Immunology Letters, 33 (1992) 157-162 0165 - 2478 / 92 / $ 5.00 © 1992 Elsevier Science Publishers B.V. All rights reserved IMLET 01812
Fast filtration enzyme immunoassay for haptens N.P. D a n i l o v a , N.I. B e c k m a n , S.A. Y a z y n i n a n d R . G . Vasilov Department of Medical Diagnostics, Institute of Biotechnology, Moscow, Russia (Received 7 February 1992; revision received 30 April 1992; accepted 30 April 1992)
An immunometric method for determination of hapten concentration in fluids has been developed. High-affinity hapten-specific enzyme-labeled monoclonal antibodies are mixed with a sample containing hapten, then the mixture is filtered through a membrane with immobilized hapten. The level of enzyme activity retained by the membrane is inversely proportional to the concentration of hapten in a sample. The assay has been developed for theophylline, digoxin and phenobarbital. The coefficient of variation is less than 5% and the test takes about 2 min. 2.
be obtained with monoclonal antibodies using immunometric methods. A solid-phase immunometric method for the measurement of digoxin concentration in serum has recently been developed [6,7]. Here we describe a new immunometric immunoassay for hapten determination in serum. The method is based on the fast filtration of a sample containing hapten (theophylline, digoxin or phenobarbital) and peroxidase-labeled antibodies through a capture phase consisting of a nitrocellulose membrane with the immobilized hapten. The amount of the hapten in a sample is calculated from the amount of enzyme activity captured by the membrane.
Much progress has been achieved in immunological techniques for the measurement of haptens in recent years due to the combination of new, well-optimized, improved assays with monoclonal antibodies with higher affinity. This greatly increased opportunities for methodological advancement [1,2]. Techniques have been designed that can be used by minimally trained personnel outside well-equipped laboratories, as well as in portable diagnostic kits [3-5]. Immunometric methods were not used widely for hapten determination when they were based on polyclonal antibodies. Good results can also Key words: Hapten; Immunoassay Correspondence to: N.P. Danilova, Department of Medical Diagnostics, Institute of Biotechnology, Nauchny proesd 8, Moscow, 117246, Russia.
Materials and Methods
Antibody purification and labeling
Monoclonal antibodies were precipitated from ascites with ammonium sulfate and purified by ion exchange chromatography on DEAE-cellulose (NaCI gradient 0-0.5 M in 0.01 M phosphate buffer, pH 8). Purified antibodies were then coupled to horseradish peroxidase (HPO). 1 mg of HPO (Sigma) was dissolved in 0.1 ml of water and 1 mg of sodium periodate in 25 #1 of water was added. The mixture was incubated for 2 h at room temperature in the dark, filtered through 0.5 cm Sephadex G-10 and mixed with 4 mg of antibodies in 1 ml of 0.1 M Na-carbonate-bicarbonate (pH 9.5). After 3 h of incubation, 0.1 mg of NaBH4 was added and the solution was incubated for another hour. Then the HPO-antibody conjugate was precipitated with 75% saturated 157
ammonium sulfate and the pellet was dissolved in water and dialyzed against PBS. The conjugates were stored at 4°C at a final concentration of 10 mg/ml.
3.2. Protein-drug conjugates Digoxin-BSA conjugates have been synthesized according to Smith  and were shown to contain 7-15 digoxin molecules per molecule of the protein. Synthesis of theophylline-BSA conjugates containing 4-20 molecules of theophylline per molecule of BSA was described earlier . Phenobarbital-BSA conjugate was prepared as follows: 100 mg of m-aminophenobarbital was dissolved in 1 ml of 1 mM NaOH and mixed with 200 mg of BSA in 0.2 ml of water. Then, 250/~1 of 25% glutaraldehyde was slowly added with stirring. After 2 h incubation at room temperature, 20 mg NaBH4 was added and the mixture was stirred for after another 1 h. The conjugate was dialyzed against water. It contained 12 molecules of phenobarbital per molecule of BSA, as determined from the UV spectrum. By varying the quantity of m-aminophenobarbital in the reaction, conjugates with different amounts of hapten were prepared (6-19 mol per mol BSA).
