Journal of Immunological Methods, 129 (1990) 159-164


Elsevier JIM05556

Development of an enzyme-linked immunosorbent assay for caffeine S.A. Fickling 1, S.M. H a m p t o n 1, D. T e a l e 2, B.A. M i d d l e t o n a n d V. M a r k s l Department of Biochemistry, Division of Clinical Biochemistry, University of Surrey, Guildfora~ Surrey GU2 5XH, U.K., and 2 Department of Clinical Biochemistry and Nutrition, St. Luke's Hospital, Guildford, Surrey GU1 3 WT, U.K.

(Received 12 May 1989, revisedreceived2 January 1990, accepted 19 January 1990)

A competitive enzyme-linked immunosorbent assay suitable for the measurement of caffeine in plasma and serum has been developed. Sheep immunised with an immunogen prepared by coupling 7-(5carboxypenthyl)l,3-dimethylxanthine to egg albumin produced antibodies with little crossreactivity with the metabolites of caffeine. The enzyme label was prepared by coupling 7-(5-carboxypentyl)-l,3-dimethylxanthine to peroxidase using the mixed anhydride method. The assay, which has a sensitivity of 0.01 /~mol/1, permits direct measurement of caffeine in plasma and serum samples. 50 plasma samples measured by ELISA and by an established radioimmunoassay showed a correlation of r = 0.97 ( P < 0.001). Key words: Caffeine; ELISA; Cross-reactivity


Caffeine (1,3,7-trimethylxanthine) is one of the most widely consumed pharmacologically active compounds. It occurs naturally in a number of beverages and is readily extracted from coffee and cocoa beans, tea leaves and kola nuts. The pharmacological actions of caffeine include stimulation of the central nervous system, respiration, cardiac muscle and skeletal muscle. Caffeine also acts on the renal tubules to produce diuresis, relaxes smooth muscle and causes coronary dilatation. Caffeine's main effect on the central nervous system is to produce a rapid and clearer flow of thought and to allay drowsiness and fatigue. High doses of caffeine cause headache, tremors, nervousness and irritability. In the body caffeine

is metabolised initially by demethylation to produce paraxanthine (1,7-dimethylxanthine) and other water soluble excretory products. Methods available for measuring caffeine in serum include gas liquid chromatography (Gurtoo and Phillips, 1973), radioimmunoassay (Cook et al., 1976) and high pressure liquid chromatography (Scott et al., 1986) which use expensive equipment, are time consuming and technically demanding. We have, therefore, developed an enzyme-linked immunosorbent assay (ELISA) for measuring caffeine in serum which does not suffer from these drawbacks and is suitable for mass screening for hypercaffeinism, an uncommon but curable disease.

Materials and methods Correspondence to: S.A. Ficlding/S.M. Hampton, Depart-

ment of Biochemistry, Division of Clinical Biochemistry,University of Surrey, Guildford, Surrey GU2 5XH, U.K.


A microtitre plate washer and plate reader were purchased from Flow Laboratories (Rickmans-

0022-1759/90/$03.50 © 1990 ElsevierScience Pubfishers B.V. (BiomedicalDivision)

160 worth, Hertfordshire) and a spectrophotometer from Cecil Instruments (Cambridge). A Probus Quatro sample processor was supplied by Quatro Biomedical (Cheshire).

Materials Polystyrene microtitre plates were obtained from Dynatech Laboratories (Billinghurst, Sussex). Orthophenylenediamine, Sephadex G-75, isobutylchloroformate, caffeine and its metabolites were obtained from Sigma (Poole, Dorset). Horseradish peroxidase was obtained from Biozyme (Gwent, Wales); B C G from Difco Laboratories (Greenford, Middlesex); W h a t m a n D E 52 from W h a t m a n (Springfield Mill, Kent) and Zeits filters from H. Erban (Suffolk). All other chemicals were obtained from B D H Chemical (Poole, Dorset). Theophylline was chemically modified by the method of Cook et al. (1976) to produce 7-(5-carboxypenthyl)-l,3-dimethylxanthine and is subsequently referred to as the caffeine ester.

Caffeine ester-protein conjugate The mixed anhydride method (Erlanger et al., 1957, 1959) was used to conjugate the caffeine ester to ovalbumin. The whole reaction was carried out at 10 ° C. Caffeine ester (2.4 mmol) was dissolved in 15 ml redistilled dioxan which had been stored over potassium hydroxide. Tri-nbutylamine (565 /~1) was added with stirring. Isobutylchloroformate (342 /~1) was then added and the reaction allowed to proceed for 30 min. Ovalbumin (0.08 mmol), dissolved in 50 ml distilled water was added to the caffeine ester solution with continuous stirring for 4 h. The mixture was dialyzed against distilled water and aliquots of dialysate were taken from estimation of their caffeine-ester content by radioimmunoassay. The amount of caffeine-ester unaccounted for in the dialysate amounted to 1 mmol. The retentate was divided into aliquots containing 0.017 m m o l of the conjugate which were lyophilised and stored at 4 ° C.

