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A homogeneous assay for biotin based on chemiluminescence energy transfer
ANALYTICAL
BIOCHEMISTRY
155,249-255
(1986)
A Homogeneous Assay for Biotin Based on Chemiluminescence Energy Transfer E.JOANWILLIAMS*
ANDANTHONYK.CAMPBELL
Departmetzf qf Medical Biochemistry, *University Hospilal of Wales, and Universit~~of Uhles College of Medicine. Heath Park. CardiJ CF4 4XW, United Kingdom Received October 23. 1985 Chemiluminescence energy transfer between aminobutylethylisoluminol (ABE&biotin and fluorescein-avidin was investigated in order to establish a homogeneous assay for serum biotin in the physiological range. ABE1 chemiluminescence was measured at pH 7.4 using microperoxidase-hydrogen peroxide and the chemiluminescence at two wavelengths (460 and 525 nm) measured simultaneously to quantify chemiluminescence energy transfer. ABEI-biotin was synthesized by a mixed anhydride reaction and purified by TLC and HPLC. Binding of ABEI-biotin to fluorescein-avidin resulted in a quenching ofthe chemiluminescence. Chemiluminescence energy transfer was demonstrated by a 2.5-fold decrease in the ratio of blue (460 nm) to green (525 nm) light emission compared with unbound ABEI-biotin. This energy transfer was used to establish an assayfor biotin in the range 1 to 10 nM by relating the concentration of biotin to the ratio of chemiluminescence monitored at 460 and 525 nm simultaneously. The assay was capable of detecting biotin in reference sera and in patients with malabsorption syndromes and chronic alcoholism. The reference range in normal subjects was I .2 to 4.3 nmol/liter mean f SD = 2.4 1 of-0.91 nmol/liter (n = 20). The quenching of the chemiluminescence of ABEI-biotin when bound to fluorescein-avidin appeared to be the result of a direct interaction between the excited state product of ABE1 and fluorescein. CI 1986 Academic press. Inc. KEY WORDS: chemiluminescence energy transfer: biotin-avidin binding: homogeneous assay.
Chemiluminescent compounds have the necessary sensitivity of detection and stability to replace ‘25I in immunoassay and 32P in recombinant DNA technology (1,2). We have shown previously that energy transfer between a chemiluminescent labeled antigen and a fluorescent labeled antibody can be used to establish homogeneous immunoassays, not requiring a separation step, for a wide range of analytes of molecular weights 3 15 to 150,000 (3-7). However, the full potential of chemiluminescence energy transfer as an analytical tool depends on the applicability of the phenomenon to binding proteins other than antibodies. Avidin is a glycoprotein which has high affinity for biotin (& = lo-l5 M) and can be readily labeled with fluorescein without damaging its binding capacity for biotin. The aim of the work reported here was first to establish 249
whether chemiluminescence energy transfer could occur between chemiluminescent labeled biotin, in the form of aminobutylethylisoluminol (ABEI)‘-biotin, and fluoresceinavidin. The work was then intended to develop a homogeneous assay enabling the rapid measurement of biotin with sufficient sensitivity for clinical samples. MATERIALS
AND
METHODS
Fluorescein-avidin, agarose-avidin, avidin, d-biotin, N-hydroxysuccinimidobiotin, and microperoxidase were obtained from Sigma (London) Chemical Company Ltd. (Poole, Dorset, U.K.). Biotin, d-[S,9-3H(N)]-, (40 Ci/ mmol) was obtained from New England Nuclear (Boston, Mass.). All other chemicals (an’ Abbreviation used: ABEI. aminobutylethylisoluminol. 0003-2697186 $3.00 Copyright 0 1986 by Academic Press, Inc. All nghtn of reproduction in any form reserved.
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WILLIAMS
AND CAMPBELL
alytical grade) were obtained from BDH Chemicals (Poole, Dorset, U.K.). Synthesis of ABEI-biotin. ABEI was synthesized by a modified Schroder method (8). The ABEI (5 mg) dissolved in 1 ml 100 mM sodium phosphate, pH 8.5, was then reacted with Nhydroxysuccinimidobiotin (3 mg) in 1 ml dimethylformamide and incubated at room temperature for 6 h (Fig. 1). Purification of ABEI-biotin. ABEI-biotin was separated from the residual reaction products on cellulose F254 TLC using butanol:acetic acid:water (12:3:5) as solvent. It was further purified by reverse-phase HPLC to remove possible contaminating biotin since the &for biotin (0.89) is very close to that of ABEI-biotin (0.86). Reversed-phase chromatography was carried out on a Spherisorb
ODS column, 5 pm (45 mm X 150 mm) using a gradient solvent system of 30% methanol in 1% aqueous acetic acid to 95% methanol in 1% aqueous acetic acid. There was a clear separation of the biotin (retention time 6.5 min) and ABEI-biotin (retention time 12.5 min). Chemiluminescence analysis. Light emission was quantified using a specially constructed digital luminometer (7). A sample of ABEI-biotin in a total volume of 100 ~1 was added to 100 ~1 of 5 pM microperioxidase in 50 IIIM sodium phosphate, pH 7.4, containing 0.0 1% bovine serum albumin. Twenty microliters of 0.175 M H202 was then added from a spring-loaded syringe into the reaction tube placed in front of a photomultiplier tube. Chemiluminescence was recorded over the first 10 s.
