59

Clinica Cltinzica Acta,

0 Elsevier/North

91 (1979) 59-65 -Holland Biomedical Press

CCA 9824

COMPETITIVE IN PLASMA

TAKASHI School

NISHIKAWA

of Medicine,

(Received

NEPHELOME-TRIG IMMUNOASSAY

*, HIROAKI

Kitasato /Jdversity,

KUBO and MASAYUKI 1, Asamkodai,

OF THEOPHYLLINE

SAITO

Sagmnilzara, Kauagaroa 22s

(Japarz)

June 30th, 1978)

Summary

We have developed an accurate, simple, and rapid method for the determination of theophylline in plasma. The principle of the method is based on inhibition of immunoprecipitation by hapten. A nephelometer is used to measure the scattered light from the immunoprecipitate. .._The macromolecule, which possesses numerous theophylline moieties and forms immunoprecipitate with anti-theophylline antibodies, can be easily prepared and used as the reagent for the assay. Theophylline in the assay mixture competitively inhibits the immunoprecipitation of the macromolecule. Therefore, theophylline can be determined by the measurement of the decrease of the scattered light. The assay is rapid (incubation time within 15 min), requires as little as 10 ~1 of plasma, and requires neither troublesome pretreatment nor separation of antibody-bound from free antigen. Estimations of theophylline levels in patient plasma specimens correlate well to those obtained by the high performance liquid chromatography (correlation coefficient 0.971). Introduction

The nephelometric immunoassay method has been widely used for the determination of protein. We expected that nephelometry would also provide a suitable method for the determination of hapten, though hapten could not be determined by the conventional nephelometric immunoassay method. Hapten is generally a monoantigenic small molecule. Immunocomplex of the monoantigenie molecule is soluble, whereas the polyantigenic molecule forms insoluble immunocomplex which precipitates and scatters the incident light. The precipitate is considered to be a complex lattice of interlocking aggregate. The monoantigenic molecule can inhtbit competitively the immunoprecipitation of the F To whom correspondence

should by addressed.

polyantigenic molecule when the antibodies can react with the both molecules. Therefore the monoantigenic molecule can be determined by the measurement of the decrease of the scattered light due to the inhibition of the precipitation. The sensitivity of the “competitive nephelometric immunoassay” method is not expected to _be as high as the sensitivities of some other immunoassay methods such as the radioimmunoassay method. However, therapeutic blood levels of SOI\~Zdrugs, such as antiepileptic drugs and theophylline, are high enough for the sensitivity of the present method, and can be easily determined by it. We used theophylline as a model molecule to be determined and used the albumin possessing theophylline moieties as a polyantigenic molecule. Theophylline is the widely used drug in the treatment of acute and chronic respiratory diseases. It was reported that plasma theophyll;ne concentration should be maintained within the relatively narrow range of 10 to 20 ,ug/ml, and that plasma theophylline determination is useful for safe and effective therapy [1,21* Materials and n&hods The Laser Nephelometer “PDQ” . (Hyland Laboratories Inc.) was used to measure the scattered light. High performance liquid chromatography (Waters Associates, Model ALC/GPC 204) was used as a reference method [ 3] for the plasma theophylline determination. Anti-theophylline antiserum was obtained from rabbit immunized with bovine serum albumin possessing theophylline moieties (T-BSA). T-BSA was prepared by the reaction of theophylline-7-propionic acid with BSA according to the procedure of the mixed anhydride method [ 41. The human serum albumin and the rabbit serum albumin, both of which possessed theophylline moieties (T-HSA and T-RSA, respectively), were prepared by the same method [ 41. The number of theophylline moieties incorporated into the albumin was determined by ultraviolet spectrophotometry. Polyethylene glycol (PEG) (Carbowax 6000, Union Carbide Corp.) was used as an agent to accelerate the immunoprecipitation [ 5,6]. T-HSA and T-RSA were dissolved in the phosphate buffer (A M Na2HP04-KH2P04, pH 7.4). Both the antiserum and the specimen were diluted with PEG-containing phosphate buffer. The antiserum, diluted usually with PEG/buffer (25 g/l), was filtered through a membrane (pore size 0.4 pm, Nucleopore Corp.) to remove contaminating particles just before assay, and pipetted into each assay cuvette of the nephelometer. Unless otherwise stated, the assay procedure was: 50 ~1 of six-fold diluted specimen or standard, 100 ~1 of T-HSA or T-RSA solution, and 1.0 ml of 60-fold diluted and filtered antiserum were pipetted sequentially into the assay cuvette and allowed to stand at room temperature. The usually employed conditions of the nephelometer were: sensitivity 2.5, computing time 5 sec. The heparinized plasma specimens were csllected from asthmatic patients who had been intravenously injected with theophylline. Results Imm unoprecipi ta tion curve The typical immunoprecipitation

