluatlon of the Molecular Weights of Glycogen Phosphorylases A -and B Uεlng Sephadex Gel Filtration, Biochemiεtr , 6:3489-3497, 1967.) Linear regreεεion analyεis of theεe data was performed using the curve fitting procedureε of the MLAB program on the National Inεtituteε of Health DEC-Syεtem 10 computer. 4. High Performance Liquid Chromatography — The apparatus used was a Model 334 MP Liquid Chromato- graph system with a Model 155-40 variable wavelength detector (Beckman/Altex) . A Spherogel TSK-3000SW column and precolumn (Altex) were used in conjunction with a 20 μ l loop at a flow rate of 0.7 ml min -1 with 0.05 M sodium phosphate, pH 7-0 containing 0.3 M NaCl as the mobile phase. Effluent absorbance was moni¬ tored at 210 nm for greater sensitivity. All buffers and standard protein εolutions were prepared in glass distilled water and filtered through a 0.22 μm filter (Nalgene). The data were analyzed and the diffusion coefficient calculated as described above. The standards and their elution volumes were: blue dextran, 10.2 ml; apoferritin, 11.0 ml; IgG, 13-3 ml; BSA di er, 14.8 ml; BSA monomer, 17.2 ml; chymotryp- εinogen-A, 21.8 ml; and cytochrome c, 22.6 ml. 5- Sedimentation Velocity Analyεiε — The S ___. n U 3 W value for the hLTAA was determined by the method of Martin and Ames (A Method for Determining the Sedimentation Behavior of Enzymeε, J. Biol. Chem., 236:1372-1379, 1961), aε previouεly deεcribed in Braatz, et al., J. Natl. Cancer Inst., 61:1035, supra, using E. coli alkaline phoεphatase, S ?π = 6.2S and bovine pancreatic α-chymotrypsin, 2.56S as standards (Gar en, A Fine Structure Genetic and Chemical Study of the Enzyme Alkaline Phosphataεe of E. coll, I. Purification and Characterization of Alkaline Phospha- taεe, Biochem. Biophys. Acta. , 38:470-483, 1969; Schlesinger, The Reversible Disεociation of the Alkaline Phosphataεe of E. coll, J. Biol. Chem., 240:4284-4292, 1965; and Smith, Sedimentation Behavior of Derivatives of α-chymotrypsin, J. Biol. Chem., 195:525-530, 1952). 6. Isoelectric Focusing — The apparatus used consisted of a 110 ml LKB 8100 A pholine Electro- focusing Column (LKB Instruments, Inc., Rockville, MD) and Isco Model 94 Electrophoreεiε Power Supply (Instruments Specialties Company, Lincoln, Neb.). A linear gradient of glycerol (5-50% prepared in freshly deionized water, 50 ml of each), containing 1% (final concentration) carrier ampholytes, was formed in the column with an Ampholine Gradient Mixer (LKB). Half of the following mixture of ampholyteε waε added to each εolution forming the glycerol gradient: 1 ml pH 3-5-5, 2.5 ml pH 4-6, and 5 ml pH 2.5-4 LKB 1809 Ampholine carrier ampholyteε. Purified and radio¬ iodinated hLTAA was mixed with the high density glycerol solution and introduced into the colum after approxi¬ mately half the gradient had been formed. The anolyte was 1 M acetic acid in 50% glycerol and the catholyte waε 2% LKB 1809 Ampholine, pH 6-8 in 5% glycerol. Focuεing waε carried out for 27 h at 4°C, at a conεtant power of 15 W, during which the voltage rose from 1000 to 1550 V and the current dropped from 15 to 9.9 mA. After focusing, the solution was pumped from the column and 0.5 ml fractions were collected and subjec¬ ted to pH and radioactivity measurement . 7. Immunoaεsays — Binding of radioiodinated hLTAA to R-201 was performed in 400 μl polyethylene Microfuge tubeε. Twenty-five (25) microliters of labelled antigen containing 15,000-20,000 cpm was mixed with 10-200 μl of a 1:2000 dilution of R-201 in a total volume of 350 μl. Dilutions and volume adjustments were made with PBS containing BSA at 1 mg/ ml. After 16 h at 4°C, 30 μl of a 50% suεpenεion of Pansorbin was added and incubated for an additional 1.5 h. The suspension was centrifuged for 1 min in a Beckman Microfuge and the supernatant was aspirated. The tip containing the pellet was removed with a razor and radioactivity was determined in a gamma counter. Incubations with Pansorbin from 1-5 h gave similar results. Binding with the Sansorbin control was insig¬ nificant. For the generation of the standard binding inhibition curve (Fig. 11) and the analysiε of