0 HN
/I\,
H
H
t s
0
ri
o=
=o
cl N-OH
SUCCINYL
BIOTIN
!
AMINOBUTYL
ETHYL
ISOLUMINOL
BIOTIN
-
ABEI
FIG. I. Synthesis of chemiiuminescent labeled biotin. ABEI-biotin.
CHEMILUMINESCENCE
ENERGY TRANSFER
251
ASSAY FOR BIOTIN
Detection of chemiluminescence energy methanol:water, 90: 10; and ethyl acetate: transfer. The ABEI of biotin can transfer en- methanol, 50:50, with Rf values for ABEIergy to the fluorescein of avidin only when biotin 0.73,0.77,0.67; ABE1 0.19,0.14,0.05; biotin binds with avidin and the two labels are and biotin 0.70, 0.74, 0.5, respectively. less than 10 nm apart (5,9). ABEI-biotin inThe purified ABEI-biotin was stored as lOOcubated with fluorescein-avidin (3 mol fluo- ~1 aliquots in sodium phosphate buffer, pH rescein/mol avidin) is bound to varying de- 7.4, at -20°C and was stable for at least 12 months when no hydrolysis to biotin and grees depending upon the biotin concentration added. Competitive binding with unlabeled ABE1 was detectable. biotin causes less ABEI-biotin to bind to fluThe structure of the purified compound was orescein-avidin. This results in less energy established by field desorption mass spectransfer from blue (460 nm) to green (525 nm), trometry (Fig. 2). The molecular ion m/z of thus increasing the chemiluminescent counts the compound was 502, which is the same as at 460 nm and hence the ratio of light emitted the molecular weight of ABEI-biotin. There was good evidence of this molecular ion in at 460 nm and 525 nm. Energy transfer was detected using a specially constructed dual- several spectra, but the peak in the m/z 525 wavelength luminometer as previously de- region was not identified. This peak may be an artifact or it may be the molecular ion at scribed (7). 502 plus a sodium ion giving a peak with RESULTS m/z 525. Characterization
ofABEI-Biotin
ABEI-biotin was characterized by four parameters: (a) purity on TLC and HPLC, (b) mass spectrometry, (c) chemiluminescence, (d) binding to avidin. The synthesized ABEI-biotin migrated on cellulose TLC in the butanol:acetic acid:water solvent system as a blue fluorescent spot situated between biotin and ABEI, as expected for this derivative. There was clear separation of ABE1 (RJ = 0.63) and ABEI-biotin (RI = 0.86) there being no detectable ABE1 in the ABEI-biotin prepared from TLC. However, since the RJ value for biotin (0.89) detected using 3H-biotin was similar to that of ABEIbiotin it was necessary to further purify the ABEI-biotin using HPLC where there was a difference of 6 min in retention times (ABEIbiotin, 12.5 min; biotin, 6.5 min; ABEI, 16 min). The final preparation of ABEI-biotin used for further experiments was applied again onto HPLC and showed no contaminating ABEI or biotin peaks. Further confirmation of the purity of the preparation was demonstrated when ABEI-biotin migrated as a single blue fluorescent spot in three other solvent systems, namely, methanol:water, 50:50;
BIOTIN
(MW
100
244)
I
80 I
60. 40.
0 50 100
100
ABEI
(MW
150
200
250
276)
300 40
!
80
30
60 I
I
I 20
40.
10
20.
t
oi
I 50
,oo
t
111, 100
ABEI-BIOTIN
150 (MW
I/ 200
250
I
300
350
502)
400 E
80. 60
jt
40. 20 0,
/ i 50
, , TOO 150
, 200
, , 250300
, 350
, 400
‘,’ 450
1.1, 500550
, 800
FIG. 2. Characterization of ABEI-biotin. Mass spectrometry of synthesized compound with m/z 502 consistent with molecular weight ofABEI-biotin: m/z 525 compound unidentified.
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WILLIAMS
AND
ABEI-biotin was chemiluminescent, producing 7.0 X lOI luminescent counts/mol in 10 s at pH 7.4 when activated by microperoxidase and hydrogen peroxide (background = 400 luminescent counts in 10 s), compared with ABEI producing 2.3 X 1016 counts/mol in 10 s at pH 7.4. The binding properties of ABEI-biotin to avidin were preserved. ABEIbiotin retained its ability to compete with 3Hbiotin in a conventional radioassay using agarose-avidin as the solid phase. Also, a heterogeneous chemiluminescent assay was established with ABEI-biotin binding to agaroseavidin as the solid-phase-binding protein (Fig. 3). ABEI-biotin was thus pure, chemiluminescent, and remained biologically active, retaining its ability to bind with avidin. Chemiluminescence Energy Transfer and a Homogeneous Assay for Biotin In order to establish energy transfer, a fluorescein-avidin dose-response curve was constructed in the presence of a fixed concentration of ABEI-biotin (Fig. 4). No separation step was necessary, chemiluminescence energy transfer being monitored by measuring light emission at 460 and 525 nm simultaneously.
01.. 0
IO nM
100 nM Biotin
' MM concentration
10 UM
100 JIM
FIG. 3. Heterogeneous chemiluminescence assay for biotin. Agarose-bound avidin (50 ~1 of 0.5 u avidin/ml) was incubated with 50 pl 60 nM ABEI-biotin and 50 ~1 of standard biotin in 50 mM sodium phosphate, pH 7.4, for 2 h at 25°C. The tubes were then centrifuged and ABE1 chemiluminescence was assayedat pH 7.4 in 50 ~1 of supernatant as described under Materials and Methods. Results represent means f SD for three determinations.
