ANALYTICAL

BIOCHEMISTRY

192,

Chromogenic Susan Chemistry

Received

M. Conyers Division,

June

207-211

(1991)

Substrates and David Naval

for Horseradish

Peroxidase

A. Kidwelll

Research Laboratory,

Washington,

D.C. 20375-5000

27, 1990

Two new detection systems for horseradish peroxidase (HRP) have been developed for the staining of membranes used in immunoassays. These systems use dimethyl or diethyl analogues of p-phenylenediamine with 4-chloro-1-naphthol to generate a blue product or 3-methyl-2-benzothiazolinone hydrazone with 4chloro-1-naphthol to generate a red product. These reagents offer increased sensitivity and lower background staining than currently available chromogenic detection substrates. In addition, the incorporation of these substrates increases the sensitivity of HRP labels to be comparable to that of alkaline phosphatase with the 5-bromo-4-chloro-3-indolyl phosphate + nitro blue tetrazolium substrate. 0 1991 Academic POW, hc.

Horseradish peroxidase (HRP*, EC1.11.1.7) is a commonly used enzyme label for immunological detection systems. HRP decomposes two molecules of hydrogen peroxide, the natural substrate, into water and oxygen. However, the specificity of HRP for the second molecule of hydrogen peroxide is low and many other electron donors may be substituted. This low specificity has allowed the development of many chromogenic substrates for HRP: for example, 4-chloro-1-naphthol (4CN) (l), o-phenylenediamine (Z), 3-amino-g-ethyl carbazole (3), 2,2’-azino-bis(3-ethylbenzthiazolone) (4), Hanker-Yates reagent (HYR) (5), 3-methyl-2-benzothiazolinone hydrazone hydrochloride (MBTH) + 3dimethylaminobenzoic acid (6), 4-aminoantipyrene + a phenol (4,7), 3,3’-diaminobenzidine (DAB) (8) and its i To whom reprint requests should be sent. ’ Abbreviations used: 4-CN, 4-chloro-I-naphthol; DEPDA, N,N-diethylphenylenediamine; DMPDA, N,N-dimethylphenylenediamine; 2-ADET, 2-amino-4-N,N-diethylaminotoluene; NSA, 1-naphthol-4sulfonic acid; TMB, 3,3’,5,5’-tetramethylbenzidine; HYR, HankerYates reagent; HRP, horseradish peroxidase; MBTH, 3-methyl-2benzothiazolinone hydrazone hydrochloride; BCIP, 5-bromo4-chloro-3-indolyl phosphate; NBT, nitro blue tetrazolium; AP, alkaline phosphatase; DAB, 3,3’-diaminobenzidine; PBS, phosphate-buffered saline. 0003.2697/91 $3.00 Copyright 0 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.

derivatives, such as 3,3’,5,5’-tetramethylbenzidine (TMB), o-dianisidine (9), and dicarboxidine (10). Many of these reagents have been compared in stability and sensitivity for detection of HRP (3,4,11). Of the above systems, only 4-CN, HYR, DAB, and 3-amino-9-ethylcarbazole produce water-insoluble dyes and are thus suitable for membrane-based immunoassays. Unfortunately, the dyes produced with all these reagents are either prone to fading or show poor detection capability. This paper will describe two new chromogenic systems for HRP detection on membranes which use the principle of the Nadi reaction (12-15). The new substrates combine 4-CN with DEPDA, DMPDA, or MBTH. These substrates form water-insoluble indamine dyes with hydrogen peroxide in the presence of HRP which offer greater sensitivity and lower background staining than the other substrate systems currently available. In addition, these substrates produce two colors, red and blue, which may have use in some immunoassays that employ two enzymes. EXPERIMENTAL