teεt sera (Fig. 12), a similar protocol was used but the total incubation volume was 1.0 ml and R-201 was uεed at a constant final dilution of 1:15,000. The antigen preparation used as the inhibitor was pool 1 from the affinity immunoadsorption described in I-C, supra. The pellet was sediment ed for 10 min at 3000 x _, the supernatant drained and the pellet counted In a gamma counter. The logit-log transformation of the binding data was used for analysis of the results according to Rodbard, et al., Rapid Calculation of Radioimmunoassay Results , J. Lab. and Clin. Med. , 74:770-781, 1969. Sera from normal or lung cancer patients were assayed for hLTAA by including 10 μl of sera during the first incubation and the amount of binding was used to determine, from the standard binding inhibition curve, their concentration of hLTAA. 8. Other Procedures — E. coll alkaline phos- phataεe and chymotrypsin activities were determined using p-nitrophenyl-phosphate (Braatz, et al., J. Natl. Cancer Inst., 61:1035, supra) and benzoly-L-tyro- εine ethyl ester (Hummel, A Modified Spectrophotometrlc Determination Of Chymotrypsin, Trypεin, and Thrombin, Can. J. Biochem. Physiol. , 37:1393-1399, 1959). C. Discussion of Results 1. Radioiodinatlon of hLTAA — Attempts at labelling the partially purified hLTAA with 125 Iodine by the chlormaine T method of Hunter and Greenwood 5 (Preparation of Iodlne-131 Labelled Human Growth Hormone of High Specific Activity, Nature, 194:495-496, 1962) were unsuccessful, with negligible incorporation of radioactivity into material with an f of 0.0-0.2 on 7% polyacrylamide gels. This failure to 0 Incorporate radioactive iodine into the protein was not corrected by the treatement of the protein with SDS prior to radioiodination, which would expose .potentially buried tryosine groupε. For this reason all subεequent radioiodinations were performed with Bolton-Hunter 5 reagent, which yielded levels of incorporation of 125 I into protein of from 0.1-2.0 μCi/μg. Electrophoresis of this radioiodinated purified antigen on 7% poly¬ acrylamide gels indicated the presence of two (2) components with R- values of 0.15 and 0.44 (Fig. 1, Q panel A). Since gel electrophoresis of the unlabelled antigen produced only one (1) component which stained with Coomassie Blue and which had an R f comparable to the slower moving component in Fig. 1, panel A, it was of interest to determine whether both of the 5 radiolabelled peaks reacted with immune serum. Fig. 1, panel B, shows the effect of immune serum on these components. The slower peak was totally excluded by the gel aε expected for immune co plexeε, whereaε the mobility of the faster peak was unchanged. In a separate experiment, the labelled components were isolated from the gel after electrophoresis and mixed with immune serum, R-201. Radiolabelled antigen which bound to antibody was separated from the free form by Pansorbin aε described in II-B-7, εupra. With a dilution of antiserum which produced 30% precipitation of the ε low peak, only 3% of the faster peak could be precipitated . Thus , the R -. = 0.44 component appeared to be a non-antigenic contaminant or denatured antigen and was removed from the labelled mixture by preparative polyacrylamide gel electrophoresis . When electro¬ phoresis was carried out for extended periods of time (generally for 1 h after the tracking dye ran off the gel) the reactive component could be resolved in two (2) peaks , as shown in Fig. 13. Even better resolution could be obtained in longer runs which eluted the fast , unreactive component from the gel . Using these condi¬ tions , both components of the reactive peak designated (hLTAA-I and hLTAA-II ) were isolated and shown by re- electrophoreεiε to be diεtinct (Fig. 14 ) . Furthermore, analytical gel electrophoreεiε of a mixture of hLTAA-I and hLTAA-II reεulted in complete εeparation of the two (2 ) componentε (data not εhown ) . Radiolabelled hLTAA-I and hLTAA-II were added back to the unlabelled εample from which they were derived, and their positions in acrylamide gels used to located unlabelled hLTAA-I and -II. Using this technique , 20-50 μg have been isolated of each for further studieε (see results with HPLC, II-C-3, infra) . 