Immunisation procedure Two Suffolk and two Soay sheep were immunised. 5 mg conjugate and 0.1 ml B C G vaccine in 1 ml sterile water were emulsified with 1 ml of non-ulcerative adjuvant (Morris) and injected in-

tramuscularly into the upper legs. The animals were boosted with half the priming dose containing no BCG, approximately nine months later but showed little immunological response. They were given a further 'priming' dose 3 months later. Blood was collected from the jugular vein 9 days following the prime, reprime or booster injections and thereafter at one monthly intervals, allowed to clot and the serum separate. It was stored at 4 ° C with the addition of 1 g/1 sodium azide.

Purification of antisera The immunoglobulin fraction was obtained from the antisera after repriming the animals. Each serum was stirred slowly on a magnetic stirrer. Saturated a m m o n i u m sulphate placed in a microburette at room temperature was added dropwise until half the original volume of the serum had been added. The mixture was centrifuged at 2500 r p m for 10 min and the supernatant discarded. The precipitate was dissolved in 0.03 M phosphate buffer, p H 7.4, and dialysed against the same buffer for 48 h. After dialysis, the protein solution was loaded onto a W h a t m a n D E 52 ionexchange column, and eluted with 0.03 M phosphate buffer, p H 7.4, 20 ml fractions being collected. The optical density of each fraction was measured at 280 nm. The immunoglobulins were eluted in fraction numbers 8-15 and these were pooled.

Preparation of peroxidase caffeine ester label The mixed anhydride (Erlanger et al., 1957, 1959) method was again used to conjugate the caffeine ester to peroxidase. Caffeine ester (83 /~mol) and 114/xl tri-n-butylamine were dissolved in 100/~1 redistilled dimethylformamide. The solution was cooled to 1 0 ° C in an iced water bath. Isobutylchloroformate (31 /tl) was added and the reaction allowed to proceed for 30 min at 10 * C. A solution of 0.625 /xmol of peroxidase dissolved in 2 ml distilled water containing 100 /~1 1 M sodium hydroxide was added to the reaction mixture with continuous stirring. The mixture was left in an ice bath for 4 h at 1 0 ° C and then overnight at 4 ° C . The reaction mixture was loaded onto a Sephadex G-75 column and ehited with 0.15 M phosphate buffered saline,




STEP I ' Coat plate w l t h

purified caffeine antibody (5uglml) diluted in O.IM blcarbonate / carbonate ph9.6




STEP 2 ' Add caffeine standards / plasma samples plus enzyme labelled caffeine










Add substrate

Stop the r e a c t i o n w i t h d i l u t e d s u l p h u r i c acid. Read o p t i c a l d e n s i t y o f w e l l s at 49Onto. ° PDSGT- P h o s p h a t e b u f f e r e d saline pI~7.6 c o n t a i n i n g 0.1% g e l a t i n and 0.05% T w e e n . o

' I n c u b a t e at 37 C f o r one h o u r in a m o i s t c h a m b e r .

Fig. 1. Competitivecaffeineenzyme-linkedimmunosorbentassayprotocol.


p H 7.4 containing 0.02% w / v thiomersal, 30 1 ml fractions being collected. The optical density of each fraction was measured at 280 n m and 420 nm.

Competitive ELISA The ELISA procedure was carried out using a Probus Quatro sample processor. A microtitre plate was coated with 5 # g / m l ion exchange purified caffeine antibody (batch no. H P / S / 3 4 0 5 R P D ) diluted in 0.1 M b i c a r b o n a t e / c a r b o n a t e buffer p H 9.6 (see Fig. 1). The plate was incubated at 37 ° C in a moist chamber and washed with 0.15 M phosphate-buffered saline, p H 7.4, containing 0.05% v / v Tween 20 and 0.1% w / v gelatin (PBSGT). All further dilutions and washing were carried out with this buffer unless otherwise stated. The samples diluted 1 / 1 0 in P B S G T were added to the appropriate well (5 /xl/well). Standard solutions of caffeine in the range 0 - 2 5 /Lmol/l (nine standards) were prepared using caffeine-free plasma diluted 1 / 1 0 with assay buffer. The addition of caffeine-free serum to the standard curve was necessary since it depressed the curve compared with a standard curve set up in buffer. After the addition of either samples or standards, 95 /xl of PBSGT plus peroxidaselabelled caffeine ester diluted 1/100,000 (100 /d/well) were added to all wells. The plate was incubated at 3 7 ° C in a moist chamber, for a further hour, washed and 150 pl of substrate (orthophenylenediamine) added to all wells. After a further 30 min incubation in a moist chamber at 37 ° C the reaction was stopped by the addition of 2.5 M sulphuric acid (50 #1). The plate was read using a Flow automated 'Multiscan Plus' at 492 nm.

Sample collection Venous blood was collected into lithium heparin tubes and centrifuged within 15 min of collection. The plasma was frozen and stored at - 2 0 ° C until required. 50 samples were obtained in this way and analysed in duplicate by radioimmunoassay and ELISA.

Clinical samples Plasma caffeine levels were measured in samples collected from 250 patients attending the outpatients department of St. Luke's Hospital, Guild ford.