4-CN, N,N-dimethyl-p-phenylenediamine monohydrochloride (DMPDA), HRP-conjugated and alkaline phosphatase-conjugated goat anti-mouse antibody, and HRP were obtained from Sigma Chemical Co. 1-Naphthol-4-sulfonic acid, sodium salt (NSA), 2amino-4-(N,N-diethylamino)toluene monohydrochloride (2-ADET), and N,N-diethylphenylenediamine monohydrochloride (DEPDA) were obtained from Kodak. MBTH was obtained from Aldrich. Commercially prepared 4-CN, BCIP-NBT, and TMB substrates were obtained from Kirkegaard and Perry. Mouse antibody was obtained from Roche. The indamine dye systems were optimized for pH, and concentration of the phenylenediamine, naphthol derivative and hydrogen peroxide. Besides 4-CN and NSA, various other naphthols and phenols were tested with the three p-phenylenediamine derivatives: DEPDA, DMPDA, and 2-ADET. 207

208

CONYERS

AND

For comparison of the substrates with HRP, various concentrations of HRP were spotted on nitrocellulose and immersed in a buffered solution of the chromogenic substrate reagents for lo-30 min. The nitrocellulose strips were then removed from the development solution, washed with distilled water, and allowed to air-dry. For comparison of AP with the BCIP-NBT substrate system to HRP with the indamine substrate system, mouse antibody in serial dilutions was spotted on nitrocellulose and blocked with 5% bovine serum albumin in phosphate buffer saline (PBS) for 1 h. The nitrocellulose was incubated overnight with either HRP- or APconjugated anti-mouse antibody (1:lOOO in PBS), thoroughly washed with PBS (HRP) or diethanolamine buffer (AP), and the enzyme substrate was added. The absorbance of the spots was read on a Shimadzu dual wavelength TLC scanner, Model CS-930, in the reflectance mode at the wavelengths indicated in Fig. 2.

KIDWELL TABLE

1

Optimized Substrates Analogues DMPDA

(1.1 mM)

Detection limits

Comments

5 Pg

very

+ 4-CN

5 Pg

no

background

MBTH (0.022 mM) + 4-CN (1.1 mM) 2-ADET (2.2 mM) + 4-CN

5 Pg

no

background light (red) color

DEPDA

(1.1

mM)

(2.2mM)

(2.2mM) DMPDA

(1.1

20Pg

moderate

moderate background some smearing moderate background some smearing high background

mM)

+ NSA

10 Pg

mM)

+ NSA

10 Pg

+ NSA

40 Pg

(22.2mM) DEPDA

(0.22

(22.2mM) 2-ADET

(2.2

(22.2mM)

mM)

low

background

+ 4-CN

(2.2mM)

background

Substrate Preparation

Note. All substrate systems contained 2.9 mM hydrogen peroxide. The MBTH + 4-CN system was buffered in 100 mM sodium citrate, pH 4; all other systems were buffered in 100 mM sodium citrate, pH 6.

To prepare the substrate systems, dissolve 0.06 M DEPDA or DMPDA (blue products) or 0.01 M MBTH (red product) and 0.11 M 4-CN in acetonitrile. A small amount of water will increase the solubility of the amine salts. The substrate solution can be prepared in advanced and stored in the refrigerator for several days. Slightly colored solutions do not appear to affect the background or the sensitivity of the assay. A 0.1 M sodium citrate buffer, pH 6 (pH 4 for MBTH with 4-CN), should be made 2.9 mM in hydrogen peroxide (1 ~1 of 30% H,O, in 10 ml of buffer). Immediately before developing the blot, the acetonitrile solution of dye precursors should be mixed with the buffer-hydrogen peroxide solution, 1:50. The solution should be homogeneous.

lists several variations on the Nadi reaction with optimum concentrations of the substrates useful for membrane staining. It is not known if these modified conditions could be used for histochemical work and thereby reduce the reported background in this application. While DMPDA + 4-CN was the most sensitive of the systems tested, DEPDA + 4-CN was of similar sensitivity and produced lessbackground, even after incubation periods of 2 h. Hence, the ideal detection system will vary according to individual assay requirements with longer, unobserved incubations using DEPDA + 4-CN and shorter (~30 min) incubations using DMPDA

