The Use of Antiavidin Antibody and Avidin -Biotin - Peroxidase Complex in Immunoperoxidase Technics

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1 The Use of Antiavidin Antibody and Avidin -Biotin - Peroxidase Complex in Immunoperoxidase Technics SU-MING HSU, M.D., LAURENCE RAINE, M.S., AND HERBERT FANGER, M.D. Hsu, Su-Ming, Raine, Laurence, and Fanger, Herbert: The use of antiavidin antibody and avidin-biotin-peroxidase complex in immunoperoxidase technics. Am J Clin Pathol 75: , Avidin has an extraordinary affinity for the smallmolecule vitamin biotin. Covalently coupling biotin or avidin to peroxidase molecules does not interfere with their normal biochemical functions. The avidin or biotin molecules, either peroxidase conjugated or unconjugated, can be brought to the antigen sites by means of an antiavidin antibody. Several immunohistochemical staining technics based on this principal have been described. The method utilizing an avidin-biotinperoxidase complex was found to be more sensitive than the unlabeled antibody (PAP) method. This method involved four sequential staining procedures: (1) primary antibody (goat antihuman antigen); (2) secondary antibody (rabbit antigoat IgG) added in relative excess; (3) goat antiavidin antibody; (4) avidin-biotin-peroxidase complex. The applications of this technic are discussed. (Key words: Immunoenzymatic technics; Avidin; Biotin.) THE IMMUNOPEROXIDASE TECHNICS have emerged as a valuable tool in both routine histopathology and research. 1 With the material obtained under controlled conditions, the specificity and sensitivity of immunoperoxidase staining have been reported as comparable or superior to those of immunofluorescence. 1 " 5 Several immunoperoxidase staining procedures have been described. Among them, the unlabeled antibody (PAP) method has been widely accepted as the most sensitive method. 717 However, the results obtained from routinely fixed specimens have often been unpredictable and negative. H ' 19 The decreased sensitivity of immunoperoxidase technics compared with immunofluorescence was attributable to antigen loss through tissue fixation or processing Efforts to enhance the sensitivity of the immunoperoxidase technics without compromising specificity has now become an important issue. Curran and Gregory 5,6 have recommended initial trypsin digestion to unmask tissue antigens. Trypsinization, in our experience, enhanced the sensitivity to some degree but without complete satisfaction. 14 Other methods, including osmication, 4 detergent treatment, 9 and freeze and thaw, 15 were used less frequently with less satisfactory results in formalin-fixed tissues. 14 Received July 31, 198; received revised manuscript and accepted for publication October 6, 198. Address reprint requests to Dr. Hsu: Department of Pathology, Rhode Island Hospital, Providence, Rhode Island 292. Department of Pathology, Rhode Island Hospital and Brown University, Providence, Rhode Island We have developed "self sandwich" and "ABC" (indirect bridged avidin biotin) immunostaining procedures that are more sensitive than the PAP method This report presents an alternative approach using antiavidin antibody and/or an avidin-biotin interaction in immunoenzymatic technics. Avidin is an egg white glycoprotein (molecular weight, 68,) that has a high affinity for the small-molecule vitamin biotin. Covalently coupling biotin to peroxidase molecules gives peroxidase the ability to bind avidin. Accordingly, the biotin-labeled peroxidase molecules can be brought to the antigen sites by means of an antiavidin antibody and avidin. Several staining procedures are described, and the staining intensities of each method are compared with that of the PAP method. The staining method using the avidin-biotin-peroxidase complex (ABC) is found to be highly sensitive and easy to perform. The applicability of this method is discussed. Reagents Materials and Methods The following reagents were obtained commercially: rabbit antihuman IgG, swine antirabbit IgG antiserum, and rabbit peroxidase-antiperoxidase complex (PAP)*; avidin-conjugated peroxidase (percent active conjugate, 54%) and avidint; goat antihuman IgG, rabbit antigoat IgG, and goat PAP and goat antiavidin antibodies^; horseradish peroxidase (type VI).8 Tissues Formalin-fixed paraffin-embedded thyroids (five) from patients who had chronic thyroiditis or Hashimoto's thyroiditis, and tonsils (five) from patients who * Dakopatts, Accurate Chemical and Scientific Corp., Hicksville, New York. t Vector Laboratories, Burlingame, California. t Cappel Laboratories, Inc., Cochranville, Pennsylvania. Sigma Chemical Co., St. Louis, Missouri /81/6/816 $.8 American Society of Clinical Pathologists 816 on 15 May 218

2 Vol. 75 No. 6 Step 1 Step 2 Step 3 Step 4 Step 5 Step 6 PAP* PAP complex 1:1 ANTIAVIDIN METHOD Table I. Staining Procedures* of the PAP and Antiavidin Methodst Antiavidin Methods Method I Method II Method III Method IV Primary antibody (goat antihuman IgG) at various dilutio Rabbit antigoat IgG, 1:5 Goat antiavidin 1:2 AA-AV-PO complex Goat antiavidin 1:2 AA-AV complex AV-PO AV 5 /xg/ml Bio-PO 5 /xg/ml * Each stage lasted 3 min and was folowed by a 2-min washing in Tris buffer. t Abbreviations: PAP, peroxidase-antiperoxidase; AA-AV-PO, antiavidin antibodyavidin-conjugated peroxidase complex; AA-AV, antiavidin antibody-avidin complex: AV, avidin; AV-PO, avidin-conjugated peroxidase; Bio-PO, biotin-labeled peroxidase; Bio-PO 5 /xg/ml Method V Goat antiavidin 1:2 AV-Bio-PO complex 817 Method VI AA-AV-Bio-PO complex AV-Bio-PO. avidin-biotin-labeled peroxidase complex; AA-AV-Bio-PO. antiavidin antibody-avitin-biotin-labeled peroxidase complex. t Rabbit PAP system was also tested in the PAP method. The preparation and the concentration of each complex were described in the text. had chronic tonsilitis were obtained for immunostaining. Serial sections (5 fim) were cut from paraffin blocks, deparaffinized, and processed to alcohol as in routine processing. Biotin-labeling of Horseradish Peroxidase Biotinyl-N-hydroxysuccinimide (BNHS) was used for introducing biotin moieties into peroxidase molecules. It was prepared according to the procedure described by Bayer and associates. 2 Dicyclohexylcarbodiinimide,.8 g, is added to 12 ml dimethylformamide solution containing 1 g biotin and.6 g N-hydroxysuccinimide. The suspension is stirred overnight at room temperature. The precipitate is filtered and then the filtrate is evaporated under reduced pressure. The residue is washed well with ether, and the product (BNHS) is recrystallized with isopropanol. BNHS was kept dessicated under vacuum until used. The biotinlabeled peroxidase was prepared by mixing 1 mg horseradish peroxidase in 1 ml of.1 M NaHC 3 with.2 ml of.1 M BNHS in distilled dimethylformamide. The reaction was incubated at room temperature for one hour and then dialyzed against several changes of phosphate-buffered saline solution (PBS). Immunostaining Procedures The unlabeled antibody (PAP) method using PAP complex was carried out as described previously. 7 " 16 To block the endogenous peroxidase activity and to reduce the nonspecific background staining, the slides were treated with methanolic hydrogen peroxide and 3% normal swine serum, respectively. The staining methods using antiavidin antibody were performed as shown in Table 1. To achieve a high staining intensity and minimal background staining, different concentrations of avidin (AV), avidin-conjugated peroxidase (AV-PO), and biotin-labeled peroxidase (Bio-PO) were tested. It was found that 5 ju,g/ml of each was optimal (methods I, III, and IV, Table 1). An alternative approach using a preformed complex (Table 1) was also tested. The concentrations of each complex, which were also determined after a series of experiments to obtain maximal staining intensity and minimal background staining, were prepared by incubating each component in Tris buffer, ph 7.6, for 3 min at room temperature before use. (1) Antiavidin antibody-av-po complex (AA-AV-PO): antiavidin antibody, 1 to 2 dilution, mixed with 1 /u,g/ml of AV- PO. (2) Antiavidin antibody-av complex (AA-AV): antiavidin antibody, 1 to 2 dilution, mixed with 1 or 2 /xg/ml of AV. (3) AV-Bio-PO