CAMPBELL
12 Fluorescein-avidin
1.2 ntl
0.12
FIG. 4. Chemiluminescence energy transfer between ABEI-biotin and fluorescein-labeled avidin. ABEI-biotin (50 ~1 of 6 IIM) was incubated with 25 ~1of 50 mM sodium phosphate, pH 7.4, and 25 ~1 of fluorescein-avidin (w) (0.12 to 120 nM) or 25 ~1 of unlabeled avidin (0) for 2 h at room temperature. Chemiluminescence was initiated as described in the text and the results were expressed as a ratio of light emitted at 460 nm and 525 nm f SD for three determinations.
The ratio of chemiluminescence counts at 460 nm/525 nm decreased from 4.25 to 1.0 over a range of fluorescein-avidin from 30 pM to 30 nM (final concentration before addition of microperoxidase). A standard curve for biotin was established at pH 7.4 (Fig. 5a) using a fluorescein-avidin concentration of 1.5 nM which gave a ratio of counts at 460 nm/525 nm of approximately 1.75. Results were expressed as a ratio of luminescent counts (Fig. 5a) or as counts per 10 s for each wavelength (Fig. 5b). Competitive binding with unlabeled biotin allowed less ABEI-biotin to bind to fluorescein-avidin. Increasing concentrations of biotin therefore caused an increase in the 460 nm/525 nm ratio. A linear response was obtained for biotin concentrations over the range 1 to 10 nM. Quenching of Chemiluminescence 6-v Fluorescein-Avidin Binding of ABEI-biotin to fluorescein-avidin resulted in a reduction in chemiluminescence. The reduction at 460 nm was 90% and at 525 nm was 30% (Fig. 5b). Fluorescein was
CHEMILUMINESCENCE
Biotin
ENERGY
nM
TRANSFER
ASSAY
FOR
Blotin
BIOTIN
253
nM
FIG. 5. Homogeneous chemiluminescence energy transfer assay for biotin. Standard biotin solution (25 ~1 of 0.5 to 10 nM) in 50 mM sodium phosphate buffer, pH 7.4, was incubated with 50 ~1 ABEI-biotin (6 nM) and 25 ~1 of fluorescein-avidin (6 nM) for 2 h at room temperature. Chemihtminescence was initiated as described in the text. (a) The results were expressed as a ratio of light emitted at 460 and 525 nm k SD for three determinations. (b) Results may also be expressed as counts per 10s for each wavelength with quenching and loss of chemiluminescent counts. n , h = 460 nm; 0, X = 525 nm.
necessary for this quenching of ABEI-biotin chemiluminescence since unlabeled avidin produced no loss of chemiluminescence when it bound ABEI-biotin. The two possible explanations for this quenching of fluorescein were investigated. First, it was possible that ABEI-biotin binding to fluorescein-avidin markedly reduced the quantum yield of fluorescein. Second, it was possible that an interaction between the fluorescein and ABE1 caused a reduction in chemiluminescence quantum yield of ABEL In order to exclude the first of these possibilities, the tluorescein excitation and emission spectra were measured in the presence and absence ofbiotin (0.5 nM5 mM). Unlabeled biotin bound to fluorescein-avidin enhanced the fluorescence by a factor of 2. In contrast, ABELbiotin reduced the fluorescence at 525 nm by approximately 30% Assay of Biotin in Serum Measurement of biotin in biological specimens was complicated at high serum concentrations by quenching of ABELbiotin che-
miluminescence. This was attributable to protein in the sample (7), the effect being removed by predilution of the sample; a lo-fold predilution was sufficient to abolish this quenching effect. A major advantage of the ratio method for detecting energy transfer is that it self-compensates for any effect of biological samples on the kinetics and quantum yield of the chemiluminescence. The value of serum biotin using these conditions was 2.4 1 + 0.9 1 nM (n = 20), consistent with other assays. The assay was also capable of detecting biotin in patients with malabsorption syndromes and in chronic alcoholics. Biotin was undetectable (co.5 nmol/liter) in one patient with celiac disease and in another who had been on total parenteral nutrition for over 6 weeks. DISCUSSION
Biotin is an essential cofactor for at least three mitochondrial carboxylases: propionyl CoA, 3-methyl crotonyl CoA carboxvlase. and pyruvate carboxylase.-Biotin-responsive multiple carboxylase deficiency is clinically characterized by alopecia, erythematous rash,
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AND CAMPBELL