Antiserum production: After repriming, all four immunised sheep produced an adequate immune response after only nine days. Antiserum collected from sheep ( H P / S / 3 4 0 5 - R P D ) 9 days after repriming, was used to establish the assay described here.

Assay validation A standard curve obtained by this caffeine ELISA method is shown in Fig. 2. The sensitivity of the assay, defined as two standard deviations from zero, was 0.01 # m o l / l . The intra-assay coefficient of variation, at a mean plasma level of 5.6 /tmol/l, was 9% (n = 10); at 20 p m o l / l it was 8% (n = 6) and at 30 # m o l / L it was 5% (n = 6) after the results had been multiplied by the appropriate


E_ Charcoal treated plasma Plasma obtained from volunteers who had fasted overnight was pooled. 100 ml were mixed overnight, using a magnetic stirrer, with 20 g activated charcoal at 4 ° C . The supernatant remaining after centrifugation (1600 × g for 15 min at 4 ° C) was filtered through a Zeits filter (Grade H P / K S ) . Aliquots of the filtrate were stored at - 20 o C until required.


z o







Caffeine pmol/l_ F i g . 2. C a f f e i n e s t a n d a r d c u r v e , m e a n ± S D ,




% crossreactivity

Theophylline Theobromine Uric acid Guanine 1,7-dimethyl uric acid 1-methyl uric acid 1-methyl xanthine 1,3-dimethyluric acid 7-methylxanthine 3-methylxanthine Paraxanthine (1,7-dimethylxanthine) Xanthine

16.0% 0.2% 0.002% 0.004% < 0.001% 0.013% 0.002% 0.011% 0.005% 0.003% 0.05% < 0.001%




Caffeine ~mol/L

the population fell within the range up to 35 t~mol/l.


Comparison of EL I SA and RIA 50 plasma samples were assessed for caffeine using the established radioimmunoassay as well as by the enzyme-linked immunosorbent assay (Fig. 3). A correlation coefficient of r = 0.97 ( P < 0.001) was obtained.

We have developed a quick, sensitive, easy to perform, enzyme-linked immunosorbent assay for caffeine in plasma and serum. The caffeine peroxidase label is easy to produce and has remained stable for at least 1 year. Cross-reactivity with the major metabolites of caffeine was low. Theophylline (1,3-dimethylxanthine), which is produced by an N-demethylation of caffeine, is a minor metabolite in man (Sved et al., 1976). It did however crossreact slightly in the caffeine ELISA, and this fact must be borne in mind when interpreting the results obtained from patients who may be taking medication containing theophylline. A good correlation was obtained between samples measured in the established caffeine radioimmunoassay and the enzyme-linked immunosorbent assay.

Clinical measurement Fig. 4 shows the distribution of plasma caffeine levels in a population of 250 outpatients. 95% of



10 15 20 25 30 35 40 45 50 55 60 65 70 75 80

Fig. 4. Distribution of caffeine levels in 250 outpatients. Each bar covers a range of 5 tt tool/1 caffeine.

dilution factor. The interassay coefficient of variation was less than 14%. The cross-reactivity of the assay with various caffeine metabolites is shown in Table I.



30iJ.mol/L RIA

References 10 f ° 0














pmol/k ELISA

Fig. 3. Correlation of 50 plasma caffeine samples using a radioimmunoassay and enzyme-linked immunosorbent assay.

Cook, C.E., Tallent, R., Amerson, E.W., Myers, M.W., Kepler, J.A., Taylor, G.F. and Dix Christenser, H. (1976) Caffeine in plasma and saliva by a radioimmunoassay procedure. Pharmacol. Exp. Ther. 199, 679-686. Erlanger, B.F., Borek, F., Beiser, S.M. and Lieberman, S. (1957) Steroid-protein conjugates. I. Preparation and char-

164 acterisation of conjugates of bovine serum albumin with testosterone and cortisone. J. Biol. Chem. 228, 713-729. Erlanger, B.F., Borek, F., Beiser, S.M. and Liebermon, S. (1959) Preparation and characterisation of conjugates of bovine serum albumin with progesterone, deoxycorticosterone and estrone. J. Biol. Chem. 234, 1090-1094. Gurtoo, H.L. and Phillips, B.M. (1973) Determination of total salicylates, phenacetin a n d / o r total N-acetyl paminophenol and caffeine in biological samples and analgesic formulations. J. Pharm. Sci. 62, 383-387.

Scott, N.R., Chakraborty, J. and Marks, V. (1984) Determination of caffeine, theophylline and theobromine in serum and saliva using high-performance liquid chromatography. Ann. Clin. Biochem. 21, 120-124. Sved, S., Hassie, R.D. and McGilveray, l.J. (1976) The human metabolism of caffeine to theophylline. Res. Commun. Chem. Pathol. Pharmacol. 13, 185-192.

Development of an enzyme-linked immunosorbent assay for caffeine.

A competitive enzyme-linked immunosorbent assay suitable for the measurement of caffeine in plasma and serum has been developed. Sheep immunised with ...
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