Colorimetric competitive inhibition method for the quantitation of avidin, streptavidin and biotin

Colorimetric competitive inhibition method for the quantitation of avidin, streptavidin and biotin

J. Biochem. Biophys. Methods 39 (1999) 1–6 Colorimetric competitive inhibition method for the quantitation of avidin, streptavidin and biotin Zhibo G...

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J. Biochem. Biophys. Methods 39 (1999) 1–6

Colorimetric competitive inhibition method for the quantitation of avidin, streptavidin and biotin Zhibo Gan, Ronald R. Marquardt* Department of Animal Science, The University of Manitoba, Winnipeg, MB, Canada R3 T 2 N2 Received 24 June 1998; accepted 3 August 1998

Abstract A colorimetric competitive inhibition assay for avidin, streptavidin and biotin was developed. The method for avidin or streptavidin was based on the competitive binding between avidin or streptavidin and a streptavidin–enzyme conjugate for biotinylated dextrin immobilized on the surface of a microtitre plate. For biotin quantitation the competition is between free biotin and the immobilized biotin for the streptavidin–enzyme conjugate. The limits of detection which was determined as the concentration of competitor required to give 90% of maximal absorbency (100% inhibition) was approximately 20 ng / 100 ml per assay for avidin and streptavidin and 0.4 pg / 100 ml per assay for biotin. The methods are simple, rapid, highly sensitive and adaptable to high throughput analysis.  1999 Elsevier Science B.V. All rights reserved. Keywords: Competitive inhibition assay; Avidin; Streptavidin; Biotin

1. Introduction Avidin–biotin technology has been applied in almost every aspects of biological sciences [1]. One of the most important applications of this technology has been the biotinylation of antibodies and other reagents used for immunoassays. Another important use of the technology has been the use of biotin-labeled DNA probes. These preparations usually need to be analyzed for extent of biotinylation. Biotin content of tissues and food *Corresponding author. Tel.: 1 1-204-474-8188; fax: 1 1-204-474-7628; e-mail: rr [email protected] ] 0165-022X / 99 / $ – see front matter  1999 Elsevier Science B.V. All rights reserved. PII: S0165-022X( 98 )00051-7

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also need to be evaluated in metabolic and nutrition studies. The concentration of avidin as a anti-nutrition factor is often considered. Several methods have been used to analyze for biotin, avidin and streptavidin. Assays using microorganisms [2], fluorescence [3,4], isotopes [5], dyes [6], enzymes [7,8] and antibodies against avidin [9] have been exploited for this purpose. Although these methods basically meet the requirements of the assay, there are drawbacks associated with each of the different methods. More suitable methods for the determination of not only biotin but also avidin and streptavidin would therefore be useful. The methods described in this paper are based on the competitive binding between avidin or streptavidin and a streptavidin–enzyme conjugate for biotinylated dextrin immobilized on the surface of a microtitre plate, or between free biotin and the immobilized biotin for the streptavidin–enzyme conjugate. The former procedure quantitates avidin or streptavidin while the latter procedure quantitates biotin. The methods are highly sensitive, simple to use, can be easily manipulated to facilitate the type of assay to be carried out and can be adapted to high throughput analysis.

2. Materials and methods

2.1. Materials Biotinylated dextrin and streptavidin–horseradish peroxidase conjugate were from Norzyme (1839 Logan Ave. Winnipeg, MB, Canada R2R 0H4). Avidin, streptavidin, d-biotin, dimethyl sulfoxide (DMSO), sodium phosphate, Tween-20 were purchased from Sigma Chemical (P.O. Box 14508, St. Louis, MO 63178-9916). Microtiter plates (Falcon 3911) were from Becton Dikinson (1950 Williams Drive, Oxnard, CA 93030). UltraBlue soluble substrate for horseradish peroxidase was from Intergen company, 25 Birch St. Milford, MA 01757.

