Biotherapy 4: 17-22, 1992. © 1992 KluwerAcademicPublishers.Printedin the Netherlands.

Serodiagnosis of cancers by ELISA of anti-histone H2B antibody M. Kamei 1, M. Kato ~, K. Mochizuki 1, K. Kuroda 1, S. Sato 1, S. Hashizume 1, K. Yasumoto z, H. Murakami 3 & K. Nomoto 4

IMorinaga Institute of Biological Science, Shimosueyoshi 2-1-1, Tsurumi-ku, Yokohama 230; 2Matsuyama Red Cross Hospital, Bunkyo-cho 1, Matsuyama 790; 3Graduate School of Genetic Resources Technology, Kyushu University, Hakozaki 6-10-1, Higashi-ku, Fukuoka 812; 4Medical Institute of Bioregulation, Kyushu University, Maidashi 3-1-1, Higashi-ku, Fukuoka 812, Japan Received 14 December1990; accepted26 December1990

Key words: anti-histone H2B, cancer, ELISA, serodiagnosis Abstract

An enzyme-linked immunosorbent assay method for serodiagnosis of cancers was developed by employing histone H2B. This method measures anti-histone H2B antibody levels in sera and includes a device for coating the plastic immunoplate with a mixture of histone H2B and diluted fetal calf serum. The coating of immunoplates with this mixture decreased apparent sensitivity of the assay compared with that in the absence of fetal calf serum, but effective reduction of nonspecific background enabled a specific assay of anti-histone H2B antibody with excellent reproducibility. By this method cancer patients were discriminated from normal healthy subjects at detection rates of 37% for lung cancer, 33% for liver cancer, 50% for pancreatic cancer, 42% for colon cancer, and 78% for cervical cancer. However, stomach and esophagus cancers showed detection rates of less than 17%, which are comparable to the values for benign diseases. It is likely that this assay method detects squamous cell carcinomas at relatively high rates.

Abbreviations: ELISA: enzyme-linked immunosorbent assay; FCS: fetal calf serum; PBS: phosphatebuffered saline; BSA: bovine serum albumin; PBS-Tween: phosphate-buffered saline containing 0.05% tween 20; PBS-BSA-Tween: phosphate-buffered saline containing 1 mg/ml bovine serum albumin and 0.05% Tween 20; ABTS: 2,2'-Azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt; S.D.: standard deviation; CEA: carcinoembryonic antigen; SCC: squamous cell carcinoma-related antigen; NSE: neuron-specific enolase.

Introduction

Serodiagnosis is extensively employed as a powerful means to detect various diseases by a variety of methods such as radioimmunoassay and ELISA because of its high sensitivity and reliability [1]. Serodiagnosis comprises two categories of measurements, depending on the materials to be determined, i.e., either marker antigens [2-8] or anti-marker antibodies [9-12]. In serodiagnoses of cancers, elevated concen-

trations of marker antigens such as oncofetal proteins [2, 3], cancer-related antigens [4, 5], isozymes [6,7] and hormones [8] have been evaluated almost exclusively to assess malignancy. Our preliminary radioimmunoimaging experiment with a human monoclonal antibody (termed HB4C5), which had been derived from a human-human hybridoma cell line [13], in athymic nude mice bearing lung cancer xenografts (LC6 cell line) gave promising results of

18 specific accumulation of radioactivity to the cancer [14]. We have also found that histone H2B has the specific antigenicity for this human monoclonal antibody, which will be described in detail elsewhere. In this context, an attempt was made to screen cancer patients by measuring anti-histone H2B antibody concentrations in sera. Determination of anti-histone antibody concentrations in sera has already been proved effective in screening patients suffering from systemic lupus erythematosus and rheumatoid arthritis [11, 12]. Although some investigators focused their attention on diagnosing cancers by determining anti-histone antibody concentrations in sera [11, 15], no appreciable positive rate for cancer detection has been attained. In this paper we describe that serodiagnosis of cancers can be performed successfully by the determination of anti-histone H2B antibody level.

