highly efficient and simple methods for the preparation of peroxidase and active peroxidase-antibody...

iNALYTICALBIoCHEMlSTRY 136,451-457(1984)

Highly Efficient and Simple Methods for the Preparation of Peroxidase and Active Peroxidase-Antibody Conjugates for Enzyme tmmunoassays

P. TIJSSEN AND E. KLJRSTAK’

Comparative Virology Research Group, Faculty of Medicine, Universite’ de Montrkal, Montreal, Quebec H3C 357, Canada

Received July 25, 1983

The periodate-mediated conjugation of horseradish peroxidase to antibody is one of the most popular methods to prepare conjugates for enzyme immunoassays of antigens or corresponding antibodies. A very simple method to obtain peroxidase, which is both about five times cheaper than the rather expensive commercial preparations and has a significant higher activity, is reported. Moreover, the conjugation method was critically investigated and considerably simplified. Con- jugates thus obtained are about three times more active than the best obtained with the original method.

KEY WORDS: peroxidase purification; NaIO,-mediated glycoprotein oxidation; peroxidase conjugation; IgG-peroxidase conjugate purification, enzyme immunoassays.

Enzyme immunoassays represent in many cases the preferred procedure for the detection of antigens or the corresponding antibodies. Among the enzymes used for EIA’ and in immunohistochemistry horseradish peroxidase (HRPO) is probably the most popular.

Here, we wish to communicate observations that the rather expensive commercial HRPO preparations contain both relatively large amounts of impurities and isozymes with quite low activities. A very simple method for HRPO purification is presented. This method is also suitable to directly purify HRPO from commercially supplied crude extracts which would lower the cost of enzyme preparation by about five times. Moreover, the conjugation by the widely used periodate method of Nak- ane and Kawaoi (1) has been simplified and optimized, and the critical steps have been further investigated. The modifications intro- duced were tested by the ELBA procedures

’ To whom reprint requests should bc addressed. ’ Abbreviations used: EIA, enzyme immunoassay;

HRPO, horseradish peroxidase, ELBA, enzyme-linked immunosorbent assay; IgG, immunoglobulin G, Con A, concanavalin A, PBS, phosphate-buffered saline.

of densonucleosis virus as reported by Tijssen et al. (2).

The original conjugation method of Nakane and Kawaoi (1) has several drawbacks though it is more efficient for HRPO than the glutaraldehyde methods of Avrameas (3) and of Avrameas and Ternynck (4). Nakane and Ka- waoi’s method (1) is quite elaborate, results in large polymers, and inactivates the enzyme to a large degree. For example, Nygren (5) noted in his investigation that their procedure inactivated 46% of the enzyme and yielded large complexes, but was five times as sensitive in spot-ELISA tests than conjugates prepared with glutaraldehyde.

The central problem in the periodate method is the sensitivity of the carbohydrate moiety of the enzyme for oxidation. In our experience this varied for HRPO from lot to lot but current methods tend to severely over- oxidize the HRPO.

Too little oxidation prevents an efficient conjugation, whereas a strong oxidation results in (i) the formation of carboxyl groups instead of aldehyde groups since the aldehyde groups are more sensitive to oxidation than the vicinal

451 0003-2697184 $3.00 Copyright 0 1984 by Academic Press, Inc. All rigbrs of reproduction in any form reserved.

452 TIJSSEN AND KURSTAK

glycols; (ii) inactivation of enzymes; (iii) formation of polymers, since strongly oxidized HRPO acts as a bridging molecule between two IgG molecules; and (iv) problems with the purification of conjugates by affinity chromatography on Con A-Sepharose. In the modified method reported here, the optimum oxidation conditions and a very simple and efficient labeling procedure to obtain highly active conjugates are presented.