Preparation of membranes
Nitrocellulose filters (Schleicher and Schuell, 0.45 /~m pore size) 88 × 88 mm were incubated in 10 ml of a solution of protein-drug conjugate (0.1-500 ktg/ml in 0.05 M Na-carbonate-bicarbonate buffer, pH 9.6) overnight at 4°C and washed 5 times with 10 ml of water. To prevent nonspecific binding, the membranes were incubated in a 1% solution of BSA in PBS for 2 h at room temperature, washed 5 times with water and dried.
An initial 1 mg/ml stock solution of each compound was prepared in water or in ethanol. Sequential 10-fold dilutions of the free hapten were made in assay buffer from 100/~g/ml to 0.1 ng/ml. Additionally, standard hapten samples were prepared in human serum at the following concentrations: theophylline 0, 10, 20/~g/ml; digoxin 0, 1, 2, 158
3 ng/ml; phenobarbital 0, 10, 20, 30 #g/ml.
3.5. Determination of working concentrations of HPO-labeled antibodies Serial dilutions of labeled antibodies in PBS containing 0.2% BSA and 0.05% Tween-20 were prepared. The dry nitrocellulose strips with immobilized antigen were placed into a Minifold filtration unit (Millipore). Then, 100 #1 of antibody solution followed by 100 /~1 of water were filtered sequentially through the membranes. In order to measure enzyme activity attached to the strip, 100 /~1 of substrate solution was passed through the membrane. The substrate solution was prepared as follows: 1 ml of the solution of 3 mg of 4-chloro-l-naphthol in 1 ml of methanol was mixed with 4 ml of 0.003% H202 in 20 mM TrisHCI. The strip then was dried, and color intensity was measured at 540 nm with 'Gamag TLC-Scanner II' (Gamag Lab., Switzerland).
Fast filtration EIA
Samples or hapten standards were mixed with 100 #1 of relevant labeled antibodies at the working dilution and the mixture was immediately filtered through the capture membrane. The membrane was washed and stained as above. For comparison purposes, we assayed clinical samples containing theophylline by HPLC , and samples containing phenobarbital by TDx (Abbott Diagnostics Inc.), according to the manufacturer's instructions. 4.
Results and Discussion
BSA-hapten conjugates with different degrees of hapten substitution from 4 to 20 mol hapten per mol protein were used in our method. The best results were obtained with conjugates containing about 10 mol of hapten per mol protein. The concentration of the conjugates for adsorption onto the nitrocellulose membrane was varied from 0.1 to 500 #g/ml. The concentration of 100 /~g/ml was found to be optimal. High-affinity monoclonal antibodies to phenobarbital (gaff 109 M - l ) , digoxin (Kaff 5 x 109 M -1) and theophylline (Karl 2 x 101° M - l ) were
Fig. I. Kinetics of the theophylline fast filtration assay. (A) Effect of incubation time on the assay. (B) Effect of filtration time on the assay. The theophylline concentration in the sample is 10 ~g/ml.
used in our assay. The antibodies were developed as described previously [10,11]. They were purified by ion exchange chromatography and labeled with HPO. The working dilution of the HPO-labeled antibodies was determined according to the titration curves; we chose a dilution which provided about 50% of the maximum color intensity of the membranes. More than 90% of peroxidase activity was discovered to be coupled to the membrane. We found that the assay kinetics are very fast. After mixing the sample with the conjugate the reaction reaches equilibrium in < 1 min (Fig. 1A) and the signal is stable for more than 24 h. Variations in the time of filtration demonstrated that in a 15 s interval an equilibrium was reached in immobilization of the monoclonal antibodies in the membrane (Fig. 1B), which seems to be due to the use of high-affinity anti-hapten antibodies in our assay. This observation is in good agreement with several recent investigations which demonstrated that high-affinity anti-hapten antibodies may have dissociation half-lives for bound hapten of a few minutes or less [5,12]. The dose-response curve for each drug was studied to find the steepest slope of calibration curves within the therapeutic intervals. The optimal sample volumes were found for theophylline, phenobarbital and digoxin which allow the drug concentration within the therapeutic range to be
BOOooncantratlon of dl~oxln, n~/ml
Fig. 2. Standard calibration curve for determination of digoxin. (Top) Absorbance profile of the membrane. The figures above the peaks correspond to the concentration of hapten in the bottom curve.