complex: prepared by incubating different concentrations of AV and Bio- PO. Variations in ratio of AV:Bio-PO affected the staining intensity dramatically. The effects of concentrations and ratios of this complex on the staining intensity are shown in Table 2. (4) AA-AV-Bio-PO complex: since the 1:4 AV-Bio-PO complex containing 2.5 Table 2. Effect of Avidin-Biotin-peroxidase Complex (AV-Bio-PO) in Various Ratios and Concentrations on the Staining Intensity* for IgG in Thyroids Avidin (Mg/ml) l Concentrations of Biotin-peroxidase (jxg/ml) * The staining intensity was graded on a semiquantitative scale of (none) to - (intense). on 15 May 218

3 818 HSU, RA1NE, AND FANGER A.J.C.P.. June 1981 Control Slides Table 3. Controls of Avidin-Biotin Interaction in Immunoperoxidase Technics* Normal Serum or Buffer Secondary Antibody AA AV-PO AA-AV-PO Immunoreagentst Staining Procedures AA-AV- AV AA-AV Bio-PO AV-Bio-PO Bio-PO * Abbreviations: AA, antiavidin antibody: AV-PO, avidin-conjugated peroxidase; biotin-labeled peroxidase complex; AA-AV-Bio-PO. antiavidin antibody-avidin-biotinlabeled peroxidase complex. AA-AV-PO. antiavidin antibody-avidin-conjugated peroxidase complex; AA-AV, antiavidin antibody-avidin complex: Bio-PO, biotin-labeled peroxidase; AV-Bio-PO, avidin- t The preparation of each complex and the concentrations used are described in the text. (jig/ml of avidin and 1 /ug/ml of Bio-PO was optimal to give a strong staining (Table 2), the AA-AV-Bio-PO complex was therefore prepared by incubating the 1:4 AV-Bio-PO complex with antiavidin antibody, 1:2. Controls Controls for the specificities for the PAP method were performed as described by Sternberger. 7 Additional controls for the biotin-avidin interaction in tissue sections were carried out as shown in Table 3. Results The PAP method was satisfactory to demonstrate IgG-containing plasma cells when sufficient concentration of primary antibody (1:5 and 1:1,) were used. Further dilution of the primary antibody presented a very weak or frequently negative staining. In many cases, a small dotlike cytoplasmic staining was noted when highly diluted antibody (> 1:2,) was used. There was a slight variation in staining intensities among specimens. This may reflect the fact that the specimens had been fixed in formalin for different durations (six to 18 hours). The results obtained from the antiavidin methods (except for method V) revealed a staining intensity comparable or inferior to that of the PAP method. An unsatisfactory result was obtained from method VI, in which an AA-AV-Bio-PO complex was used. The use of an AV-Bio-PO complex (method V), however, dramatically increased the staining intensity (Figs. 1 and 2). It was noted that the staining intensity increased as the proportion of Bio-PO in complex increased, and reached its plateau when a ratio of 1:4 of avidin:bio- PO was achieved (Table 2). Increases in the proportion of avidin diminished the staining intensity. In general, the use of a 1:4 AV-Bio-PO complex allowed the detection of IgG plasma cells even when the primary antibody was diluted as much as 1:8, (Table 4). The concentration of the AV-Bio-PO complex consisting of 2.5 /xg/ml of avidin and 1 /u.g/ml of Bio-PO was found to be optimal without increased background staining. In all methods tested, the distributions of IgG plasma cells were found to be similar, with no visible difference in background or collagen tissue staining. Control studies indicated no nonspecific binding derived from avidin, avidin-po, biotin-po, or complexes used with appropriate dilutions. Increases in concentrations of AV-PO to 2 /Ag/ml, and AV-Bio-PO complex to 4:16 /^.g/ml, resulted in nonspecific background staining. However, the AV-Bio-PO complex with a concentration of 2.5 /u,g/ml AV and 1 /xg/ml Bio-PO was sufficient for intense staining, and the problem of nonspecific background staining was practically eliminated. Discussion Possible explanations for the high sensitivity of antiavidin antibody methods are listed as follows. (1) Antiavidin antibody, avidin, AV-PO, and Bio-PO are apparently smaller than the PAP complex. The tissue penetration or diffusion of these reagents should be on 15 May 218

