Original Article Immunohistochemical Analysis of Human Adenocarcinoma-Associated Antigen YH20 6 Detected by a Monoclonal Antibody Akira Yachi, Kohzoh Imai, Takao Endo and Yuji Hinoda A monoclonal antibody YH206(IgM, k) was prepared using human lung adenocarcinoma A549 cell line as an immunogen. The histological distribution of antigenic determinant of the YH206antigen was highly restricted to adenocarcinomas of various organs including the lung, the pancreas and the stomach, but it did not react with 10 cases of colonic adenocarcinomas by the immunoperoxidase technique. It did not stain non-cancerous tissues of 16 different organs, except for the renal tubules and for the secreting glands of the pancreas with which the antibody faintly reacts. It did not react with either granulocytes, lymphocytes or red blood cells. Positive staining was observed in various fetal tissues. Periodic acid and neuraminidase treatments on tissue sections suggested that the chemical nature of the antigenic determi- nant of YH206antigen was carbohydrate in nature which might be maskedby sialic acids. Key Words: Adenocarcinoma-associated antigen, Immunohistochemistry, Monoclonal antibody There have been some reports concerning MATERIALS AND METHODS monoclonal antibodies (MoAbs) to human lung adenocarcinoma associated antigens . None of Monoclonal antibody YH206 these antibodies, however, were found to detect The MoAb YH206 was obtained from the fusion between murine myeloma X63-Ag8.653 corresponding antigen(s) in the sera of lung cancer cells and splenocytes of BALB/c mouse, which patients. Recently, Iguro et al. described that the was immunizedwith humanlung adenocarcinoma new monoclonal antibody, designated as CSLEX- A549 cell line . The method of preparation has 1, could detect the corresponding antigen in the sera of cancer patients including lung cancer. The been described elsewhere . Testing of the mono- antigenic determinant of this antigen was found clonal antibody with rabbit antisera to mouse Ig to be sialylated Lewisx carbohydrate moiety. classes and subclasses (Bionetics, Kensington, MD) in radial immunodiffusion showed that the MoAb More recently, we established an interesting mono- YH206isIgM, K. clonal antibody (IgM), designated as YH206, which could detect the circulating antigen(s) in Tissue processing and fixa tion the sera of patients with various cancers (manu- Normal tissues as well as benign and malignant script submitted for publication). In this paper tissues were obtained from patients who under- we would like to describe the immunohistochemi- went surgery. Fetal tissues were obtained from 16- cal analysis of the corresponding antigen (YH206 and 24-week-old fetuses. A portion of each speci- antigen) detected by the MoAbYH206. men was fixed with 10%buffered formaldehyde. Twoconsecutive 5 /im-thick paraffin sections were obtained from each sample. One of the paraffin From Department of Internal Medicine (Section 1), Sapporo Medical College, Sapporo. Received for publication September 25, 1985. Reprint request to: Akira Yachi, MD,Department of Internal Medicine, Sapporo Medical College, S-l, W-16, Chuo-ku, Sapporo 060,Japan. JapJMedVol 25,No 2 (May 1986) 127 Yachi et al sections was stained with hematoxylin and eosin as a criterion of positive reactions of the tissue and was studied for its histologic features, while sections with the monoclonal antibody. Unreactive the other section was used as a substrate for tissue sections were tested further with concen- immunoperoxidase analysis. trated (5x) and diluted (10x) solutions of the Immunoperoxidase technique monoclonal antibody to avoid false negative re- A conventional procedure for the immunoper- actions attributable to either a low concentration oxidase technique was employed for the staining of monoclonal antibody or to a prozone-like effect. The specificity of the reaction was control- reactions with the monoclonal antibody. The sec- led by testing tissue sections with myeloma tions were first incubated with 0.6% H2O2 in methanol for 20 min to block endogenous per- protein secreted by the murine myeloma P3-X63- Ag8 cells and with RPMI1640 medium containing oxidase, and were then overlayed with the mono- 10% fetal calf serum. Immunoperoxidase staining clonal antibody for 30 min at room temperature in a moist chamber. After washing three times for ously6).of blood smears was performed as described previ- 5 min in cold phosphate