2. Immunoreactivity of Iodinated hLTAA Forms I and II — Although the resultε shown in Fig . 1 suggeεted that both peaks I and II should react with R-201, it was necessary to directly examine this point . Fig . 2 showε that both peakε I and II bound appreciably above the controlε to varying dilutionε of R-201. The εimilar shapes of the curves suggest the similarity of the antigenic combining sites contained within each component . Also demonstrated In this chart is that neither hLTAA-I nor hLTAA-II reacted with a commercial antiserum to alpha- 1-antichymotryps in (ACT). Thlε distinguishes these proteins from hLTAA-2b, another protein recognized by R-201 and R-152, which has been shown to be cross-reactive with plasma ACT. 3- Biochemical Analysis of hLTAA-I and hLTAA-II — Further characterization was then performed on both hLTAA-I and hLTAA-II. Gel filtration of each on an analytical column of Sephadex G-200 produced identical elution profiles, with both peaks eluting just prior to the dimer of BSA, indicating a molecular weight of approximately 140,000-150,000 (Fig. 15). The diffusion coefficient of the antigen was determined by comparing its elution position from Sephadex G-200 with that of a series of standards. A plot of the reciprocal of the diffusion coefficient of these standard proteins versus their elution positions (V /V ) yielded a smooth curve, from which was interpolated a D _-..u,-, , . w, of 4.2 x 10 " 7 cm 2 s -1 for the antigen. High performance liquid chromatography (HPLC) was also performed on unlabelled hLTAA-I and hLTAA-II (isolated as described above). A Spherogel-TSK 3000SW (Altex) size exclusion column was used which yielded profiles similar to those obtained with Sephadex G-200 (Fig. 16). Again, both antigens eluted just prior to the elution of the dimer of BSA, with elution times of 20 min. Radiolabelled antigen eluted as a single, symmetrical peak with the same retention time of 20 min (data not shown). Panel C of Fig. 16 shows the HPLC chromatogram of the sample used for iodination in these stuies, from which hLTAA-I and hLTAA-II were isolated. At leaεt three (3) minor contaminant ε are detected which are removed by gel electrophoreεis. As before with Sephadex, a series of standard proteins - 12 - was chromatographed and a plot of 1/D vs V /V con¬ structed. From this curve, a value for the diffusion coefficient for the hLTAA was calculated to be 4.3 x 10 " 7 cm 2 s "1 , In close agreement with the value obtained using Sephadex G-200. From the average of the diffusion coefficients obtained above, 4.25 x 10~ 7 cm 2 s "1 , we calculated o the Stokes radius of the antigen to be 51 A, accor- - ding to Siegal, et al., in Biochem. Biophyε . Acta., 112:246-362 (1966). Analysis of hLTAA-I and -II by velocity sedimentation in 5-20% sucroεe gradientε (Fig. 17), showed each to behave as a single sedimenting species. When compared with two (2) standard proteins, E. coli alkaline phoεphatase (6.2 S) and bovine pancreatic α- chymotrypsin (2.56 S), hLTAA-I and -II were found to have indiεtinguiεhable εedlmentatlon coefflcientε of 4.6 + 0.4 S and 4.5 +.0.2 S, reεpectivel . The ability to separate hLTAA-I and -II by acrylamide gel electrophoresis, despite their very similar size as evidenced by gel filtration and sedimentation velocity analyεiε, suggests then that they differ in charge. To address this point, isoelectric focusing was performed on each (Fig. 18). As expected from Its greater electrophoretic mobility in acrylamide gels, peak II was shown to have a greater net negative charge. Peak II focused around pH 3-2 with a smaller component at pH 2.6, whereas hLTAA-I focused primarily around pH 4.7, with a minor component at pH 3.1. Analysis by SDS gel electrophoresis consis¬ tently showed hLTAA-II to be a single component (Fig. 19). The pattern obtained was independent of o: :?