MATERIALS AND METHODS

HRPO type VI, lots 31F-9505 and 62F- 9545, and HRPO type II, lot 12 1 F-9600, were obtained from Sigma Chemical Company, and HRPO grade I, lots 12323 18 and 1358506, were purchased from Boehringer-Mannheim GmbH. DEAE-Sepharose (CLdB), PBE-94 and Polybuffer 96, and Con A-Sepharose were from Pharmacia Fine Chemicals. Sodium m- periodate and sodium borohydride were obtained from Sigma Chemical Company, and purified IgG (anti-rabbit IgG produced in goat) was purchased from Miles Laboratories Ltd.

PuriJication of HRPO. HRPO was purified, first by chromatofocusing, but later by ion- exchange chromatography, since the latter gave exactly the same results, but is cheaper and simpler. For chromatofocusing, 5 ml of degassed PBE-94 gel was packed in a small column and equilibrated with 200 ml of 0.025 M ethanolamine-acetic buffer, pH 9.4 HRPO (20 mg) is dissolved in 2 ml of the same buffer and applied to the column. The adsorbed proteins are eluted with lo-times-diluted Poly- buffer 96 equilibrated to pH 6.0 with acetic acid. Purification on DEAE-Sepharose was obtained by equilibrating this ion-exchanger with 2.5 mM sodium phosphate buffer, pH 8.0, followed by degassing, and pouring a column of about 7 ml bed volume. HRPO (30 mg/2 ml) was applied in the same buffer, and an ionic strength gradient (75 ml, 2.5-350 mM phosphate, pH 8.0) was used to fractionate the HRPO preparation.

Conjugation. In order to establish the optimum concentration of NaI04 to achieve a

relatively slow and limited oxidation of HRPO, the following conditions were chosen to be constant: (i) pH 8.1, by solubilizing HRPO in 0.1 M sodium bicarbonate to which an equal volume of m-periodate solution (double concentration in distilled water) was added; (ii) 2 h incubation at 20°C; (iii) a small, closed tube to avoid pH fluctuations; and (iv) darkness during incubation.

The oxidation was, after the incubation, slowed down by the addition of 0.1 vol of 0.1 M sodium carbonate. IgG (molecular ratio HRPO/IgG of 1.2) in 0.1 M sodium carbonate buffer, pH 9.2, was added to the HRPG sample and the combined sample was transferred im- mediately to another tube (or closed Pasteur pipet fitted with a glasswool filter) followed by dry Sephadex G-25 (l/6 of combined weight of HRPO and IgG samples). The tube was closed and left at room temperature for 3 h or overnight, depending on reactant concentrations (see Discussion).

The conjugate was then eluted from the Sephadex and stabilized by the addition of l/20 vol of a fresh NaBH4 solution (5 mg/ml in 0.1 mM NaOH), repeated after half an hour with an addition of 3/20 vol of another freshly prepared NaBH4 solution and left for 1 h.

Purification of conjugates. Saturated ammonium sulfate was prepared by dissolving the salt in water at 40°C and overnight cooling to room temperature. The pH was raised to 7.2 by the addition of NH40H, while checking the pH value each time with a 20-times-diluted solution to avoid measurement errors (about 2 pH unit error with concentrated solution). The precipitate formed during a l-h incubation of the conjugate preparation after the addition of ammonium sulfate was collected by centrifugation, washed with 50% ammonium sulfate, and recentrifuged. The pellet, which contained only free IgG and conjugate, was then equilibrated with PBS (dialysis, or passage over Sephadex G-25).

Free IgG was separated from the conjugate by affinity chromatography on Con A-Se- pharose as described elsewhere (2).

Analysis. The RZ values, customarily used

ISOLATION AND CONJUGATION OF PEROXIDASE 453

to indicate purity of commercial preparations of HRPO, equal the absorbance ratio at 403 and 275 nm. As pointed out earlier by Kurstak et al. (6), this is merely indicative of hemin content and not a good measurement of purity since each isozyme has a different RZ. Specific enzyme activities (i.e., activity per unit weight) were measured as reported earlier (7,2) and normalized to commercial pure HRPO (put at 100%).

The rate of m-periodate consumption during oxidation was measured by the method of Dixon and Lipkin (8).