+ 4-CN. RESULTS

Experimental

AND

DISCUSSION

Results

The older histochemical literature discusses detection of several oxidizing enzymes using a staining system termed indophenol blue (12). This system, comprising a 1-naphthol derivative and an appropriate analogue of p-phenylenediamine, was historically used for the detection of cytochrome oxidase in tissue sections. As early as 1885, the production of indophenol blue (also called the Nadi reaction) has been studied and modified for optimum sensitivity in cell staining. Because the Nadi reaction produces a high background color and a counter stain was necessary to increase sensitivity (12-15), it has not found much favor. The procedures described in this literature were unsuitable for the staining of membranes used in immunoassays since very poor sensitivity with high background levels are observed. However, these conditions may be modified to produce a superior membrane stain. Table 1

The MBTH system is less likely to produce a background and the reagents are more stable on long term storage. However, it produces a red precipitate which is more difficult to detect visually at low concentrations compared to the blue precipitates produced with the diamines. Also, like the other diamines, the sensitivity of MBTH + 4-CN decreases as the reagent concentration is varied but the concentrations ranges appear to be more critical. Increasing the MBTH or 4-CN causes higher background and decreasing either component leads to poorer detection limits. Therefore, the diamine systems are recommended over the MBTH system unless a contrasting color is desired (such a staining a single membrane with two enzyme systems; AP with BCIP-NBT for a blue color and HRP with MBTH-4CN for a red color) or background development becomes a problem. Other diamine and naphthol components also were tested as listed in Table 1.2-ADET demonstrated comparably poor sensitivity and, even at low concentra-

CHROMOGENIC

SUBSTRATES

FOR

HORSERADISH

Alternative

4-Chloro-1-naphthol + DEPDA

$2

I)*

i

4-Chloro-1-naphthol

Hanker-Yates Reagent

Commercial CChloro-l-naphthol

Tetramethylbcnzidine

,,

“-,

FIG. 1. Comparison of HRP substrates. HRP was spotted creasing concentrations onto nitrocellulose and placed in the cated solutions until the color was fully developed. The strips photographed after 30 min. Fading of the tetramethylbenzidine was evident even before the photograph could be taken.

tions, caused high background discoloration. NSA, as the naphthol component, has an advantage over 4-CN in that it is more water soluble and less likely to form precipitates in storage. However, although satisfactory, the sulfonic acid in NSA imparted some water solubility to the dye formed. This caused poorer localization and color smearing. Porstmann et al. report that the presence of nonionic surfactants stabilize the HRP and therefore increase detection limits (11). Although we investigated the benefits of the detergents, their presence at the critical micelle concentration did not make any contribution to the sensitivity of the assay nor did they appear detrimental in solubilizing the dyes formed.

Detection

and Stability

Enzyme

Systems

The use of alkaline phosphatase (AP, EC3.1.3.1) labels with the substrate system BCIP-NBT (5bromo-4chloro-3-indolyl phosphate + nitro blue tetrazolium) offers high sensitivity for solid phase enzyme detection (16). Therefore, AP has been used more frequently than HRP as a labeling enzyme. Figure 2 is a graph of the results of the comparison of AP using BCIP-NBT and HRP using 4-CN vs HRP using the chromogenic substrate described in this paper. As can be seen the system DEPDA + 4-CN is clearly superior to 4-CN and only slightly poorer than AP with BCIP-NBT. However, a strict comparison of AP and HRP enzyme-antibody conjugates may not be valid since a different number of enzyme molecules may be conjugated per antibody. The Chemistry

at deindiwere spots

209

PEROXIDASE

Behind

the Detection

Systems

A thorough understanding of the chemistry of these detection systems is not necessary to employ them as described above. However, the following discussion is provided for those who wish to understand the rational behind the development of these detection systems and possibly modify them for other colors or greater sensitivity. HRP is oxidized and activated in the presence of hydrogen peroxide. Activated HRP will oxidize electron donors such as substituted phenylenediamines and MBTH to cationic electrophiles (see Fig. 3). These electrophiles will react with many electron-rich, aromatic compounds to form colored dyes. The rate of production of the electrophilic form of the electron donor is dependent only on the electron donor and activated HRP and is independent of the second aromatic component of the

