Journal of Biochemical and Biophysical Methods, 13 (1986) 205-210
Elsevier BBM 00563
A spectrophotometric assay for nanogram quantities of biotin and avidin R a y m o n d S. Niedbala, Franklyn Gergits III and Keith J. Schray Dept. of Chemistry, Lehigh University, Seeley G Mudd Bldg., Bethlehem, PA 18015, U.S.A.
(Received 1 March 1986) (Accepted 1 July 1986)
Summary Parameters and conditions of an enzyme based assay for biotin and avidin are presented. Biotinylated glucose-6-phosphate dehydrogenase when complexed with avidin becomes inactivated. Thus it was possible to construct a competitive assay system for biotin. The assay is sensitive between 100-500 ng/ml and could detect as little as 10 ng in 0.1 ml with a between run error of 2.4%. It requires a 60 rain incubation at 21°C and 5 rain to assay. The avidin assay, based on the degree of inactivation of biotinylated-glucose-6-phosphatedehydrogenase in relation to the concentration of avidin, could detect as little as 0.25 ng in 0.1 ml or 2.5 ng/ml with an assay time of 10 rain with a between run error of 3.9%. Both assays are rapid with significant improvements over other non-isotopic methods in sensitivity and comparable to radioisotopic methods in sensitivity with the added advantage of ease of method. Key words: Avidin; Biotin; Glucose-6-phosphate dehydrogenase
Introduction The high affinity of avidin for biotin, K D = 10 -13 M, makes this couple useful for conjugation of non-associating components, cytologic identification of antigens, and amplification of analyte detection [1-3]. The biotinylation and avidinylation of proteins is performed under mild conditions preserving full biological activity in many cases. Prepared avidin and biotin conjugates have a long shelf life and are stable under extreme experimental conditions . The use of avidin and biotin in detection of analytes can improve sensitivity by bridging components or inhibiting biological activity upon binding and is finding wide spread use [5,6]. Correspondence address: Raymond S. Niedbala, Hoffmann-La Roche Inc., Diagnostics Division, 340 K.ingsland St, Nutley, NJ 07110, U.S.A. 0165-022X/86/$03.50 © 1986 Elsevier Science Publishers B.V. (Biomedical Division)
206 Biotin and avidin are not only useful because of their high affinity for one another, but also function physiologically. Biotin, vitamin H, functions as a cofactor in carboxylase reactions and its deficiency can result in alopecia, dermatitis, metabolic acidosis, ketosis, and organic aciduria . Although the physiologic role of avidin has not yet been defined one of its functions may be to act as an antibacterial agent. Thus assay methods for avidin and biotin are not only useful for quantifying biotinylated or avidinylated conjugates but also to the clinician for diagnosis. Assay methods for avidin and biotin previously described have utilized isotopically labeled biotin, colorimetric determination using dyes, or competitive binding enzymatic systems. The most sensitive of these methods have been the radiolabeled and colorimetric systems [8,9]. The competitive binding enzymatic system employed the use of biotinylated glucose-6-phosphate dehydrogenase (G6PDH). However, the sensitivity of the assay was limited since 96% of enzymatic activity was lost during biotinylation . Recently, active site blocking agents have been used to improve biotinylation of G6PDH . Thus it has become possible to develop an assay superior to previous assays in either simplicity, sensitivity, or both.