buffered saline (PBS), peroxidase-conjugated rabbit anti-mouse IgM anti- Indirect immunofluorescence technique serum (DAKOa/s Immunochemicals, Copenhagen, This technique was performed according to Denmark) was applied for an additional 30 min. standard procedure. FITC-conjugated rabbit anti- After a final wash with cold PBS, the sections were mouse IgM antiserum (DAKOa/s Immunochemi- reacted for 60 sec in a solution containing 40 mg cals, Copenhangen,Denmark)was used as a second 3,3'-diaminobenzidine and 0.01% hydrogen per- antibody. Cells were observed under fluorescent oxide in 100 ml 0.05M Tris-HCl buffer (pH 7.6), microscope (Zeiss, MC65, FRG). after which they were stained with 0.2% Methyl Periodic acid and neuraminidase treatment on Green for 3 to 5 min and were observed with a tissue sections light microscope. One hundred microliters of 1% periodic acid A distinct brown staining of the cells was used solution in PBS(pH 7.2) were placed on each sec- Table 1. Tissue distribution of the antigenic determinant recognized by the monoclonal antibody YH206. C a n ce r tissu e N o rm al tissu e O rga n T y p e O r g a n A d u l t F e t u s L u n g a d e n o ca rc in o m a + + * (9 /1 3 )* * L u n g - (0 /8 ) + + (1 / 1 ) alv eo lar c el l c ar ci nom a + + (2 / 2 ) C o lo n - (0 /9 ) + (1 / 1 ) e pi d e r m o i d c a r c i no m a F + (1 / 6 1 K id n e y F + (3 /4 ) + + (1 /1 ) lar g e ce ll ca rc in o m a F + (1 / 5 ) L iv e r - (0 /4 ) - (0 / 1 ) sm a ll c ell c ar cin o m a - (0 / 6 ) P a n c re a s F + (3 / 3 ) + (1 /1 ) S m all in te stin e - (0 /4 ) F + (1 / 1 ) Pan cre a s a d e n o ca rc in o m a + + (3 / 4 ) S to m a c h - (0 /8 ) + (1 /1 ) S to m a c h a d e n o ca rc in o m a + (6 / 9 ) A d re n al glan d - (0 /1 ) n .t.* si gne t-r ing c ell ca rci nom a - (0 / 3 ) B ra in - (0 /1 ) n .t . E so p h a g u s - (0 / 2 ) n .t . ch o lan g io ca rc in o m a + (1 / 1 ) L iv e r H e ar t - (0 /1 ) n .t . he pa t oc e ll ul a r c ar ci n om a - (0 / 2 ) L y m p h n o d e - (0 /2 ) n .t . B re a st scirrh o u s ca rc in o m a - (0 / 2 ) S k ele tal m u sc le - (0 /1 ) n .t . S p in a l c o rd - (0 / 1 ) n .t . - (o /i o ) C o lo n a d e n o ca rc in o m a S p le e n - (0 / 2 ) n .t. K id n ey Gr aw it z 's t um or - (0 / 1 ) T h y ro id gla n d - (0 / 1 ) n .t. G all b la d d er a d e n o ca rc in o m a - (o /- i ) *staining intensity: ++; strongly positive, +; positive, F+; faintly positive, -; negative :no. positive/no, tested **no. positive/no. tested ***not tested 128 JapJMedVol 25, No 2 (May 1986) Immunohistology of adenocarcinoma antigen tion for 10 to 20 min at room temperature. One Tokyo) or 0.1 U/ml endoglycosidase H (Seika- hundred microliters of 1 U/ml neuraminidase gaku-Kogyo, Tokyo) for one hour at 37°C as de- (Sigma, No. N-3001, USA) in 0.01M acetate buffer scribed by Zola et al. solution (pH 5.1) were placed on the section for 12 hours at 37°C. After three washes with PBS, RESULTS each section was stained by immunoperoxidase Staining pattern of cancerous tissues method as described above. MoAb YH206 stained the majority of lung adenocarcinomas and alveolar cell carcinomas, Enzymetreatment on the cultured cells One milliliter of the cultured A549 cells (2 x but did not stain or weakly stained the other 10 /ml) was incubated with a solution of either histplogic types of lung carcinoma. Adenocar- 0.1 U/ml of neuraminidase F (Seikagaku-Kogyo, cinomas of the pancreas and the stomach were Tokyo), 0.5 mg/ml of protease V8 (Miles, USA), also stained, whereas 10 cases of colonic adeno- 2 mg/ml of glycosidase mixed (Seikagaku-Kogyo, carcinomas were negative (Table 1). The predomi- Fig. 1. Staining pattern of cancerous tissues by peroxidase technique. secretionApical portionof gastricof lungadenocarcinomaadenocarcinoma (b)(a) wereand apicalstained. portionOriginal andmagnification:intra-luminal a,x90; b,x40. JapJMedVol 25, No 2 (May 1986) 129 Yachi et al nant staining pattern obtained in most lung adeno- apical portion and intraluminal secretion were carcinoma specimens wasa labelling of the apical essentially stained (Fig. lb). surfaces of tumor cells (Fig. la). Lung alveolar cell carcinomas (2 cases) showed cytoplasmic staining Staining pattern of noncancerous and fetal tissues In adult lung tissues, any portions including pattern. In gastric adenocarcinoma specimens. epithelial cells of the bronchus or the alveolus Fig. 2. Staining pattern of noncancerous tissues.