Absorbance

Comparison

The optimized HRP assays were compared for their color intensity and dye stability on nitrocellulose to some of the commercially marketed and published methods of HRP detection. All commercial reagents were prepared by manufacturers instructions and photographs were taken after 30 min to emphasis stability and sensitivity. Representative results from these systems are shown in Fig. 1. As is evident from Fig. 1, the indamine substrates proposed in this paper are superior to many of the alternative HRP detection systems. Although not shown in Fig. 1, DEPDA + 4-CN offers sensitivity equal to that of the other indamine substrates.

1000

500

250

Primary

125

63

AntIbody

32

Cone

16

a

(ng)

4-w at 580 “ml DEPD/\+4-ON a, 520 “IT BCIP*NsT 8, 550 nm FIG. 2. Comparison of enzyme systems and substrates. Detection of spotted mouse antibody was performed with a second enzyme labeled anti-mouse antibody. The antibody enzyme ratio of the second antibody may vary in the two HRP and AP conjugates which could affect the reported comparison.

210

CONYERS

AND

R= Methy 1 Ethyl

A pheny lenedicmine tbrwmiish Peroxidose HydrogenPeroxi&

MBTH

Cl

D

VN\R Blue Dye 0

MBTH Electrophile

Red Dye

FIG. 3. 4-CN.

The

dyes

formed

from

the

reaction

of electrophiles

with

dye. Thus the color formed and its degree of localization can be varied widely by modification of the second aromatic component. Likewise, the enzyme turnover number, i.e., how fast the HRP produces a molecule of dye, is dependent only on the electron donor and its concentration. Some of the fastest turnover numbers known for HRP are for the diamines and MBTH (17). Since a highly electrophilic species is generated, it may react with the HRP protein and deactivate the enzyme. Therefore, a fine balance must be reached to produce an electrophilic enough species for a fast second coupling, provide a high turnover number, and not deactivate the HRP. 4-CN could react with MBTH and the phenylenediamine in one of two positions: the S-position and with replacement of the chlorine in the 4-position (18). With DEPDA, one dye is produced, as indicated by thin-layer and gas chromatography. That dye is identical with indamine blue produced chemically (20) from DEPDA and 1-naphthol as determined by gas chromatography/ mass spectrometry analysis. NSA produced two dye species, presumably by substitution in both the 2- and 4-positions. A study of the reaction of MBTH with numerous aromatic amines (19) and phenols (20) has been made in conjunction with spots tests to detect these materials. Although the electrophilic form of MBTH was generated chemically, the chemistry of the reaction and the dyes produced is identical regardless of the source of the electrophilic MBTH. Therefore, this literature was relied upon to select likely candidate phenols for testing to determine the localization of the dye produced in an immunoassay. Phenylenediamines have been well-studied in conjunction with color photography. Briefly, in color photography light activates silver halide crystals and these

KIDWELL

are then reduced to silver metal by a developer, which is removed. The remaining silver halides may oxidize a phenylenediamine to an electrophilic form, which then couples to an immobilized aromatic compound producing a colored dye. The most widely used phenylenediamine, is diethylphenylenediamine which produces shades of blue and red with most aromatic compounds (21-23). Thus the color of the stain may be varied by modification of the coupling components. The aromatic coupling component was chosen to react with the electrophile at the pH where the diamine is stable and the enzyme is active. For a phenol or naphthol to be reactive with an electrophile, it must be ionized. The pK, of unsubstituted phenol and naphthol is near 10. At this pH, the diamine is quickly oxidized by air producing a strong background. Electron-withdrawing groups will lower the pK, of the phenol or naphthol and allow the reaction to proceed at a more neutral pH. However, electron-withdrawing groups will also deactivate the phenol or naphthol. A monochloro substituent was a compromise on electron withdrawing and deactivation. 4-CN was chosen over substituted phenols because of their stench, although all gave intense, stable dyes with good localization properties and similar colors. CONCLUSIONS