2.2. Methods 2.2.1. Coating of a microtiter plate with different concentrations of biotinylated dextrin The wells in a 96-well microplate except for the first column of eight wells, were coated using biotinylated dextrin in phosphate-buffered saline (PBS, 0.01 M phosphate and 0.15 M NaCl, pH 7.2) at a concentration of 0.5 or 5 mg / 100 ml per well. The plate was incubated in a humid atmosphere overnight at 378C and was then washed three times using PBST (PBS with 0.05% Tween-20). 2.2.2. Assay procedure for avidin, streptavidin and biotin PBS (50 ml) was added to each well in the plate. The sample solution (50 ml avidin, streptavidin or biotin) which is referred to as the competitor was applied to the wells of the second column followed by successive doubling dilutions of the avidin or streptavidin solution or five-fold dilutions of the biotin solution in wells up to and including those in column 11. The additional 50 ml of solution that was present in

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column-11 wells was discarded. Streptavidin–horseradish peroxidase in PBS (50 ml of 1:4000 dilution of the stock) was added to each well followed by incubation at room temperature for 30 min. The final volume in each well was 100 ml. The blank set of wells with no coated biotinylated dextrin and no competitor (column 1) served as the negative control while the last set of wells (column 12) with coated biotinylated dextrin but no competitor served as the positive control. The plate was then washed five times using PBST and 100 ml of mltraBlue soluble substrate was added to each well of the plate. The plate was incubated at room temperature for about 30 min and the absorbency was read at 655 nm using a microplate reader (Bio-Rad laboratories Inc. Mississauga, ON, Canada. Model 450).

3. Results

3.1. Effect of coating concentration on the sensitivity of assay The sensitivity of the assay was affected by the concentration of biotinylated dextrin coated on the microplate (Table 1). The results demonstrated that the wells that had the lower coating concentration had a much higher sensitivity for avidin, streptavidin and biotin but required a longer time for color development with the time being 30 and 10 min, respectively, for wells coated with low and high concentrations of the biotinylated dextrin.

3.2. Assay for avidin and streptavidin The assay for avidin and streptavidin involved competition between one of these compounds and a streptavidin–horseradish peroxidase conjugate for binding by the immobilized biotin (Fig. 1). Unknown values can be quantitated from the standard curve. The limits of detection for avidin and streptavidin, as determined by the concentration or competitor required to give 90% of maximal absorbency, was approximately 20 ng / 100 ml per assay.

Table 1 Effect of coating concentration on the sensitivity of the assay Analyte

Avidin Streptavidin Biotin a

Amount of analyte required for 50% inhibition (ng / 100 ml)a Coating concentration (0.5 mg / 100 ml per well)

Coating concentration (5 mg / 100 ml per well)

41 43 0.03

700 840 9

Standard errors for all comparisons were less than 5% of mean values for triplicate analysis.

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Fig. 1. A typical standard curve of avidin ( ? - - ? ) or streptavidin (s - - s). See Section 2.2.2 for details of the assay procedure. The values represent a mean of triplicate analysis. The absorbency 6SD of the positive control was 2.4560.03 absorbency units.

3.3. Assay for biotin The binding of the streptavidin–peroxidase complex by the immobilized biotin was inhibited by free biotin in the sample. Therefore, a standard curve can be established and unknown values can be determined from this curve (Fig. 2). The limits of detection for biotin, as determined by the concentration of competitor required to give 90% of maximal absorbency, was approximately 0.4 pg / 100 ml per assay.