Materials and Methods
The following materials were obtained from the Sigma Chemical Co.: avidin, biotin, N-hydroxysuccinimido biotin, glucose-6-phosphate dehydrogenase (G6PDH; E.C. 188.8.131.52) having a specific activity of 270 U / m g of protein, glucose-6-phosphate (G6P), NADPH, and NADP. All other materials and reagents were of the highest quality obtainable. Experiments were carried out in 10 mM EDTA/20 mM Tris buffer (pH 8.0) unless stated otherwise. The biotinylation of G6PDH was carried out by methods described elsewhere . Briefly, to 72 U of enzyme enough G6P and NADPH was added to give final concentrations of 18 mM and 0.44 mM, respectively. After incubation for 10 rain at 21° C 40 # g / m l of N-hydroxysuccinimido biotin was added and incubated for 2 h at 21° C. During this period aliquots were removed and assayed for enzymatic activity. The solution was applied to a Sephadex G-25 column with eluted fractions containing protein dialyzed overnight at 4°C. These fractions were assayed for biotin content and enzymatic activity . The number of biotins bound per enzyme were determined by the method of Green . The method relies on an absorbance change which occurs when a dye, hydroxyazobenzoic acid, is displaced from avidin by biotin. The method is reliable in 10 mM EDTA/20 mM Tris and applicable to measuring protein bound biotin as first suggested by Green. Biotinylated G6PDH was stored in 10 mM EDTA/20 mM Tris at 4°C, and was stable for at least 30 days. The competitive assay for biotin was set up as follows: to 0.5 #g of avidin in 0.1 ml of 10 mM EDTA/20 mM Tris buffer (pH 8.0) various concentrations of biotin were added and incubated for 30 rain at 21° C. Biotinylated enzyme, 2.9 #g/ml, was then added and incubated for 30 min at which time the solution was assayed for
207 enzymatic activity for 5 min. Avidin was assayed by taking 0.3 #g of biotinylated G 6 P D H in 0.1 ml 10 m M E D T A / 2 0 m M Tris (pH 8.0), adding various concentrations of avidin, incubating 10 min at 21°C, and assaying enzymatic activity for 5 min. Results from duplicate experiments were plotted to construct standard curves with the between run error calculated from the difference between numerical values for each point.
Results The biotinylation of G 6 P D H resulted in 100% of enzymatic activity conserved . Enzyme with two different biotinylation levels was used in the assay methods. The preparation of b i o t i n - G 6 P D H used for the assay of biotin and avidin were prepared on two separate days. The ratio of biotin to enzyme was approximately 8 : 1 for the assay of biotin and 5 : 1 for the avidin assay. The basis for the assay procedures is the fact that biotinylated G 6 P D H is inactivated following complexation by avidin as originally shown by Ngo . The time dependence of this inactivation is shown in Fig. 1. The extent of inactivation for a given incubation time is dependent on the avidin concentration as shown in Fig. 2, and forms the basis for both assays. Fig. 2 constitutes a standard curve for assay of avidin. The assay procedure for avidin, requiring a 10 min incubation at
I( ¢ o
TIME ( r a i n )
Fig. 1. Time dependence of avidin mediated inactivation of biotinylated G6PDH. Biotinylated G6PDH, 2.0 ~g, was mixed with 10 ~g of avidin in 1 ml of 10 mM EDTA/20 mM (pH 8.0) buffer, incubated at 21°C, with aliquots removed from 0 to 30 min, and assayed for enzymatic activity.
LOG (AVIDIN)ng/ml Fig. 2. Parameters used for the assay of avidin. To 0.25 ng to 10/~g of avidin in 0.1 ml 10 mM E D T A / 2 0 mM Tris buffer (pH 8.0), 0.3 /~g of biotinylated G6PDH was added, incubated 10 rain at 21°C, and assayed for 5 min at 340 nm.
21°C and 5 min to assay, was sensitive to 0.25 ng in 0.1 ml or 2.5 n g / m l . The between run variation was 3.9% and the measured error when transforming % inactivation to real numbers using Fig. 2 was 26%. The biotinylated G 6 P D H used in
LOG (BlOTIN)ng/ml Fig. 3. Parameters used for the assay of biotin. To 0.1 ng to 25/~g of biotin in 0.1 ml 10 mM EDTA/20 m M Tris buffer (pH 8.0), 0.5 /~g of avidin was added and incubated 30 rain at 21°C. Biotinylated G6PDH, 0.29/~g, was added, incubated 30 rain at 21°C, and assayed at 340 nm for 5 rain.
209 the avidin assay was inactivated ~ 60% in 10 rain while that used in the biotin assay was inactivated - 70% in 60 rain. The avidin level selected for use in the assay of biotin is an optimal concentration providing a high degree of enzyme inactivation while allowing a desirable level of sensitivity to competing biotin. The assay of biotin is rapid and measured spectrophotometrically via enzyme activity for 5 rain after a 60 rain incubation at 21°C. The entire assay procedure required 65 min without any special conditions other than 10 mM EDTA, and was linear between 100-500 ng/ml or could detect as little as 10 ng in 0.1 ml with a between run error of 2.4%, see Fig. 3.