Materials and methods

ELISA Calf thymus histone H2B was purchased from Boehringer Mannheim GmbH, Germany. ELISA was carried out using the histone H2B as the coating antigen. Histone H2B was dissolved to a concentration of 50 tzg/ml in 43 mM sodium carbonate buffer, pH 9.6, containing 0.05% FCS (Armour Pharmaceutical Co., Kankakee, IL, USA) and 0.02% sodium azide. Wells of immunoplates (Nunc MaxiSorp, 96 wells, Kamstrup, Roskilde, Denmark) received 100/zl aliquots of this histone H2B solution and were coated with the proteins by incubation for 1 h at 37°C. Wells for a 'minus histone' control, taken routinely to ensure histone H2B dependency of the assay, were coated in the same manner with the above solution from which histone H2B was excluded. After washing the wells with PBS, 360/xl aliquots of PBS containing 10% FCS and 0.02% sodium azide were added and incubated for 1 h at 37°C in order to block possible remaining sites for adsorbing proteins on the plastic surface. Sera from patients and normal healthy subjects were diluted 200 times with PBS-BSATween before dispensing to the wells of immuno-

plate. The immunoplate having 50/xl aliquots of the diluted sera in the wells was incubated for 1 h at 37°C followed by extensive washing with PBSTween. A 100/xl aliquot of the peroxidase-conjugate of affinity purified goat anti-human IgG (gamma chain specific; Tago Inc., code No. 2390, Burlingame, CA, USA) diluted 1000 times with PBSBSA-Tween, at which concentration of the conjugate the assay attained saturating level of reaction, was added to each well and incubated for 1 h at 37°C. After washing the wells with PBSTween three times, activity of peroxidase bound in each well was assayed. Enzyme reaction of the assay was started by the addition of a 100/~1 aliquot of ABTS substrate solution to the well. The substrate solution was prepared by mixing the A and B stock solutions described below at a ratio of 19:1 just before use. The stock solution A consisted of 12.9 g of citric acid monohydrate, 27.6 g of disodium hydrogenphosphate dodecahydrate, and 0.1ml of 30% H 2 0 2 in a total volume of 1000 ml. The stock solution B contained 6 mg/ml of ABTS (Wako Pure Chemical Ind. Co., Osaka, Japan) in H20. The reaction was performed for 15 min at 25°C and stopped by the addition of a 100/~1 aliquot of 1.5% oxalic acid. Absorbance at 415 nm was read within 10min after stopping the reaction with an automatic microplate reader (Toso Model MPR-A4, Tokyo, Japan).

Results and discussion

Immunoplate coating with histone H2B In ELISA to determine antibody, where the plastic immunoplate is coated with a protein antigen, the coating of antigen has usually been carried out with a solution of pure antigen, i.e., devoid of other protein materials which could be adsorbed onto the plastic surface. After coating with the antigen, possible remaining sites on plastics for adsorbing proteins are blocked with appropriate blocking agents, such as FCS, BSA, etc. However, we often encountered the difficulty that enough blockade could not be attained in 'minus-antigen' control wells which were coated

19 first with the coating buffer alone in the absence of antigen followed by the blockade as described above, and the assay sometimes resulted in higher intensities of coloration in wells of 'minusantigen' control rather than those coated with the antigen (Table 1). However, when the control wells were coated first with the blocking agent instead of the coating buffer alone, much lower background intensities were attained. Since it seemed likely that plastics would adsorb proteins more efficiently on the first occasion of contact with the solution than the second, we tried to coat plastic immunoplates with the blocking agent in the presence and absence of antigen in order to reduce nonspecific background, especially in the 'minus-antigen' control, and results of E L I S A were compared with those of the conventional method. Table 1 shows that the presence of FCS decreased nonspecific background of the assay effectively, and significant improvements were attained in histone H2B dependency of the assay as well as minimized fluctuation of the determination in 'minushistone' H2B controls, proving the assay to be more reliable for detection of cancer-dependent anti-histone H2B antibody in sera. Although absolute A415values determined (i.e., the apparent sensitivity of the assay) were reduced by the presence of FCS in the histone H2B solution for coating, these results were expected because of the reduced amount of bound histone H2B on