ataxia, acidosis, and low serum biotin concentrations ( 10). Low serum biotin concentrations have also been demonstrated as a complication of parenteral nutrition (11). Only a few cases of spontaneous biotin deficiency have been reported, caused by excessive intake of raw egg white which contains the biotin binder avidin. Current methods for biotin estimations include microbiological assays ( 12,13) of poor specificity and radiolabeled competitive binding assays ( 14,15). Schroder et al. ( 16) developed a competitive protein binding assay for biotin monitored by chemiluminescence using isoluminol-labeled biotin. The method was sensitive to biotin concentrations of 50 nM but not sensitive enough for serum estimations. The results described in this paper demonstrate that chemiluminescence energy transfer can occur between an ABEI-labeled ligand and a fluorescein-labeled binding protein other than antibody. This phenomenon has been used to establish a homogeneous assay for biotin which was considerably more sensitive than reported fluorimetric assays (17,18). The detection limit of the assay for biotin reported here was approximately 25 fmol in a 25-~1 sample (1 nM). This is a value similar to that obtained by conventional radioassays and bioassays. The advantages of the chemiluminescence energy transfer assay are that no preincubation of sample nor a separation step is required, and the inconvenience of handling radioactive materials is eliminated. A striking feature of these experiments was the demonstration of more than 90% quenching of chemiluminescence counts when ABEIbiotin bound to fluorescein-avidin. This phenomenon was not observed with fluoresceinlabeled antibody bound to a range of ABEIlabeled antigens (MW 315-150,000) such as progesterone, CAMP, cGMP, adenosine, and IgG (5). The quenching was not observed with unlabeled avidin. Furthermore, it has been reported that avidin bound to an isoluminollabeled biotin slightly enhanced chemiluminescence (16). The question therefore arose whether this quenching when ABEI-biotin bound to fluorescein-avidin was caused by a
decrease in the fluorescence quantum yield of the fluorescein or whether an interaction between the fluorescein and ABEI molecules caused a reduction in the chemiluminescence quantum yield of ABEI. Although a small quenching of fluorescein fluorescence at 525 nm (- 30%) was observed when fluoresceinavidin bound ABEI-biotin, this was insufficient to explain the quenching of the chemiluminescence. The enhancing effect of biotin on fluorescein-avidin fluorescence and yet reduction of fluorescence caused by ABEI-biotin strongly suggest that the excited product of the ABE1 chemiluminescence reaction is close enough to interact directly with fluorescein, resulting in an overall reduction in chemiluminescence quantum yield. Mathematical description of radiationless resonance energy transfer was first described by Forster in 1948 (9). Effective energy transfer requires that the donor and acceptor molecules are within 5-10 nm (9,19) of each other. Avidin (MW 66,000) is labeled with three fluorescein molecules and has four biotin binding sites grouped in two pairs at opposite ends of the avidin molecule (20). The quenching effect suggests that the ABEI on biotin may be close enough to a fluorescein molecule on the avidin such that direct molecular orbital interaction can take place. ACKNOWLEDGMENT We thank Mr. M. Rossiter, Department of Chemistry, University College, Cardiff for mass spectroscopy of synthesized compounds. REFERENCES 1. Campbell, A. K., Hallett, M. B., and Weeks, I. (1985) Methods Biochem. Anal. 31,3 1l-4 16. 2. Weeks, I., Beheshti, I., McCapra, F., Campbell, A. K., and Woodhead, J. S. (1983) Clin. Chem. 29,14741479.
Weeks, I., Campbell, A. K., and Woodhead, J. S. (1983) C’lin. Chem.29, 1480-1483. 4. Pate], A., Davies, C. J., Campbell. A. K., and McCapra, F. (1983) Anal. Biochem. 129, 162-169. 5. Campbell, A. K., and Pate], A. (1983) Biochem. J. 216, 185-194. 3.
CHEMILUMINESCENCE
ENERGY TRANSFER
6. Patel, A., and Campbell, A. K. (1983) C/in. Chem. 29, 1604-1608. 7. Campbell, A. K., Roberts, A., and Patel, A. ( 1985) in Alternative Immunoassays (Collins, W. P., ed.), pp. 153-l 83, Wiley, New York. 8. Schrader, H. R.. Boguslaski, R. C., Carrico, R. J., and Butler, T. R. (1978) in Methods in Enzymology (DeLuca, M. A., ed.), Vol. 57, pp. 424-445, Academic Press, New York. 9. Forster, T. (1948) Ann. Phys. (Leipzig) 2, 55-15. 10. Theone, J., Baker, H., Yoshino, M., and Sweetman, L. (198 I) N. Engl. J. Med. 304, 817-820. I I. Mock, D. M., Delorimer, A. A., Liebman, W. M.. Sweetman, L., and Baker, H. (1981) N. Engl. J. Med. 304,820-823. 12. Bhagavan, H. N., and Coursin, D. B. ( 1967) Amer. J. Clin. Nutr. 20, 903-906.
ASSAY FOR BIOTIN
255
13. Baker, H.. Frank, O., Matoich, V. B., Pasher, I., Aaronson, S., Hunter, S. H., and Sobotka, H. (1962) Anal. Biochem. 3,3 l-39. 14. Sanghri, R. S., Lemons, R. M., Baker, H.. and Theone, J. G. (1982) Clin. Chim. Acta 124,85-90. 15. Horsburgh, T.. and Gompertz, D. ( 1978) Clin. Chim. .4cta 82, 2 15-223. 16. SchrMer, H. R., Vogelhut. P. O., Carrico, R. J.. Boguslaski, R. C., and Buckler, R. T. (1976) Anal. Chem. 81, 1933-1937. 17. Lin, H. J., and Kirsch. J. F. (1977) Anal. Biochem. S&442-446. 18. Al-Hakiem. M. H. H., Landon. J., Smith, D. S., and Nargessi, R. D. (1981) Anal. Biochem. 116, 264261. 19. Stryer, L. (1978) Annu. Rev. Biochem. 41, 819-846. 20. Green, N. M. (1975) Adv. Protein Chem. 29,85-133.