The avidin-biotin system has been used for numerous applications designed to amplify sensitivity and specificity of analyte detection. In creating such systems the concentration of biotin or avidin must be determined accurately in minimal time in order to determine such things as degree of biotinylation. Green pioneered much of the fundamental characterization of avidin and was the first to develop an assay for it using [14C]biotin for detection of 1.0 /~g avidin and 0.01 /~g Of biotin . However, the assay involves the usual problems of radiolabeled components such as handling, disposal, shelf life, and commercial availability of labeled biotin. A similar method for assaying avidin has been used by O'Mailey which involves the use of labeled biotin which is bound to avidin, precipitated after interaction with avidin antibody and counted . Although more sensitive than the method of Green and linear between 0.1-0.5 #g avidin, it still requires labeled biotin and considerable handling. A spectrophotometric assay of biotin and avidin has been used relying on the change in absorbance upon displacement of dye bound to avidin . This method which employs no radiolabeled component is facile and can detect 50 /tg/ml of avidin and 0.65 ttg/ml of biotin. However, the dye binds non-specifically in the presence of albumin. The enzymatic assay of biotin previously developed by Ngo et al. could detect 40-60 mg/ml of biotin and 25-95 /tg/mi of avidin in 2 min . However this work employed the use of G6PDH which had a 26-fold decrease in enzymatic activity during biotinylation. The assay procedures for avidin and biotin presented here have utilized an improved biotinylation procedure. The improvement has increased enzyme detectability due to decreased inactivation of the enzyme. This has allowed orders of magnitude improvement in sensitivity over those previously reported using this system . (Discrepancies in text and figure presentations in the first publication of the enzymatic method prevent precise statement of the improvement factor represented by this work.) It should be noted that the biotinylated G6PDH preparations used in the assays presented in this report are heterogeneous with respect to the number of biotins per enzyme. Biotinylated G6PDH used for the assay of biotin contained a biotin/G6PDH ratio of 8 : 1 while biotin/G6PDH used for assay of avidin is 5 : 1. It is not clear if this heterogeneity affected sensitivity, however it is possible it may
210 affect the rate of e n z y m e inactivation resulting from b i o t i n - e n z y m e - a v i d i n c o m plexation. It is advised that a s t a n d a r d curve be generated with each b i o t i n - G 6 P D H p r e p a r a t i o n . This is d u e to the varying degree o f b i o t i n y l a t i o n a n d enzyme inactivation that c a n occur if the c o n c e n t r a t i o n s of N A D P H a n d G 6 P are altered d u r i n g b i o t i n y l a t i o n . F o r example, when the ratio of b i o t i n to G 6 P D H ( 1 0 5 : 1 ) a n d i n c u b a t i o n time was fixed d u r i n g b i o t i n y l a t i o n , b u t the c o n c e n t r a t i o n s of N A D P H a n d G 6 P varied (0.44 # M N A D P H , 16.5 /~M G6P, vs 2.7/~M N A D P H , 35.5 /~M G6P), the resulting degree of e n z y m e i n a c t i v a t i o n d u r i n g b i o t i n y l a t i o n varied from 95.5% to 82% with 10.9 a n d 21.9 biotins b o u n d p e r e n z y m e ( u n p u b l i s h e d data). Since the i m p r o v e d b i o t i n y l a t i o n p r o c e d u r e is the suggested key to i m p r o v e m e n t of the assays p r e s e n t e d in this paper, it is critical to follow the p r o t o c o l for b i o t i n y l a tion p r e s e n t e d to o b t a i n viable assay c o m p o n e n t s which are sensitive to avidin inactivation. Nonetheless, the assay p r e s e n t e d is c o n s i d e r a b l y simpler than isotopic assays yet achieves m u c h greater sensitivity t h a n o t h e r n o n - i s o t o p i c assays.
Simplified description of the method and its advantages A conjugate of biotin and glucose-6-phosphate dehydrogenase is synthesized (ca. 6:1 ratio). A concentration dependent inactivation of the enzyme activity by avidin may be used to construct an avidin standard curve for assay purposes. A biotin concentration dependent prevention of this inactivation at any given avidin concentration yields a standard curve for a biotin assay. The assay times are fifteen and seventy minutes respectively and require a spectrophotometric measurement of enzyme activity.
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