:s0 ,~] i
~ Ii , '
i i ~'
"i i '/
t , noise le~eE -
raw data f i l e
- 51r~LE L
1000 concentration of phenobarbltal,
Fig. 4. Standard calibration curve for determination of phenobarbital.
Fig. 3. Standard calibration curve for determination of theophylline. measured effectively (Figs. 2-4). The assay parameters for each drug are summarized in Table 1. We determined the specificity o f the assay by adding incremental a m o u n t s o f structurally similar c o m p o u n d s or drug metabolites to samples containing a fixed a m o u n t o f free hapten. The
mixture was then assayed for hapten concentration. The a m o u n t o f each c o m p o u n d required to give a 20% increase in the a p p a r e n t hapten concentration is shown in Tables 2-4. As these results indicate, the assay has excellent specificity for all drugs, and relatively high concentrations o f structurally similar c o m p o u n d s d o not substantially affect the clinical accuracy o f the method. L o w cross-reactivity is maintained by use o f m o n o c l o n a l antibodies. The precision and accuracy o f the determinations was estimated by assaying different dilutions o f control sera in triplicate. Each measurement was repeated 20 times. Within-run and be-
TABLE l Parameters of assay for determination of theophylline, digoxin and phenobarbital Measured compound
Antibody dilution from J0 mg/ml
Theophylline Phenobarbital Digoxin
10-20/.tg/ml 10-30/tg/ml 1-2 ng/ml
2 x 101oM - l 109 M - 1 5 x l09 M -I
l: 1000 1:500 h400
3 ,ul l0 #l 100/A
TABLE 2 Theophylline immunoassay specificity Compound
Uric acid Xanthine Hypoxanthine 1-Methyluric acid 3-Methylxanthine 1,3-Dimethyluric acid Caffeine (1,3,7-trimethylxanthine)
> 10000 > 10000 > 10000 > 500 > 1000 > 1000 > 10000
Concentration of compound required to produce a 20% error in apparent value of 15 /tg/ml theophylline control serum.
TABLE 3 Phenobarbital immunoassay specificity Compound
p-Hydroxyphenobarbital Hexobarbital Barbital Pentobarbital Secobarbital
> 300 > 10000 > 10000 > 1000 > 10000
Concentration of compound required to produce a 20% error in apparent value of 20 #g/ml phenobarbital control serum.
TABLE 4 Digoxin immunoassay specificity Compound
Digoxigenin Gitoxin Progesterone Testosterone Cholesterol
250 ng/ml 1000 ng/ml > 100 #g/ml > 100/~g/ml > 5000 #g/ml
aConcentration of compound required to produce a 20% error in apparent value of 2 ng/ml digoxin control serum.
t w e e n - r u n v a r i a t i o n o f the assay was less t h a n 5%. T o establish the v a l i d i t y o f the assay 39 samples c o n t a i n i n g t h e o p h y l l i n e were a s s a y e d b y fast filtration e n z y m e i m m u n o a s s a y a n d high-pressure liquid c h r o m a t o g r a p h y , a n d p h e n o b a r b i t a l in 50 samples was q u a n t i f i e d b y the p r e s e n t assay a n d by T D x . T h e results c o r r e l a t e d well with each o f the c o m p a r a t i v e assays: r = 0.97 a n d r = 0.96, respectively. T h e results o f the assay can be r e a d n o t only with a s p e c t r o p h o t o m e t e r , b u t also visually, in a s e m i - q u a n t i t a t i v e way. Thus, we have designed a simple a n d fast enzyme i m m u n o a s s a y for d e t e r m i n a t i o n o f h a p t e n in fluids. This m e t h o d meets the essential requirements: high r e s o l u t i o n within the t h e r a p e u t i c interval a n d r e p r o d u c i b i l i t y (coefficient o f v a r i a t i o n less t h a n 5%). This m e t h o d is well suited for decentralized sites such as e m e r g e n c y r o o m s a n d physicians' offices, where r a p i d testing is desirable, b u t it m i g h t also be easily a u t o m a t i z e d a n d used for large-scale d e t e r m i n a t i o n .
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