BIOCHEMISTRY
155,249-255
(1986)
A Homogeneous Assay for Biotin Based on Chemiluminescence Energy Transfer E.JOANWILLIAMS*
ANDANTHONYK.CAMPBELL
Departmetzf qf Medical Biochemistry, *University Hospilal of Wales, and Universit~~of Uhles College of Medicine. Heath Park. CardiJ CF4 4XW, United Kingdom Received October 23. 1985 Chemiluminescence energy transfer between aminobutylethylisoluminol (ABE&biotin and fluorescein-avidin was investigated in order to establish a homogeneous assay for serum biotin in the physiological range. ABE1 chemiluminescence was measured at pH 7.4 using microperoxidase-hydrogen peroxide and the chemiluminescence at two wavelengths (460 and 525 nm) measured simultaneously to quantify chemiluminescence energy transfer. ABEI-biotin was synthesized by a mixed anhydride reaction and purified by TLC and HPLC. Binding of ABEI-biotin to fluorescein-avidin resulted in a quenching ofthe chemiluminescence. Chemiluminescence energy transfer was demonstrated by a 2.5-fold decrease in the ratio of blue (460 nm) to green (525 nm) light emission compared with unbound ABEI-biotin. This energy transfer was used to establish an assayfor biotin in the range 1 to 10 nM by relating the concentration of biotin to the ratio of chemiluminescence monitored at 460 and 525 nm simultaneously. The assay was capable of detecting biotin in reference sera and in patients with malabsorption syndromes and chronic alcoholism. The reference range in normal subjects was I .2 to 4.3 nmol/liter mean f SD = 2.4 1 of-0.91 nmol/liter (n = 20). The quenching of the chemiluminescence of ABEI-biotin when bound to fluorescein-avidin appeared to be the result of a direct interaction between the excited state product of ABE1 and fluorescein. CI 1986 Academic press. Inc. KEY WORDS: chemiluminescence energy transfer: biotin-avidin binding: homogeneous assay.
Chemiluminescent compounds have the necessary sensitivity of detection and stability to replace ‘25I in immunoassay and 32P in recombinant DNA technology (1,2). We have shown previously that energy transfer between a chemiluminescent labeled antigen and a fluorescent labeled antibody can be used to establish homogeneous immunoassays, not requiring a separation step, for a wide range of analytes of molecular weights 3 15 to 150,000 (3-7). However, the full potential of chemiluminescence energy transfer as an analytical tool depends on the applicability of the phenomenon to binding proteins other than antibodies. Avidin is a glycoprotein which has high affinity for biotin (& = lo-l5 M) and can be readily labeled with fluorescein without damaging its binding capacity for biotin. The aim of the work reported here was first to establish 249
whether chemiluminescence energy transfer could occur between chemiluminescent labeled biotin, in the form of aminobutylethylisoluminol (ABEI)‘-biotin, and fluoresceinavidin. The work was then intended to develop a homogeneous assay enabling the rapid measurement of biotin with sufficient sensitivity for clinical samples. MATERIALS
AND
METHODS
Fluorescein-avidin, agarose-avidin, avidin, d-biotin, N-hydroxysuccinimidobiotin, and microperoxidase were obtained from Sigma (London) Chemical Company Ltd. (Poole, Dorset, U.K.). Biotin, d-[S,9-3H(N)]-, (40 Ci/ mmol) was obtained from New England Nuclear (Boston, Mass.). All other chemicals (an’ Abbreviation used: ABEI. aminobutylethylisoluminol. 0003-2697186 $3.00 Copyright 0 1986 by Academic Press, Inc. All nghtn of reproduction in any form reserved.
250
WILLIAMS
AND CAMPBELL
alytical grade) were obtained from BDH Chemicals (Poole, Dorset, U.K.). Synthesis of ABEI-biotin. ABEI was synthesized by a modified Schroder method (8). The ABEI (5 mg) dissolved in 1 ml 100 mM sodium phosphate, pH 8.5, was then reacted with Nhydroxysuccinimidobiotin (3 mg) in 1 ml dimethylformamide and incubated at room temperature for 6 h (Fig. 1). Purification of ABEI-biotin. ABEI-biotin was separated from the residual reaction products on cellulose F254 TLC using butanol:acetic acid:water (12:3:5) as solvent. It was further purified by reverse-phase HPLC to remove possible contaminating biotin since the &for biotin (0.89) is very close to that of ABEI-biotin (0.86). Reversed-phase chromatography was carried out on a Spherisorb
ODS column, 5 pm (45 mm X 150 mm) using a gradient solvent system of 30% methanol in 1% aqueous acetic acid to 95% methanol in 1% aqueous acetic acid. There was a clear separation of the biotin (retention time 6.5 min) and ABEI-biotin (retention time 12.5 min). Chemiluminescence analysis. Light emission was quantified using a specially constructed digital luminometer (7). A sample of ABEI-biotin in a total volume of 100 ~1 was added to 100 ~1 of 5 pM microperioxidase in 50 IIIM sodium phosphate, pH 7.4, containing 0.0 1% bovine serum albumin. Twenty microliters of 0.175 M H202 was then added from a spring-loaded syringe into the reaction tube placed in front of a photomultiplier tube. Chemiluminescence was recorded over the first 10 s.