4. Discussion A variety of methods for avidin and biotin using radioactive tags [5], fluorescence [3] and dyes [6] are based on the interaction between biotin and avidin in a liquid medium. The solid-phase competitive–inhibition assay described in this study involves competitive binding between a streptavidin–peroxidase conjugate and avidin or streptavidin for a biotin–dextrin complex that was immobilized on the surface of a microtiter plate well, or between free biotin and immobilized biotin for the streptavidin–peroxidase conjugate. Interferences caused by the presence of other compounds in the assay mixture are readily removed by the washing step. As a result, background interference is minimal

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Fig. 2. A typical standard curve of biotin. See Section 2.2.2 for detail of the assay procedure. The values represent a mean of triplicate analysis. The absorbency 6SD of the positive control was 2.2360.02 absorbency units.

and would be much less than that obtained with the conventional procedures as contaminants cannot be simply removed when these methods are used. The competitive inhibition method developed in this study had a detection limit of 0.4 pg per assay which is lower than that of the microbiological method [2], the most sensitive method currently in use. The detection limit for avidin or streptavidin was 20 ng / 100 ml per assay (0.5-h incubation) compared to a detection limit of 100 ng / 100 ml (1-h incubation) obtained with the Bayer microplate procedure [7]. These results demonstrated that the assay is highly sensitive, especially for biotin. It is, therefore, particularly useful for samples that have a low content of biotin and are difficult to concentrate. The use of a streptavidin–enzyme conjugate instead of an avidin–enzyme conjugate should yield superior results as it has a lower degree of nonspecific adhesion and the reaction can be carried out at a lower pH [10]. The indicator enzyme in the streptavidin (avidin)–enzyme conjugate could also be alkaline phosphatase, b-galactosidase, etc. instead of horseradish peroxidase while the substrates for the enzymes could be chromogenic, chemiluminescent or fluorescent. In addition, the amount of biotin–dextrin coated on the plate can be varied depending on the desired level of the sensitivity required. Those characteristics make the assay highly versatile.

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Overall the method is simple to carry out as it involves the highly specific interaction between biotin and avidin or streptavidin and can be performed in a few steps in a relatively short period of time with little or no background interference. This assay has overcome the disadvantages of radioactive hazard [5], fluorescent interferences [3,4], low sensitivity [6] and time required for its assay. It is suitable for high throughput analysis and amenable to automation.

Acknowledgements This research was supported by a Strategic Grant from the National Sciences and Engineering Research Council of Canada and the University of Manitoba.

References [1] Wilchek M, Bayer EA. Application of avidin–biotin technology. In: Wilchek M, Bayer EA, editors. Methods in enzymology 184. New York: Academic Press, 1990. pp. 14–45. [2] Carlucci AF. Amphidinium carterae assay for biotin. In: McCormick DB, Wright LD, editors. Methods in enzymology 18A. New York: Academic Press, New York, 1970. pp. 379–383. [3] Lin HJ, Kirsch JF. A sensitive fluorometric assay for avidin and biotin. Anal Biochem 1977;81:442–6. [4] Al-Hakiem MHH, Landon J, Smith DS, Nargessi RD. Fluorometric assays for avidin and biotin based on biotin-induced fluorescence enhancement of fluorescein-labeled avidin. Anal Biochem 1981;116:264–7. [5] Green NM. Avidin 1. The use of [ 14 C] biotin for kinetic studies and for assay. Biochem J 1963;89:585– 91. [6] Green NM. A spectrophotometric assay for avidin and biotin based on binding of dyes by avidin. Meth Biochem J 1965;94:23c–4c. [7] Bayer EA, Ben-Hur H, Wilchek M. A sensitive enzyme assay for biotin, avidin and streptavidin. Anal Biochem 1986;154:367–70. [8] Huang Z, Haugland RP, Szalecka D, Haugland RP. A simple and sensitive enzyme-mediated assay of biotin. BioTechniques 1992;13(4):543–6. [9] Korenman SG, O’Malley BW. Newer methods of avidin assay. In: McCormick DB, Wright LD, editors. Methods in enzymology 18A. Academic Press, New York, 1970. pp. 427–430. [10] Bayer EA, Ben-Hur H, Wilchek M. Isolation and properties of streptavidin. In: Wilchek M, Bayer EA, editors. Methods in enzymology 184. New York: Academic Press, 1990. pp. 80–90.