ι the concentration of acrylamide, and the M r waε calculated aε 81,700, based on the average of values (89,000, 76,000, and 80,000) obtained uεing 7-5, 10.0 and 12.5% acrylamide gels, respectively. Peak I on the other hand was found to be composed of a major component, identical in size to peak II, and two (2) minor components of M p = 50,000 and 27,700.. These results were likewise independent of acrylamide concentrations . 4. Development of a Radioimmunoassay — The radioimmunoassay for quantitating the antigen in clinical specimens was developed using hLTAA-I as the trace-labelled antigen. Form hLTAA-I was selected on the basis of its greater availability. Subse- quent experiments gave identical results using both forms. Labelled antigen was incubated overnight with antiserum at 4°C, and the bound and free antigen was separated by Pansorbin as described in II-B-7, supra . A standard binding inhibition curve was obtained, as shown in Fig. 11, by including known amountε of unlabelled antigen in the overnight incubation. The logit-log transformation of the binding data indicated a useful range of 1-100 ng/ml. 5. Detection of hLTAA In Sera of Lung Cancer Patients — Three (3) groups of sera were tested: 15 from normal individuals, 13 from Stage I lung cancer patients, and 15 from Stage III lung cancer patients. The antigen concentration in each serum, obtained by interpolation from Fig. 11 of the amount of inhibition each produced in the binding asεay, is shown in Fig. 12. With normal sera, there was a clustering of low values with a mean (+ S.D.) of 17 __ 22 ng/ml. . Elevated and more divergent values were obtained with both Stage I and Stage III lung cancer sera. The means (+ S.D.) for these groups were 187 + 289 and 277 + 252 ng/ml, respectively. By arbitrarily selecting a cutoff of 83 ng/ml, which is three (3) standard deviations above the mean of normals, 7/13 Stage I sera, 12/15 Stage III sera and 0/15 normal sera exceeded this value. As predicted from the negative results with Ouchterlony double diffusion,, all but one of the sera had hLTAA levelε of lesε than 1 μg/ml. However, a large proportion of the lung cancer sera had levels of antigen that were clearly higher than those in normal sera. The ability to discriminate Stage I from Stage III disease is less certain, although the reεultε suggest that later stageε of the disease are associated with higher and more frequent elevations of circulating antigen. 6. Clinical RIA Procedure for hLTAA — To a series of 0.4 ml polyethylene microcentrifuge tubes were added 0.1 ml of a 1:320 dilution of normal rabbit serum. 1. To a set of four of these tubes the following were added, mixing well after each addition: a. 0.1 ml of radioiodinated hLTAA containing approximately 2000 cpm. b. sufficient PBS (0.01 M sodium phosphate, pH 7.4, 0.15M sodium chloride) containing 1 mg/ml bovine serum albumin (BSA), such that the final volume iε 1.0 ml. This set will give an esti- mate of the non-εpecific binding (NSB). 2. To another εet of four tubes were added the following, mixing well after each addition: a . 0. 1 ml of a 1 : 1000 dilution of antiserum to hLTAA (R-201 ) . b . same as in la and lb . This set will give an estimate of the maximum binding (MB) . 3. To the remaining tubes were added the following, mixing well after each addition : a. 0. 01 ml of patient 's serum, or b . 0. 01-0.1 ml of a standard hLTAA solution of varying concentrations such that a range of 10-100 nanograms of hLTAA is added to each tube . c . same as in 2a and 2b . The tubes were rested at 4°C for 16 hours, after which 0.05 ml of a 50% suspension of inactivated Staphylococcuε aureuε cellε containing Protein A (Pansorbin, Chalblochem-Behring Corp.) were added to each tube with mixing. The tubes again were rested at 4°C for 1.5 hours, followed by centrifuging for 2 minutes at 12,000 rp in a microcentrifuge . The supernatant was discarded, and the tip containing the pellet removed with a razor. The radioactive content of the pellet was measured by a gamma counter. A standard curve was constructed using the values obtained with the hLTAA standard solutions by plotting the binding values for each standard against the log of its concentration. From this standard curve, the hLTAA concentration in each serum εample was interpolated from the individual binding values.