4 Vol. 75 No. 6 ANTIAVIDIN METHOD easier than that of the PAP complex in the PAP method. 8 (2) The binding of antiavidin antibody to the sites of antigen is through the action of the bridge antibody. The principle of this technic is similar to that of the PAP method of Sternberger. One bridge antibody can bind one PAP complex, which contains three peroxidase molecules. 16 However, one bridge antibody can also bind one antiavidin antibody, which maximally can bind two avidin molecules. (3) Avidin provides four active binding sites for biotin. Thus, two avidin molecules can possibly bind eight Bio-PO molecules. This would therefore increase the staining intensity. 819 However, the antiavidin methods, except that using the AV-Bio-PO complex (method V), generally did not show superior results when compared with the PAP technic. In trials of methods I and II in which an AVPO or AA-AV-PO complex was used, the unsatisfactory result can be attributed to the difficulty in conjugation of avidin with peroxidase. The unconjugated avidin or inactive conjugate may compete with the active AV-PO conjugates for the binding sites of antiavidin antibody and lead to decreased sensitivity. In trials of methods III and IV that involved the use of an AA-AV complex or a sequential addition of avi- FIG. 1. (A) Thyroid, nonspecific chronic thyroiditis, immunostained for IgG plasma cells. Unlabeled (PAP) method. The dilution of primary antibody used was 1:2,. Note the very weak staining of the plasma cells. Counterstain, hematoxylin, x 15. (B) Higher magnification of 1A. x6. FIG. 2. (A) An adjacent section, immunostained for IgG plasma cells by the antiavidin antibody method in which an avidin-biotinperoxidase complex was used. Same dilution of primary antibody (1:2,) was applied. The plasma cells stained strongly positive. Counterstain, hematoxylin. xl5. (B) Higher magnification of 2A. x6. on 15 May 218

5 82 HSU, RAINE, AND FANGER A.J.C.P. June 1981 Methodt PAP I II III IV V VI Table 4. Summary of the Staining Intensities* of Thyroids Immunostained for IgG by the PAP and Antiavidin Methods 5 / / / / 1, / / / * The intensity of staining was graded on a semiquantiative scale of (none) to - (intense). Dilutions of Primary Antibody (1 x) 2, / / / 4, / 8, / 16, / t For staining procedures of each method, see Table 1. PAP, unlabeled antibody. din and Bio-PO, the antibody-antigen (avidin) interaction may have sterically interfered with the subsequent reaction between avidin and Bio-PO. The formation of a huge complex (AA-AV-Bio-PO) in method VI is probably not desirable, because the tissue penetration would be extremely difficult. The use of AV-Bio-PO complex (method V) not only shortened the reaction time but also greatly enhanced the staining intensity (Figs. 1 and 2). It has been shown that by adjusting the BNHS:amino group ratio, certain numbers of the amino group in the peroxidase molecules can be substituted. 8 It is apparent that multiple biotin molecules can be present on the peroxidase molecules. This would increase the ability of the peroxidase molecule to bind several molecules of avidin. Thus, during the formation of the complex, not only can avidin act as a bridge between Bio-PO molecules, but also the Bio-PO molecules can serve as a link between avidin molecules. Consequently, a latticelike complex containing several peroxidase molecules (more than three) is likely to be formed. The binding of this complex to the antiavidin-antibody therefore enhances staining intensity. Obviously, the best complex to use for this purpose would contain no free avidin molecules and would have enough Bio-PO to saturate all of the biotin binding sites in the complex. We have reported an ABC (indirect bridged avidin biotin) staining technic in which a 4:1 avidin:bio-po ratio was recommended. 