0 HN
/I\,
H
H
t s
0
ri
o=
=o
cl N-OH
SUCCINYL
BIOTIN
!
AMINOBUTYL
ETHYL
ISOLUMINOL
BIOTIN
-
ABEI
FIG. I. Synthesis of chemiiuminescent labeled biotin. ABEI-biotin.
CHEMILUMINESCENCE
ENERGY TRANSFER
251
ASSAY FOR BIOTIN
Detection of chemiluminescence energy methanol:water, 90: 10; and ethyl acetate: transfer. The ABEI of biotin can transfer en- methanol, 50:50, with Rf values for ABEIergy to the fluorescein of avidin only when biotin 0.73,0.77,0.67; ABE1 0.19,0.14,0.05; biotin binds with avidin and the two labels are and biotin 0.70, 0.74, 0.5, respectively. less than 10 nm apart (5,9). ABEI-biotin inThe purified ABEI-biotin was stored as lOOcubated with fluorescein-avidin (3 mol fluo- ~1 aliquots in sodium phosphate buffer, pH rescein/mol avidin) is bound to varying de- 7.4, at -20°C and was stable for at least 12 months when no hydrolysis to biotin and grees depending upon the biotin concentration added. Competitive binding with unlabeled ABE1 was detectable. biotin causes less ABEI-biotin to bind to fluThe structure of the purified compound was orescein-avidin. This results in less energy established by field desorption mass spectransfer from blue (460 nm) to green (525 nm), trometry (Fig. 2). The molecular ion m/z of thus increasing the chemiluminescent counts the compound was 502, which is the same as at 460 nm and hence the ratio of light emitted the molecular weight of ABEI-biotin. There was good evidence of this molecular ion in at 460 nm and 525 nm. Energy transfer was detected using a specially constructed dual- several spectra, but the peak in the m/z 525 wavelength luminometer as previously de- region was not identified. This peak may be an artifact or it may be the molecular ion at scribed (7). 502 plus a sodium ion giving a peak with RESULTS m/z 525. Characterization
ofABEI-Biotin
ABEI-biotin was characterized by four parameters: (a) purity on TLC and HPLC, (b) mass spectrometry, (c) chemiluminescence, (d) binding to avidin. The synthesized ABEI-biotin migrated on cellulose TLC in the butanol:acetic acid:water solvent system as a blue fluorescent spot situated between biotin and ABEI, as expected for this derivative. There was clear separation of ABE1 (RJ = 0.63) and ABEI-biotin (RI = 0.86) there being no detectable ABE1 in the ABEI-biotin prepared from TLC. However, since the RJ value for biotin (0.89) detected using 3H-biotin was similar to that of ABEIbiotin it was necessary to further purify the ABEI-biotin using HPLC where there was a difference of 6 min in retention times (ABEIbiotin, 12.5 min; biotin, 6.5 min; ABEI, 16 min). The final preparation of ABEI-biotin used for further experiments was applied again onto HPLC and showed no contaminating ABEI or biotin peaks. Further confirmation of the purity of the preparation was demonstrated when ABEI-biotin migrated as a single blue fluorescent spot in three other solvent systems, namely, methanol:water, 50:50;
BIOTIN
(MW
100
244)
I
80 I
60. 40.
0 50 100
100
ABEI
(MW
150
200
250
276)
300 40
!
80
30
60 I
I
I 20
40.
10
20.
t
oi
I 50
,oo
t
111, 100
ABEI-BIOTIN
150 (MW
I/ 200
250
I
300
350
502)
400 E
80. 60
jt
40. 20 0,
/ i 50
, , TOO 150
, 200
, , 250300
, 350
, 400
‘,’ 450
1.1, 500550
, 800
FIG. 2. Characterization of ABEI-biotin. Mass spectrometry of synthesized compound with m/z 502 consistent with molecular weight ofABEI-biotin: m/z 525 compound unidentified.
252
WILLIAMS
AND
ABEI-biotin was chemiluminescent, producing 7.0 X lOI luminescent counts/mol in 10 s at pH 7.4 when activated by microperoxidase and hydrogen peroxide (background = 400 luminescent counts in 10 s), compared with ABEI producing 2.3 X 1016 counts/mol in 10 s at pH 7.4. The binding properties of ABEI-biotin to avidin were preserved. ABEIbiotin retained its ability to compete with 3Hbiotin in a conventional radioassay using agarose-avidin as the solid phase. Also, a heterogeneous chemiluminescent assay was established with ABEI-biotin binding to agaroseavidin as the solid-phase-binding protein (Fig. 3). ABEI-biotin was thus pure, chemiluminescent, and remained biologically active, retaining its ability to bind with avidin. Chemiluminescence Energy Transfer and a Homogeneous Assay for Biotin In order to establish energy transfer, a fluorescein-avidin dose-response curve was constructed in the presence of a fixed concentration of ABEI-biotin (Fig. 4). No separation step was necessary, chemiluminescence energy transfer being monitored by measuring light emission at 460 and 525 nm simultaneously.
01.. 0
IO nM
100 nM Biotin
' MM concentration
10 UM
100 JIM
FIG. 3. Heterogeneous chemiluminescence assay for biotin. Agarose-bound avidin (50 ~1 of 0.5 u avidin/ml) was incubated with 50 pl 60 nM ABEI-biotin and 50 ~1 of standard biotin in 50 mM sodium phosphate, pH 7.4, for 2 h at 25°C. The tubes were then centrifuged and ABE1 chemiluminescence was assayedat pH 7.4 in 50 ~1 of supernatant as described under Materials and Methods. Results represent means f SD for three determinations.