The quality of the conjugates obtained by the methods presented here was compared with that obtained with the method reported by Nakane and Kawaoi (1) using unpurified commercial HRPO type VI by ELISA of densonucleosis virus according to the procedure of Tijssen et al. (2).

RESULTS

Preliminary experiments showed that (i) it was necessary to use double-distilled water for all solutions of peroxidase; (ii) deionized water may inhibit the enzyme to unpredictable de- grees; (iii) blocking of amino groups of HRPO is not necessary, since very few or no free amino groups are present (9), an observation in agreement with that of Wilson and Nakane (10) and which may explain the difficulties encountered with the conjugation of this enzyme to immunoreactants with glutaraide- hyde; and, (iv) that commercial “pure” peroxidase should be further purified.

Purification of HRPO type II (RZ according to supplier 1.7, but by our measurements 1.15) by chromatofocusing and DEAE-Sepharose ion-exchange chromatography yielded enzyme fractions in the same relative quantitative proportion with similar RZ and activity. Therefore, the simpler and cheaper DEAE- Sepharose was employed further. A large fmc- tion of the HRPO preparation, dissolved in 2.5 mM phosphate buffer, passed unretained. This fraction contained about 75% of the original hemin present, but more than 90% of the

activity, and generally had an RZ between 3.2 and 3.3. The application of the ionic strength gradient resulted in elution of a second fraction, which contained about 15% of the hemin originally applied, at 5 InM phosphate buffer. The isozyme in this fraction had an RZ higher than in the first fraction (3.30-3.40), but a specific activity which was at least four times lower. Finally, a heterogeneous group of fractions eluted between 40 and 300 mM, which contained less than 10% of the hemin content and about 3% of the original activity. This purification scheme could, therefore, be simplified to stepwise elution: (i) the desired HRPO dissolved in 2.5 I’nM sodium phosphate buffer, pH 8.0, passed directly through the DEAE-Sepharose column equilibrated against the same buffer (this yielded highly pure peroxidase with a specific activity 1 O-20% higher than commercial “pure” peroxidase); (ii) a second isozyme could be recovered by apply- ing 20 mM of the same buffer (though this enzyme has a higher RZ it is less suited for enzyme immunoassays); and (iii) rest fractions eluted with a highly concentrated (350 mM) buffer; (these fractions contained, besides a very low peroxidase activity, the great majority of contaminants). Table 1 gives the results of a typical purification experiment using the commercial crude extract. The further purification of commercial pure peroxidase using the same system showed that the RZ obtained in the first fraction could be somewhat higher than those found for the crude extracts. The RZ of such fractions generally were between 3.45 and 3.55, recovery ofactivity was usually higher than 95%, and, most importantly, the specific activity increased by about 25%. The second isozyme, recovered with 20 mM phosphate buffer, makes up much less of the total activity than with the crude extracts. However, there is a relatively large fraction of nonactive proteins which absorb only in the uv region. Typical results obtained with the purification of commercial pure HRPO with an RZ of about 3.0 are given in Table 2.

The oxidation of HRPO by sodium periodate had several effects (Table 3): (i) a de-


TABLE 1

PURIFWATION OF HORSERADISH PEROXIDASE FROM A COMMERCIAL CRUDE EXTRACT

Fraction

Recovery (%) Percentage of total Relative specific activity (W) activity of 3 compared to commercial

Rz 275 nm 403 nm fractions pure pcroxidase

1 3.21 26.1 73.0 92.16 111.5 2 3.35 5.2 15.3 4.50 27.0 3 0.36 27.5 8.7 3.35 3.8

Note. Sigma lot 12 1F - 9600 (marked RZ 1.7, measured RZ 1.15); on DEAE-Sepharose (7 ml; 52 optical density units at 403 nm applied).

crease of the absorbance by HRPO at 403 nm accompanied by a change of color from brown to green for the samples with very high concentrations of periodate; (ii) the number of molecules of periodate consumed per molecule of peroxidase increased progressively with higher concentrations of peroxidase; and (iii) a decrease in the specific activity of the enzyme, particularly at higher periodate concentrations. The activity lost at high periodate concentrations could not be regained after dialysis and reduction of the preparation.