Claims: WHAT IS CLAIMED IS: 1. A human lung tumor-associated antigen (hLTAA) associated with lung tumors comprising small cell carcinomas, large cell undifferentiated carcinomas, εquamous cell carcinomas and adenocarcinomaε, charac¬ terized by a diffusion coefficient (D 0 ) of 4.2-4.3 x 10 "7 cm 2 s _1 , a sedimentation coefficient (S 2Q ) of 4.5-4.6S; and a Stokes radius of 51 A; immunological reactivity with a xeno¬ antiserum containing heterlogous specieε antibodieε raiεed in a heterlogouε εpecieε animal aε an Immune reεponse to a crude extract of at least one of said lung tumors; and no antigen crosε-reactivity with α,-antichymotrypεin. 2. A method for the iεolation and purifica¬ tion of the hLTAA of Claim 1, from a crude " extract of εaid lung tumor compriεing complexlng hLTAA with, and eluting hLTAA from, heterlogouε εpecieε antibodies in insolubilized form. 3. The method of Claim 2, wherein the crude extract Is pretreated to remove extraneous protein material. 4. The method of Claim 3, wherein the crude extract is pretreated by gel filtration and/or ion- exchange chromatography. 5 - An asεay εyεtem for use in a serological immunoaεsay for the hLTAA of Claim 1, comprising hLTAA and antibody specific to hLTAA, wherein either the hLTAA or the antibody is fluorescent-, enzyme-, or radioisotope-labelled. 6. The invention of Claim 5, wherein the immunoassay is a radioimmunoassay and the assay mixture comprises radiolabelled hLTAA and unlabelled hLTAA antibody. 7. The invention of Claim 6, wherein the hLTAA is radiolabelled with radioactive iodine. 8. A reagent for use in lung cancer diag- noεiε compriεing heterlogous specieε antibodies to the hLTAA of Claim 1. 9. The invention of Claim 8, wherein the antibodieε are fluorescent-, enzyme-, or radioiεotope- labelled. 10. The invention of Claim 8, wherein the antibodies are coupled to a solid support. 11. A diagnostic kit for use in the diagnoεis of lung cancer including a first reagent compriεing unlabelled heterlogous specieε antibodies and a second reagent comprising labelled heterlogous species OMPI V _ V -O , ^ϋ NATXOt antibodies , εaid antibodies having immunological reactivity to the hLTAA of Claim 1. 12. The Invention of Claim 11, wherein the unlabelled antibodies are coupled to a solid support , 13 - The invention of Claim 11, wherein the labelled and unlabelled antibodies are derived from two different het erologous species . 14. A method for detecting the presence of the lung tumor antigen (hLTAA) of Claim 1 in serum comprising interacting the serum or a fract ion thereof with insolubllized anti-hLTAA antibody to bind hLTAA present in the serum t o the antibody and form an antibody/antigen complex ; interacting labelled anti-hLTAA antibody with this complex to form an antibody /antigen/labelled antibody complex; and measuring the amount of labelled anti-hLTAA antibody in this complex. 15. A radioimmunoassay for determining the concentration of hLTAA in an unknown comprising : admixing radiolabelled purified hLTAA and unlabelled hLTAA antibody with the unknown; after equilibrium is attained, recover¬ ing radiolabelled hLTAA complexed with antibody ; and measuring the amount of radiolabelled hLTAA recovered and comparing It to a standard which correlates- the amount of radiolabelled hLTAA recovered with the concentration of unlabelled competitor hLTAA in the unknown. - - 16. The invention of Claim 11, wherein the unknown is a human serum sample. 17. The invention of Claim 12, wherein the radiolabel is 125 I. l8. The invention of Claim 5, wherein the hLTAA is coupled to a solid support.

Inventors: BRAATZ JAMES ANTHONY, MCINTIRE KENNETH ROBERT, PRINCLER GERALD LEE, BRAATZ, JAMES, ANTHONY, MCINTIRE, KENNETH, ROBERT, PRINCLER, GERALD, LEE
Applicants: US COMMERCE, UNITED STATES OF AMERICA, AS REPRESENTED BY THE SE
Application year: 1983
Main classification: G01N33
IPC codes: A61B10, A61K39, A61K51, C12Q1, G01N33