CAMPBELL
12 Fluorescein-avidin
1.2 ntl
0.12
FIG. 4. Chemiluminescence energy transfer between ABEI-biotin and fluorescein-labeled avidin. ABEI-biotin (50 ~1 of 6 IIM) was incubated with 25 ~1of 50 mM sodium phosphate, pH 7.4, and 25 ~1 of fluorescein-avidin (w) (0.12 to 120 nM) or 25 ~1 of unlabeled avidin (0) for 2 h at room temperature. Chemiluminescence was initiated as described in the text and the results were expressed as a ratio of light emitted at 460 nm and 525 nm f SD for three determinations.
The ratio of chemiluminescence counts at 460 nm/525 nm decreased from 4.25 to 1.0 over a range of fluorescein-avidin from 30 pM to 30 nM (final concentration before addition of microperoxidase). A standard curve for biotin was established at pH 7.4 (Fig. 5a) using a fluorescein-avidin concentration of 1.5 nM which gave a ratio of counts at 460 nm/525 nm of approximately 1.75. Results were expressed as a ratio of luminescent counts (Fig. 5a) or as counts per 10 s for each wavelength (Fig. 5b). Competitive binding with unlabeled biotin allowed less ABEI-biotin to bind to fluorescein-avidin. Increasing concentrations of biotin therefore caused an increase in the 460 nm/525 nm ratio. A linear response was obtained for biotin concentrations over the range 1 to 10 nM. Quenching of Chemiluminescence 6-v Fluorescein-Avidin Binding of ABEI-biotin to fluorescein-avidin resulted in a reduction in chemiluminescence. The reduction at 460 nm was 90% and at 525 nm was 30% (Fig. 5b). Fluorescein was
CHEMILUMINESCENCE
Biotin
ENERGY
nM
TRANSFER
ASSAY
FOR
Blotin
BIOTIN
253
nM
FIG. 5. Homogeneous chemiluminescence energy transfer assay for biotin. Standard biotin solution (25 ~1 of 0.5 to 10 nM) in 50 mM sodium phosphate buffer, pH 7.4, was incubated with 50 ~1 ABEI-biotin (6 nM) and 25 ~1 of fluorescein-avidin (6 nM) for 2 h at room temperature. Chemihtminescence was initiated as described in the text. (a) The results were expressed as a ratio of light emitted at 460 and 525 nm k SD for three determinations. (b) Results may also be expressed as counts per 10s for each wavelength with quenching and loss of chemiluminescent counts. n , h = 460 nm; 0, X = 525 nm.
necessary for this quenching of ABEI-biotin chemiluminescence since unlabeled avidin produced no loss of chemiluminescence when it bound ABEI-biotin. The two possible explanations for this quenching of fluorescein were investigated. First, it was possible that ABEI-biotin binding to fluorescein-avidin markedly reduced the quantum yield of fluorescein. Second, it was possible that an interaction between the fluorescein and ABE1 caused a reduction in chemiluminescence quantum yield of ABEL In order to exclude the first of these possibilities, the tluorescein excitation and emission spectra were measured in the presence and absence ofbiotin (0.5 nM5 mM). Unlabeled biotin bound to fluorescein-avidin enhanced the fluorescence by a factor of 2. In contrast, ABELbiotin reduced the fluorescence at 525 nm by approximately 30% Assay of Biotin in Serum Measurement of biotin in biological specimens was complicated at high serum concentrations by quenching of ABELbiotin che-
miluminescence. This was attributable to protein in the sample (7), the effect being removed by predilution of the sample; a lo-fold predilution was sufficient to abolish this quenching effect. A major advantage of the ratio method for detecting energy transfer is that it self-compensates for any effect of biological samples on the kinetics and quantum yield of the chemiluminescence. The value of serum biotin using these conditions was 2.4 1 + 0.9 1 nM (n = 20), consistent with other assays. The assay was also capable of detecting biotin in patients with malabsorption syndromes and in chronic alcoholics. Biotin was undetectable (co.5 nmol/liter) in one patient with celiac disease and in another who had been on total parenteral nutrition for over 6 weeks. DISCUSSION
Biotin is an essential cofactor for at least three mitochondrial carboxylases: propionyl CoA, 3-methyl crotonyl CoA carboxvlase. and pyruvate carboxylase.-Biotin-responsive multiple carboxylase deficiency is clinically characterized by alopecia, erythematous rash,
254
WILLIAMS
