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. Dani lova, N.I. Beckman, S.A. Yazynin and 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)

1. Summary

An immunometric method for determination of hapten concentration in fluids has been devel- oped. High-affinity hapten-specific enzyme-la- beled monoclonal antibodies are mixed with a sample containing hapten, then the mixture is fil- tered through a membrane with immobilized hap- ten. The level of enzyme activity retained by the membrane is inversely proportional to the con- centration of hapten in a sample. The assay has been developed for theophylline, digoxin and phe- nobarbital. The coefficient of variation is less than 5% and the test takes about 2 min.

2. Introduction

Much progress has been achieved in immuno- logical techniques for the measurement of hap- tens in recent years due to the combination of new, well-optimized, improved assays with mono- clonal antibodies with higher affinity. This greatly increased opportunities for methodological ad- vancement [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.

be obtained with monoclonal antibodies using im- munometric methods. A solid-phase immunomet- ric method for the measurement of digoxin con- centration in serum has recently been developed [6,7].

Here we describe a new immunometric immu- noassay for hapten determination in serum. The method is based on the fast filtration of a sample containing hapten (theophylline, digoxin or phe- nobarbital) and peroxidase-labeled antibodies through a capture phase consisting of a nitrocellu- lose 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.

3. Materials and Methods

3.1. Antibody purification and labeling

Monoclonal antibodies were precipitated from ascites with ammonium sulfate and purified by ion exchange chromatography on DEAE-cellu- lose (NaCI gradient 0-0.5 M in 0.01 M phosphate buffer, pH 8). Purified antibodies were then cou- pled 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-bicar- bonate (pH 9.5). After 3 h of incubation, 0.1 mg of NaBH4 was added and the solution was incu- bated for another hour. Then the HPO-antibody conjugate was precipitated with 75% saturated

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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 [8] and were shown to contain 7-15 digoxin molecules per molecule of the pro- tein. Synthesis of theophylline-BSA conjugates containing 4-20 molecules of theophylline per molecule of BSA was described earlier [9]. Pheno- barbital-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 phe- nobarbital per molecule of BSA, as determined from the UV spectrum. By varying the quantity of m-aminophenobarbital in the reaction, conju- gates with different amounts of hapten were pre- pared (6-19 mol per mol BSA).

3.3. 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-bicarbo- nate buffer, pH 9.6) overnight at 4°C and washed 5 times with 10 ml of water. To prevent nonspeci- fic binding, the membranes were incubated in a 1% solution of BSA in PBS for 2 h at room tem- perature, washed 5 times with water and dried.

3.4. Hapten standards

An initial 1 mg/ml stock solution of each com- pound was prepared in water or in ethanol. Se- quential 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 pre- pared in human serum at the following concentra- tions: theophylline 0, 10, 20/~g/ml; digoxin 0, 1, 2,

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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 immo- bilized antigen were placed into a Minifold filtra- tion unit (Millipore). Then, 100 #1 of antibody so- lution 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 pre- pared 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 Tris- HCI. The strip then was dried, and color intensity was measured at 540 nm with 'Gamag TLC-Scan- ner II' (Gamag Lab., Switzerland).

3.6. Fast filtration EIA

Samples or hapten standards were mixed with 100 #1 of relevant labeled antibodies at the work- ing dilution and the mixture was immediately fil- tered through the capture membrane. The mem- brane was washed and stained as above.

For comparison purposes, we assayed clinical samples containing theophylline by HPLC [9], and samples containing phenobarbital by TDx (Abbott Diagnostics Inc.), according to the manu- facturer'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 con- taining about 10 mol of hapten per mol protein. The concentration of the conjugates for adsorp- tion 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 pheno- barbital (gaff 10 9 M - l ) , digoxin (Kaff 5 x 10 9 M -1) and theophylline (Karl 2 x 101° M - l ) were

2000,

1000.

~- v w

2000

1000

. , , , - .

m , , , , , , ,

1' 2' 3 h 1 2 3 4 5m~' t

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 puri- fied by ion exchange chromatography and la- beled with HPO. The working dilution of the HPO-labeled antibodies was determined accord- ing to the titration curves; we chose a dilution which provided about 50% of the maximum col- or intensity of the membranes. More than 90% of peroxidase activity was discovered to be coupled ~a to the membrane.