12 The excess avidin in that 4:1 complex supplies the free biotin binding sites, which can therefore bind to the antibody previously conjugated with biotin. Since biotin is an important coenzyme for transcarbamylation, it may be present in sufficient quantities to bind the 4:1 complex in some tissues. However, in the antiavidin antibody method utilizing the 1:4 complex, there are probably no free biotin binding sites in the complex; thus, nonspecific or unwanted binding through intrinsic biotin may be eliminated. The antiavidin antibody method would be preferred to the indirect bridged avidin biotin method when the problem of unwanted binding through intrinsic biotin is concerned. In conclusion, using antiavidin antibody and an AV-Bio-PO complex provides an alternative approach to the localization of antigens in paraffin sections. In case of very weak or equivocal staining results obtained by the PAP method, this antiavidin antibody method should be done to clarify the results. We plan to use this technic also to localize two antigens within the same tissue section; since biotin can be conjugated to other types of enzymes," such as alkaline phosphatase and glucose oxidase, the reaction products could be red, blue, violet, or black, depending on which enzymes or substrates are used. Further characterization of the AV-Bio-PO complex is necessary to understand the mechanism of reactions in the antiavidin antibody method and to produce increased sensitivity. Because the AV-Bio-PO complex is probably extraordinarily large, it will penetrate only with great difficulty. It is hoped that pretreatment of tissue sections with trypsin and the use of an avidin-biotin-microperoxidase complex would further increase the staining intensity. References 1. Avrameas S: Enzyme markers: their linkage with proteins and use in immunohistochemistry: a review. Histochem J 4: , Bayer E, Skutelsky E, Wilchek M: The avidin-biotin complex in affinity cytochemistry. Methods Enzymol 62:38-315, Burns J, Hambridge M, Taylor CR: Intracellular immunoglobulins. A comparative study on three standard tissue processing methods using horseradish peroxidase and fluorochrome conjugates. J Clin Pathol 27: , Busachi CA, Ray MB, Desmet VJ: An immunoperoxidase technique for demonstrating membrane localized HBsAg in paraffin sections of liver biopsies. J Immunol Methods 19: 95-99, Curran RC, Gregory J: The unmasking of antigens in paraffin sections of tissue by trypsin. Experientia 33:14-141, Curran RC, Gregory J: Demonstration of immunoglobulin in on 15 May 218

6 Vol. 75. No. 6 ANTIAVIDIN METHOD 821 cryostat and paraffin sections of human tonsils by immunofluorescence and immunoperoxidase techniques. J Clin Pathol 31: , DeLellis RA, Sternberger LA, Mann RB, et al: Immunoperoxidase technics in diagnostic pathology. Am J Clin Pathol 71: , Guesdon JL, Ternynck T, Avrameas S: The use of avidin biotin interaction in immunoenzymatic techniques. J Histochem Cytochem 27: , Hall JG, Binbeck MSC, Robertson D, et al: The use of detergents and immunoperoxidase reagents for the ultrastructural demonstration of internal immunoglobulin in lymph cells. J Immunol Methods 19: , Hsu SM: Clinical applications of immunoenzymatic techniques. R I Med J 62: , Hsu SM, Hsu PL, Nayak R: Warthin's tumor: an immunoperoxidase study of its lymphoid stroma. Hum Pathol 12: , Hsu SM, Raine L, Fanger H: Use of avidin-biotin-peroxidase complex (ABC) in immunoperoxidase techniques: A comparison between ABC and unlabeled antibody (PAP) procedures. J Histochem Cytochem 29:577-58, Hsu SM, Raine L, Martin H: Spironolactone bodies. An immunoperoxidase study with biochemical correlation. Am J Clin Pathol 75:92-95, Hsu SM, Ree HJ: Self-sandwich methods. An improved immunoperoxidase technic for the detection of small amounts of antigens. Am J Clin Pathol 74:32-4, Nakane PK: Recent progress in the peroxidase-labeled antibody method. Ann NY Acad Sci 254:23-211, Sternberger LA: Immunocytochemistry. Englewood Cliffs, Prentice-Hall, Taylor CR: Immunoperoxidase techniques. Arch Pathol Lab Med 12: , Warnke R: Alteration of immunoglobulin-bearing lymphoma cells byfixation.j Histochem Cytochem 27: , Warnke R, Pederson M, Williams C, et al: A study of lymphoproliferative diseases comparing immunofluorescence with immunohistochemistry. Am J Clin Pathol 7: , 1978 on 15 May 218