the plastic surface. The results also indicated that significantly high levels of anti-histone H2B antibodies were contained in sera of cancer patients compared with those of normal subjects. Histone H 2 B and serum dependencies o f E L I S A When the plastic immunoplate was coated with varied concentrations of histone H2B in the presence of 0.05% FCS, A415 readings of the assay with sera from cancer patients increased as the coating concentration of histone H2B became higher and reached plateaus around 50 p~g/ml (Figure 1). These results show that the reaction of this assay is saturable and histone H2B-dependent. On the other hand, when sera from normal healthy subjects were similarly examined, negligible increases of histone H2B-dependency were observed (Figure 1). U n d e r the conditions of histone H2B coating at 50 ~ g / m l concentration, deviations of individual A415 values determined with normal healthy sera from their average were within a range of 2 × S.D. However, sera from cancer patients showed much higher values of A415 (Figure 1). These results indicate that serodiagnosis of cancers can be performed effectively with this assay system in which the immunoplate is coated with a mixture of histone H2B and FCS at concentrations of 5 0 / z g / m l and 0.05%, respectively.

Table 1. Effect of FCS in histone H2B solution for coating immunoplates on ELISA of anti-histone H2B antibody in sera. Wells of an immunoplate were coated with the indicated concentrations of histone H2B in the presence and absence of 0.05% FCS, blocked with 10% FCS, and then proceeded for the assay with sera as described under Materials and methods. Serum sample

Reaction (A415) Minus FCS

Plus FCS

Histone H2B Concentration (/.tg/ml) 0 10

0

10

Normal No. 26 No. 73 No. 86 No. 89

0.190 0.194 0.223 0.166

0.142 0.229 0.197 0.166

0.076 0.115 0.115 0.098

0.101 0.131 0.132 0.120

Cancers No. 76 (Colon ca.) No. 85 (Liver ca.) No. 102 (Lung ca.)

0.341 0.442 0.573

0.511 0.449 0.423

0,i92 0.187 0.152

0.365 0.320 0.326

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With this assay system anti-histone H2B antibody levels in sera were examined in various cancer patients and normal healthy subjects, as well as those of systemic lupus erythematosus and rheumatoid arthritis patients: the results are summarized in Figure 3. The results show that the following percentages of cancer patients can

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Fig. 3. ELISA of anti-histone H2B antibody in sera from cancer patients and normal healthy subjects. The assay was performed as described under Materials and methods with sera diluted by 200-fold. The broken line shows the average +2 S.D. level of the determination on 45 sera from normal healthy subjects.

21 be discriminated from normal healthy subjects by their high (i.e., exceeding the average by more than +2 S.D. of the normal healthy level) anti-histone H2B antibody contents: 37% for lung cancer; 33% for liver cancer; 42% for colon cancer; 50% for pancreatic cancer and 78% for cervical cancer, whereas 14% and 22% of systemic lupus erythematosus and rheumatoid arthritis patients respectively were found positive by this assay. Costa and Monier reported that the detection rates of systemic lupus erythematosus and rheumatoid arthritis were 59.8% and 5.2% respectively in their ELISA system, using histones as antigen [11]. This discrepancy may be ascribed to the differences in experimental conditions: they used total histones, including histone H1, H2A, H2B, H3, and H4 as the antigen, whereas only histone H2B was employed in our system. An extremely high detection rate of 78% for cervical carcinoma, which in most cases belong to squamous carcinoma, is noteworthy. The false positive detection rate for benign diseases was 19% by this assay, which is similar to the results of currently prevailing serodiagnoses measuring tumor markers, such as CEA [16] and SCC [17]. Since anti-histone H2B antibody levels in normal sera are very limited (below 0.2 in A415) , the occurrence of anti-histone H2B antibody at elevated concentrations in cancer patients (in some cases as high as 15 x S.D. above the average of normal sera) should be valid and helpful for diagnosis of cancers (Figure 3).