We found that the assay kinetics are very fast. After mixing the sample with the conjugate the aa0c reaction reaches equilibrium in < 1 min (Fig. 1A) and the signal is stable for more than 24 h. Varia- z400- tions in the time of filtration demonstrated that in a 15 s interval an equilibrium was reached in im- mobilization of the monoclonal antibodies in the 200o- membrane (Fig. 1B), which seems to be due to the use of high-affinity anti-hapten antibodies in xe0o- our assay. This observation is in good agreement with several recent investigations which demon- rz00- strated that high-affinity anti-hapten antibodies may have dissociation half-lives for bound hap- ten of a few minutes or less [5,12]. BOO-

The dose-response curve for each drug was stu- died to find the steepest slope of calibration curves within the therapeutic intervals. The opti- mal sample volumes were found for theophylline, phenobarbital and digoxin which allow the drug concentration within the therapeutic range to be

ooncantratlon 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.

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2000

1000

O~f~

Tr~ck - t , noise le~eE - O 0 1 ~ L raw data f i l e - 51r~LE L

o io 2o C0N~TRATION OF THEOPHYLLINE%j~tg/ml

Fig. 3. Standard calibration curve for determination of theo- phylline.

measured effectively (Figs. 2-4). The assay para- meters for each drug are summarized in Table 1.

We determined the specificity o f the assay by adding incremental amounts o f structurally simi- lar c o m p o u n d s or drug metabolites to samples containing a fixed amoun t o f free hapten. The

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2000-

1900-

I600-

1700 -

1600 -

1 5 0 0 -

I 4 0 0 "

I 3 0 0 -

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i 0

, 5 i 0

i ~ I 20 , i i

i ' :i i , ,

i i i ~' "i / i i '

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concentration of phenobarbltal, j u g / m 1

Fig. 4. Standard calibration curve for determination of pheno- barbital.

mixture was then assayed for hapten concentra- tion. The amoun t o f each c o m p o u n d required to give a 20% increase in the apparent hapten con- centrat ion is shown in Tables 2-4. As these re- sults indicate, the assay has excellent specificity for all drugs, and relatively high concentrat ions o f structurally similar c o m p o u n d s do not substan- tially affect the clinical accuracy o f the method. Low cross-reactivity is maintained by use o f monoc lona l antibodies.

The precision and accuracy of the determina- tions was estimated by assaying different dilu- tions o f control sera in triplicate. Each measure- ment was repeated 20 times. Within-run and be-

TABLE l

Parameters of assay for determination of theophylline, digoxin and phenobarbital

Measured compound Therapeutic range Antibody affinity Antibody dilution Sample volume from J0 mg/ml

Theophylline 10-20/.tg/ml 2 x 101o M - l l: 1000 3 ,ul Phenobarbital 10-30/tg/ml 109 M - 1 1:500 l0 #l Digoxin 1-2 ng/ml 5 x l09 M -I h400 100/A

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TABLE 2

Theophylline immunoassay specificity

Compound Concentration (#g/ml) a

Uric acid > 10000 Xanthine > 10000 Hypoxanthine > 10000 1-Methyluric acid > 500 3-Methylxanthine > 1000 1,3-Dimethyluric acid > 1000 Caffeine (1,3,7-trimethylxanthine) > 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 Concentration (#g/ml) a

p-Hydroxyphenobarbital > 300 Hexobarbital > 10000 Barbital > 10000 Pentobarbital > 1000 Secobarbital > 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 Concentration a

Digoxigenin 250 ng/ml Gitoxin 1000 ng/ml Progesterone > 100 #g/ml Testosterone > 100/~g/ml Cholesterol > 5000 #g/ml

aConcentration of compound required to produce a 20% error in apparent value of 2 ng/ml digoxin control serum.

tween-run va r ia t ion o f the assay was less than 5%. To establ ish the va l id i ty o f the assay 39 sam- ples con ta in ing theophyl l ine were assayed by fast f i l t ra t ion enzyme i m m u n o a s s a y and high-pressure l iquid c h r o m a t o g r a p h y , and p h e n o b a r b i t a l in 50 samples was quant i f ied by the present assay and by TDx. The results cor re la ted well wi th each o f the compara t i ve assays: r = 0.97 and r = 0.96, re- spectively.

The results o f the assay can be read no t only with a spec t ropho tomete r , but also visually, in a semi-quant i ta t ive way.

Thus, we have designed a simple and fast en- zyme i m m u n o a s s a y for de t e rmina t ion o f hap ten in fluids. This m e t h o d meets the essential require- ments: high reso lu t ion within the therapeut ic in- terval and reproduc ib i l i ty (coefficient o f va r i a t ion less than 5%). This m e t h o d is well sui ted for de- centra l ized sites such as emergency rooms and physic ians ' offices, where r ap id test ing is desir- able, bu t it might also be easily au toma t i zed and used for large-scale de te rmina t ion .

References

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