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ataxia, acidosis, and low serum biotin concentrations ( 10). Low serum biotin concentrations have also been demonstrated as a complication of parenteral nutrition (11). Only a few cases of spontaneous biotin deficiency have been reported, caused by excessive intake of raw egg white which contains the biotin binder avidin. Current methods for biotin estimations include microbiological assays ( 12,13) of poor specificity and radiolabeled competitive binding assays ( 14,15). Schroder et al. ( 16) developed a competitive protein binding assay for biotin monitored by chemiluminescence using isoluminol-labeled biotin. The method was sensitive to biotin concentrations of 50 nM but not sensitive enough for serum estimations. The results described in this paper demonstrate that chemiluminescence energy transfer can occur between an ABEI-labeled ligand and a fluorescein-labeled binding protein other than antibody. This phenomenon has been used to establish a homogeneous assay for biotin which was considerably more sensitive than reported fluorimetric assays (17,18). The detection limit of the assay for biotin reported here was approximately 25 fmol in a 25-~1 sample (1 nM). This is a value similar to that obtained by conventional radioassays and bioassays. The advantages of the chemiluminescence energy transfer assay are that no preincubation of sample nor a separation step is required, and the inconvenience of handling radioactive materials is eliminated. A striking feature of these experiments was the demonstration of more than 90% quenching of chemiluminescence counts when ABEIbiotin bound to fluorescein-avidin. This phenomenon was not observed with fluoresceinlabeled antibody bound to a range of ABEIlabeled antigens (MW 315-150,000) such as progesterone, CAMP, cGMP, adenosine, and IgG (5). The quenching was not observed with unlabeled avidin. Furthermore, it has been reported that avidin bound to an isoluminollabeled biotin slightly enhanced chemiluminescence (16). The question therefore arose whether this quenching when ABEI-biotin bound to fluorescein-avidin was caused by a
decrease in the fluorescence quantum yield of the fluorescein or whether an interaction between the fluorescein and ABEI molecules caused a reduction in the chemiluminescence quantum yield of ABEI. Although a small quenching of fluorescein fluorescence at 525 nm (- 30%) was observed when fluoresceinavidin bound ABEI-biotin, this was insufficient to explain the quenching of the chemiluminescence. The enhancing effect of biotin on fluorescein-avidin fluorescence and yet reduction of fluorescence caused by ABEI-biotin strongly suggest that the excited product of the ABE1 chemiluminescence reaction is close enough to interact directly with fluorescein, resulting in an overall reduction in chemiluminescence quantum yield. Mathematical description of radiationless resonance energy transfer was first described by Forster in 1948 (9). Effective energy transfer requires that the donor and acceptor molecules are within 5-10 nm (9,19) of each other. Avidin (MW 66,000) is labeled with three fluorescein molecules and has four biotin binding sites grouped in two pairs at opposite ends of the avidin molecule (20). The quenching effect suggests that the ABEI on biotin may be close enough to a fluorescein molecule on the avidin such that direct molecular orbital interaction can take place. ACKNOWLEDGMENT We thank Mr. M. Rossiter, Department of Chemistry, University College, Cardiff for mass spectroscopy of synthesized compounds. REFERENCES 1. Campbell, A. K., Hallett, M. B., and Weeks, I. (1985) Methods Biochem. Anal. 31,3 1l-4 16. 2. Weeks, I., Beheshti, I., McCapra, F., Campbell, A. K., and Woodhead, J. S. (1983) Clin. Chem. 29,14741479.
Weeks, I., Campbell, A. K., and Woodhead, J. S. (1983) C’lin. Chem.29, 1480-1483. 4. Pate], A., Davies, C. J., Campbell. A. K., and McCapra, F. (1983) Anal. Biochem. 129, 162-169. 5. Campbell, A. K., and Pate], A. (1983) Biochem. J. 216, 185-194. 3.
CHEMILUMINESCENCE
ENERGY TRANSFER
6. Patel, A., and Campbell, A. K. (1983) C/in. Chem. 29, 1604-1608. 7. Campbell, A. K., Roberts, A., and Patel, A. ( 1985) in Alternative Immunoassays (Collins, W. P., ed.), pp. 153-l 83, Wiley, New York. 8. Schrader, H. R.. Boguslaski, R. C., Carrico, R. J., and Butler, T. R. (1978) in Methods in Enzymology (DeLuca, M. A., ed.), Vol. 57, pp. 424-445, Academic Press, New York. 9. Forster, T. (1948) Ann. Phys. (Leipzig) 2, 55-15. 10. Theone, J., Baker, H., Yoshino, M., and Sweetman, L. (198 I) N. Engl. J. Med. 304, 817-820. I I. Mock, D. M., Delorimer, A. A., Liebman, W. M.. Sweetman, L., and Baker, H. (1981) N. Engl. J. Med. 304,820-823. 12. Bhagavan, H. N., and Coursin, D. B. ( 1967) Amer. J. Clin. Nutr. 20, 903-906.
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13. Baker, H.. Frank, O., Matoich, V. B., Pasher, I., Aaronson, S., Hunter, S. H., and Sobotka, H. (1962) Anal. Biochem. 3,3 l-39. 14. Sanghri, R. S., Lemons, R. M., Baker, H.. and Theone, J. G. (1982) Clin. Chim. Acta 124,85-90. 15. Horsburgh, T.. and Gompertz, D. ( 1978) Clin. Chim. .4cta 82, 2 15-223. 16. SchrMer, H. R., Vogelhut. P. O., Carrico, R. J.. Boguslaski, R. C., and Buckler, R. T. (1976) Anal. Chem. 81, 1933-1937. 17. Lin, H. J., and Kirsch. J. F. (1977) Anal. Biochem. S&442-446. 18. Al-Hakiem. M. H. H., Landon. J., Smith, D. S., and Nargessi, R. D. (1981) Anal. Biochem. 116, 264261. 19. Stryer, L. (1978) Annu. Rev. Biochem. 41, 819-846. 20. Green, N. M. (1975) Adv. Protein Chem. 29,85-133.