Tissue-type specificity of ELISA for lung cancer A detailed study was carried out with sera from patients with varied tissue-types of lung cancer. As shown in Table 2, a detection rate as high as 50% was attained for the squamous carcinoma, Table 2. Positive detection rates for lung cancers of different tissue-type by ELISA. Sera from patients with lung cancers of which tissue-types had been diagnosed histologically were assayed for anti-histone H2B antibody levels as described under Materials and methods.

Histology

Number of sample

Positive rate (%)

Adenocarcinoma Squamous cell ca. Small cell ca. Large cell ca.

19 16 3 1

26 50 33 0

to which about a half of the total lung cancer population belong. On the other hand, the adenocarcinoma, to which another major population of lung cancer belong, showed a detection rate of 26%. One characteristic feature of this assay is that squamous cell carcinomas are detectable at high rates, as are the cases of lung and cervical cancers. Although SCC has been used effectively as a tumor marker for specific detection of squamous carcinomas in uterus and lung, it is known that the cancers at advanced stages are exclusively detectable with SCC [18]. The serodiagnosis method described in this paper assays antihistone H2B antibody. It may be possible to detect cancers at early stages of development by measuring anti-tumor antibody instead of tumor markers, since the antibody raised in the patient could permit amplification of the detection signal and enable a more sensitive serological detection of cancers, even if antigen concentrations in sera are still too low to be detected. Studies in this aspect are under way in our laboratories.

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22 8. Odell WD, Wolfsen A, Yoshimoto Y, Weitzman R, Fisher D, Hirose F. Ectopic peptide synthesis: a universal concomitant of neoplasia. Trans Assoc Am Phys 1977; 90: 204-27. 9. Sarngadhara MG, Popovic M, Bruch L, Schupbach J, Gallo RC. Antibodies reactive with human T-lymphotropic retroviruses (HTLV-III) in the serum of patients with AIDS. Science 1984; 224: 506-8. 10. Kanao K, Honda M, Ishihara S, Minami E, Tohe Y, Yagi E, Sakata Y, Mukuno H. Measurement of hepatitis B surface antibody (HBsAb) by radioimmunoassay. The Medical Journal of Sumitomo Hospital 1976; 3: 76-80, in Japanese. 11. Costa O, Monier J-C. Antihistone antibodies detected by ELISA and immunoblotting in systemic lupus erythematosus and rheumatoid arthritis. J Rheumatol 1986; 13: 722-5. 12. Rubin RL, Waga S. Antihistone antibodies in systemic lupus erythematosus. J Rheumatol I987; i4 (Suppl 13): 118-26. 13. Murakami H, Hashizume S, Ohashi H, Shinohara K, Yasumoto K, Nomoto K, Omura H. Human-human

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hybridomas secreting antibodies specific to human lung carcinoma. In Vitro Cell Develop Biol 1985; 21: 593-6. Hashizume S, Sato S, Matsuyama S, Tamaki S, Hanada K, Murakami H, Nakano K, Kusakabe K. Accumulation of ~25I-labelled human monoclonal antibody (HB4C5), specific to lung cancer, into transplanted human lung cancer in nude mouse. In: Proceedings of The Second Annual Meeting of Japanese Association for Animal Cell Technology. Tokyo: Kodansha, 1990: 167-72. Klajman A, Kafri B, Shohat T, Drucker I, Moatem T, Jaretzky A. The prevalence of antibodies to histones induced by procainamide in old people, in cancer patients, and in rheumatoid-like disease. Clin Immunol Immunopathol 1983; 27: 1-8. In: Instruction manual for IMx CEA DAINAPAKT M kit; DAINABOT Corp, Tokyo, Japan. In: Instruction manual for IMx SCC DAINAPAKTM kit; DAINABOT Corp, Tokyo, Japan. Kato H, Morioka H, Aramaki S, Torigoe T. Radioimmunoassay for tumor-antigen of human cervical squamous cell carcinoma. Cell Mol Biol 1979; 25: 51-6.

Serodiagnosis of cancers by ELISA of anti-histone H2B antibody.

An enzyme-linked immunosorbent assay method for serodiagnosis of cancers was developed by employing histone H2B. This method measures anti-histone H2B...
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