The detection of HRP can be improved with a dye system that combines derivatives of p-phenylenediamine or MBTH with 4-chloro-1-naphthol and H,O,. This system offers increased sensitivity and much lower background intensity than the currently available chromogenic substrates. In addition, using these substrates to detect HRP-labeled antibodies has been demonstrated to be comparable to the detection limits of APlabeled antibodies with the BCIP-NBT substrate. REFERENCES 1. Tijssen, P. (1985) Practice and Theory of Enzyme Immunoassays, pp. 474-477, Elsevier Science, Amsterdam. 2. &hall, R. F., Jr., Fraser, A. S., Hansen, H. W., Kern, C. W., and Tenoso, H. J. (1978) Clin. Chem. 24,1801-1804. 3. Hosoda, H., Takasaki, W., Oe, T., Tsukamoto, R., and Nambara, T. (1986) Chem. Pharm. BuU. 34,4177-4182.

4. Porstmann, 5. 6.

B., Porstmann, T., and Nugul, E. (1981) J. Clin. Chem. Clin. Biochem. 19,435-439. Hanker, J. S., Yates, P. E., Metz, C. B., and Rustioni, A. (1977) J. Histochem. 9,789-792. Ngo, T. T., and Lenhoff, H. M. (1980) And. Bzbchem. 105,389-

397. 7. Gallati, V. H. (1977) J. Clin. Chem. Clin. Biochem. 15, 699-703. 8. Graham, R. C., Jr., and Karnovsky, M. J. (1966) J. H&o&em. Cytochem. 14,291. 9. Gallati, H., and Brodbech, H. J. (1981) J. Clin. Chem. C&z. Bio&em.

20,221-225.

CHROMOGENIC 10. Paul,

K. G., Ohlsson,

SUBSTRATES

P. I., and Jonsson,

N. A. (1982)

Anal.

FOR Bio-

them. 124, 102-107. 11. Porstmann, B., Porstmann, T., Gaede, D., Nugel, E., and Egger, E. (1981) Clin. Chim. Actu 109, 175-181. 12. Guthrie, J. D. (1931) J. Amer. Chem. SOC. 53, 242-244. 13. Gabe, Paris.

M. (1976)

Histological

Techniques,

pp. 643-650,

Masson,

HORSERADISH 17. Chance,

211

PEROXIDASE

B. (1951)

12,153-191.

Adu. Enzymol.

18. Fieser, L. F., and Williamson, p. 326. D.C. Heath, Lexington,

K. L. (1979) MA.

Organic

19. Sawicki, E., Stanely, T. W., Hauser, T. R., Elbert, J. L. (1961) Anal. Chem. 33, 722-725. 20. Friestad,

H. O., Ott,

D. E., and Gunther,

Experiments, W., and Noe,

F. A. (1969)

Anal.

Chem.

41,1750-1754.

14. Nachlas, M. M., Crawford, A. M. (1958) J. Histochem.

D. T., Goldstein, T. P., and Seligman, Cytochem. 6,445-456.

21. Evan, R. M., Hanson, W. T., Jr., and Brewer, W. L. (1953) Principles of Color Photography, pp. 251-311, Wiley, New York.

15. Kodousek, Fat. Med.

P. (1968)

22. Friedman, J. S. (1944) History of Color 404, American Photographic Publishing,

16. Leary, Acad.

R., and Vacha, 52, 139-145.

J. J., Brigati, D. J., and Sci. USA 80,4045-4049.

Ward,

Acta

Uniu.

Pulack.

D. C, (1983)

Olomuc.

Proc.

N&l.

23. Kosar, J. (1965) New York.

Light

Sensitive

Systems,

Photography, Boston, MA. pp. 358-404,

pp. 345Wiley,