The efficiency of the conjugation procedure is presented in Table 4. This table shows that there was an optimum periodate concentration for oxidation at about 4-8 mM resulting in a conjugation efficiency of about 95%. Pu- rification of conjugates prepared at this periodate concentration did not run into problems with the isolation procedure reported earlier

(2). However, at periodate concentrations of 32 mM or more, 30 to 50% of the HRPO conjugates was not retained on the Con A- Sepharose column, probably due to extensive oxidation of the carbohydrate to which concanavalin A has an affinity. Indirect, noncom- petitive ELISA assays in which densonucleosis virus was immobilized on the solid-phase, and in which a solution with a constant anti-virus antiserum concentration (dilution 25,000 times) was brought into contact with the virus to form immune complexes, anti-immunoglobulin labeled by the original method of Nakane and Kawaoi ( 1) and by the modified method presented here were titrated. An average increase in titer by the new method of 2.9 (five experiments) was obtained; some experiments in which the original method gave much lower titers than others prepared with the same technique were disregarded. This

TABLE 2

PURIFICATION OF HOFLSERADISH PEROXIDASE FROM A COMMERCIAL PURE PREPARATION

Fraction

Recovery (W) Percentage of total activity Relative specific activity recovered from original (W) compared to

Rz 275 nm 403 nm sample applied original

1 3.50 78.3 95.6 96.4 124.0

2 too low 0.7 1.5. N.D. N.D. concentrations

3 0.27 9.8 0.8 N.D. N.D.

Note. Sigma lot 3lF-9505 (marked RZ 3.1, measured RZ 2.88); on DEAE-Sepharose (7 ml; 30 optical density units at 403 nm applied). N.D., not done.


TABLE 3

EF’FEC~S OF OXIDATION OF HORSERADISH PEROXIDASE LI

NaIO, OD,, MM) atkr2h

mol NaIO, consumed/mol

peroxidase

Percentage specific enzyme activity

(compared to original)

Control 0.50 loo 0.1 0.50 N.D. loo 0.2 0.49 N.D. 99.5

0.5 0.47 N.D. 98.5 I 0.46 N.D. 96.8 2 0.43 N.D. 95.6

4 0.40 23 89.4 8 0.38 44 79.8

16 0.36 98 71.3 32 0.32 234 54.9

‘Average of three experiments. N.D., not done (due to relatively high interference of peroxidase absorbance at 223 nm).

demonstrated, however, that large fluctuations in the success of conjugation is obtained with the original method.

DISCUSSION

The purification of HRPO on DEAE-Se- pharose of commercial “pure” peroxidase was found to be necessary to remove inactive proteins. These may be contaminating proteins or inactive apoenzyme. The apoenzyme has a pl which is considerable lower than that of the isozyme purified (6.8 vs 8.7) (9). Moroz et al. ( 10) earlier reported the presence of contaminating proteins in commercial “pure” preparations, but removed these contaminants with a significantly more complicated and less efficient method. Our purification method raised the specific activity by about 25%, which has a direct impact on the final enzyme immunoassay titers. Background staining will also be lowered. As an additional cost-saving factor, the use of antiserum will be more eco- nomical.

The purification of HRPO from crude extracts by this simple method may lower the costs very significantly. It is possible to purify an amount of HRPO, corresponding to a cost of about $500.00, for one-fifth of the cost in less than 1 h. Moreover, the HRPO purified in this way will have higher specific activities

than the commercial preparations and be de- void of significantly less active isozymes.