curve obtained

is shown in Fig. 1. Various

61

PEG 259/l

I

n: incorporated T per al bumm

!

\

PEG log/l

I? t 50 = 8

L ‘/ 0

o/

/-A

PEG 0 g/l

A/

/ A

polyhaptenic,

molecule

( mg/ml 1

00’

-_ 5 time (min)

&

10

-15

Fig. 1. Immunoprecipitation curve obtained with a constant amount of thr> antibodies. Fifty ~1 of 6-fold diluted pooled plasma, 100 ~1 of various concenlreltions (0 to 0.4 mglml, i.e. 0 to 0.04 mg/cuvette) of polyhaptenic molecule, and 1.0 ml of 60-fold diluted antiserum were mixed. Scattered light was measured at 15-min incubation time. ‘I’-HSA: human serum albumin possessing theophylline moieties; T-HSA: rabbit serum albumin possessing theophylline moieties; number in parentheses: average number of theophylline moieties incorporated into albumin. Fig. 2. Effects of PEG and of temperature on the immunoprecipitation. The PEG/buffer both 60-fold dilution of the antiserum and g-fold dilution of the pooled plasma.

was used for

concentrations of T-HSA or T-RSA were mixed with a constant amount of the antiserum, and allowed to stand for 15 min, then the cuvette was put into the cuvette holder of the nephelometer to measure the scattered light. Maximal precipitate formed in the zone of equivalence, and a lesser amount. formed in the zones of antibody excess and of antigen excess. The effect of the number of incorporated theophylline moieties per albumin was tested with T-RSA. As Fig. 1 shows, nephelometric effect increased with the number. T-HSA gave tne precipitation curve with the highest and sharpest peak among the tested polyhaptenic molecules. T-HSA was used in the experiment reported hereafter, as it was most suitable for the assay. Effects of PEG and of temperature Various amounts of PE6 were dissolved in the buffer. The immunoprecipitation was performed with these PEG/buffer solutions at the equivalent dose of ‘I’-HSA (0.1 mg/ml). As Fig. 2 shows, addition of PEG resulted in an increase of scattered light, and appeared to significantly increase the sensitivity of the assay and to shorten the analyzing time. After both the antiserum and the plasma specimen were diluted with the PEG/buffer, the solutions became slightly turbid because PEG decreased the solubility of the plasma proteins ;[7] as well as that of the immunoprecipitate. In order to remove the turbidity and to lower the blank reading of the scattered light, the diluted antiserum was allowed to stand for 30 min and then filtered. Exposure of the antiserum to PEG/buffer (20 to 30 g/l) for 30 min before the filtration gave good results of