The second part of this study, to make current enzyme immunoassays for antigens more efficient, concerned the chemical labeling of peroxidase to enzyme in terms of both efficiency and retention of enzyme activity. It should be realized that retention of enzyme activity is more important than retaining immunological activity, on the condition that a lower immunological activity is based on the blocking of paratopes rather than a decrease in affinity of the individual paratopes for the antigen or IgG epitopes. A decrease in affinity would, namely, increase elution of antibody during washing. Such an affinity decrease is not likely to occur. However, the conjugation of inactive enzyme molecules to active IgG molecules will directly lower the total enzyme activity which can be obtained in enzyme immunoassays since such conjugates will com- pete with the conjugates having active enzymes. In contrast, inactive IgG molecules conjugated with active enzyme will not in- terfere.

The conjugation experiments showed that the sodium periodate concentration is critical for an efficient conjugative and retention of enzyme activity. In our hands the use of distilled water (as in the procedure of Wilson and Nakane (11) led to widely varying results. This

TABLE 4

EFFICIENCY OF ENZYME CONJUGATION'

Fraction of HRPO Percentage specific NaIO, conjugated * enzyme activity (mW (ODw3 umts) (compared to original)

1 0.714 98.7 2 0.836 94.3 4 0.935 89.6 8 0.950 78.5

16 0.922 64.1 32 0.897 46.0

’ Average of three experiments. b Fraction of HRPO conjugated was established by am-

monium sulfate precipitation (at 50% saturation; free peroxidase precipitates at 70-90s).


is probably due to a very pronounced influence of pH (fastest at pH 3-5) on oxidation by sodium periodate (12). The aldehyde groups generated by oxidative cleavage of the carbohydrate moieties are even more sensitive to oxidation than the original sugar, resulting in carboxyl acids. This overoxidation might explain why less peroxidase is coupled at higher periodate concentrations. It is, therefore, im- perative to have as low periodate concentrations as coupling efficiency permits. An important side effect of the sodium periodate treatment is that even moderate concentrations of this salt lead to the oxidation of amino acid residues (13,14). For example, methionine is readily converted into methionine sulfoxide (13) which may alter the confor- mation of protein molecules (methionine is usually buried in hydrophobic clusters whereas methionine sulfoxide is much more hydro- philic). One of the reasons that the use of double-distilled water to prepare the solutions is important is that the presence of weakly ox- idizing metals may be sufficient to oxidize aldehyde groups. Another factor is the high sensitivity of peroxidase activity to impurities in water (bacteria, bacteriostatic agents, polysty- rene, etc).

The dry Sephadex G-25 in the second step served several purposes. The fast uptake of liquid by these beads increased the HRPO and IgG concentrations, decreased the relative periodate concentration (since this salt can enter the beads), and consumed the periodate present in these beads. The increase of HRPO and IgG concentrations of about two-fold ac- celerates the rate of conjugation formation about four times, since, according to the law of mass action,

conjugate formation = k[IgG][HRPO],

where k is associative rate constant. Three hours suffice if the HRPO concen-

tration is 2 mg/ml or more, otherwise incubation times have to be adjusted according to this simple formula (e.g., if 1 mg/ml of HRPO

would be present, then 12 h would be re- quired).

It is also important to realize that though almost all HRPO is conjugated this will usually not be the case for IgG. The HRPO will be distributed over the IgG molecules according to the Poisson distribution, at least at low ra- tios. This means that at a HRPO/IgG moleo ular ratio of about 1 a maximum close to 40% of the IgG will be conjugated with one HRPO only. Increasing the HRPO/IgG ratio beyond this optimum ( 11) will lower the free IgG fraction but increase even more the fraction of IgG conjugated with two enzyme molecules, and this might in turn lead to a significant IgG inactivation. In the procedure presented here a slightly higher molecular ratio than 1 was used to correct for the fraction of HRPO which would not bind at all.

The stabilization of the Schiffs bases was performed similar to methods used for re- ductive methylation ( 15). Since borohydride is hydrolyzed almost instantaneously in aqueous solutions, this was repeated once, but much shorter incubation times were used than described with the other m-periodate methods.

Purification of the conjugates proved es- sential to obtain high titers in enzyme immunoassays (16,2). With the convenient Con A-Sepharose affinity chromatography method, it was possible only in cases where the sugar moiety was not too much degraded by periodate, i.e., in cases where the periodate concentration was lower than 15 mM. The recommended procedure is given as an appendix.

APPENDIX: RECOMMENDED PROCEDURE (TIME: 1 DAY)

1. Activation of HRPO A. Dissolve 5 mg of purified HRPO in 0.5

ml freshly prepared 0.1 M sodium bicarbonate (prepared in distilled water) in a small tube.

B. Add 0.5 ml of 8-16 IX’IM (depending on lot of HRPO) NaIO, , close tube and leave for 2 hours in dark at 2O’C.


2. Conjugation REFERENCES A. Prepare 15 mg IgG in 0.1 M sodium

carbonate, pH 9.2 (l-2 ml). B. Add dry Sephadex G-25 to Pasteur pipet

(bottom closed) fitted with glasswool filter (amount of Sephadex equals l/6 of combined weight of HRPO and IgG samples), and con- taining HRPO and IgG samples. Leave for 3 hours (see Discussion). 3. Stabilization

1.

2.

3. 4.

5.

6.

Nakane, P. K., and Kawaoi, A. (1974) J. Histochem. Cyfochem. 22, 1084-1091.

Tijssen, P., Su, D.-M., and Kurstak, E. (1982) Arch. Viral. 74, 277-291.

Avrameas, S. (1969) Immunochemistry 6,43-52. Avrameas, S., and Temynck, T. (1971) Immuno-

chemistry& 1175-1179. Nygren, H. (1982) J. Histochem. Cytochem. 30,407-

412.

A. Elute conjugate from Sephadex. B. Add l/20 volume of freshly prepared 5

mgfml NaBH4 in 0.1 mM NaOH and after 30 min add 3/20 volume of another freshly prepared NaBH4 solution. Leave for 1 hour. 4. Purijkation

A. Precipitate conjugate and free IgG with equal volume of saturated ammonium sulfate solution, collect pellet and equilibrate against PBS.

B. Prepare small Con A-Sepharose column, apply sample, elute free IgG, and desorb conjugate by the addition of 0.01 M methyl a-D mannopyranoside in PBS.

C. Store conjugate after addition of equal volume of glycerol at -20°C.

7.

8.

9.

10.

II.

Kurstak, E., Tijssen, P., and Kurstak, C. (1977) in Comparative Diagnosis of Viral Diseases (Km&k, E., and Kurstak, C., eds.), vol. 2, pp. 403-448, Academic Press, New York.

Ngo, T. T., and Lenhoff, H. M. (1980) Anal. B&hem. 105, 389-397.

Dixon, J. S., and Lipkin, D. (1954) Anal. Chem. 26, 1092-1093.

Welinder, K. G. (1979) Eur. J. B&hem. 96, 483- 502.

Moroz, L. A., Joubert, J. R., and Hog, J. C. (1974) J. Immunol. 112, 1094.

Wilson, M. B., and Nakane, P. K. (1978) in Im- muno5uorescence and Related Techniques (Knapp, W., Holubar, K., and Wick, G., eds.), pp. 2 15-22 1, Elsevier, Amsterdam/New York.

Dyer, J. R. (1956) Methods Biochem. Anal. 3, 11 l- 152.

ACKNOWLEDGMENT

This work was supported by a grant to Professor E. Kurstak from the Natural Sciences and Engineering Re- search Council Canada.

12.

13.

14.

15.

16.

de la Llosa, P., El Abed, A., and Roy, M. (1980) Canad. J. Biochem. 58, 745-748.

Yamasaki, R. B., Osuga, D. T., and Feeney, R. E. (1982) Anal. Biochem. 126, 183-189.

Biocca, S., Calissano, P., Barra, D., and Fasella, P. M. (1978) Anal. Biochem. 87, 334-342.

Kurstak, E., and Kurstak, C. (1974) in Viral Im- munodiagnosis (Kurstak, E., and Morisset, R., eds.) pp. 3-30, Academic Press, New York.

highly efficient and simple methods for the preparation of peroxidase and active peroxidase-antibody...

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