62

the assay. The six-fold diluted plasma specimen did not need to be filtered since the blank readings (T-HSA was absent in the mixture) obtained with many normal human plasma specimens on several assay occasions after these treatments were 1.2 to 2.5. These values were negligible. The immunoprecipitation was performed at 4”C, 25’C and 37*C. As Fig. 2 shows, the rate of the precipitation was slow at 4”C, while the rates at 25’C and at 37*C were rapid and similar. Therefore,‘the assay could be performed at room temperature. The particles that :formed in the first 15 min at 25% in PEG/buffer (25 g/l) The particles were so large were fairly uniform on microscopic examination. that they could not pass through the membrane (pore size 0.4 pm). The partitles were dispersed well in the first few hours; it took more than 10 h to sediment. Dose-response curve Nephelometric effect of theophylline on the immunoprecipitation was studied. The standard theophylline solutions were prepared by adding known amounts of theophylline to the pooled human plasma. The standards were diluted G-fold with PEG/buffer (25 g/l). The antiserum was diluted 60-fold with PEG/buffer (25 g/l), allowed to stand for 40 min and filtered. The T-HSA solution (0.1 mg/ml), which would form maximal precipitate in the absence of theophylline (Fig. l), was prepared. The diluted standard, the T-HSA solution, and the filtered antiserum were then mixed at room temperature. Theophylline inhibited the precipitation quantitatively as Fig. 3 shows. Dose-response curves (solid line in Fig. 4) was obtained by plotting the log&transformed ratio (scattered light at each dose/scattered light at zero dose) against log-transformed plasma theophylline concentration. Nephelometric reading at 15-min incuba-bion time was employed for the curve preparation. Logit[scattered light ratio] decreased linearly with log[theophylline dose] at low concentrations of theophylline. When theophylline was further added, the dose-response curve tended to flatten gradually. It was not clear whether the particles were decreased in

pi asma t heophyi I I ne Wml

Fig. 3. Effect of theophylline on the immunoprecipitation T-HSA was equivalent to the antibodies.

of the complex

(T-HSA/antibodyj.

Added

63

\

80 . 60 40 .

\

\

(0.2 mg/mI)

‘A.

T-HSA i 0.05 my/ml)

-0

.

T-HSA

>.,

I

\

T-H!% ( CL1mq/ml )

-.\..

23 LOG

plasma

theophyl line

-

40

(ygml)

Fig. 4. Dose-response curves obtained with an equivalent amount of T-HSA (0.1 mglml), with an excess of T-HSA (0.2 mg/ml) and with a deficiency of T-HSA (0.05 mg/ml). Relative sscattered light (??I of the maximal among each T-HSA concentration series) was plotted against theophylline dose on the logit-log graph.

number or in size when theophylline inhibited the precipitation. When the T-HSA solutions of 0.2 mg/ml and of 0.05 mg/ml were added to give antigen excess zone and antibody excess zone (Fig. l), the dose-response curves (two dashed lines in Fig. 4) were obtained and they were disordered. It indicated that these conditions were not suitable for accurate or sensitive assay, and that the T-HSA solution should be prepared to give equivalent zones. Correlation between the presented method and the high performance liquid chromatography Theophylline concentrations in plasma specimens were determined from the standard dose-response curve on the logit-log graph. As Fig. 5 shows, the values correlated well to those obtained by the high-performance liquid chromatography with a correlation coefficient of 0.971. The coefficient of variation for concentration of 16.0 pg/ml was the plasma at average (n = 10) theophylline 6.5%. Compensation for intrinsic turbidity in plasma The plasma specimen and the antiserum themselves had light scattering abilities. When the normal clear sera gave nephelometric blank readings of 1.5 to 2.2, the four turbid sera with high lipids gave those of 5.5 to 18.0. These values were significant. The interferance could be avoided by filtration of the diluted turbid sera. However, filtration of each specimen was troublesome and impractical. Another way to avoid the interference was subtraction of the blank read-

= 0.971 = 1,10x

a

1 Oo

.

10 plasma

.

.

20

30

theophyl!lne(pg/ml),

- 1.40

. 40

50 HPLC

Fig. 5. Correlation between the competitive chromatography for the plasma theophylline

nephelometric determination.

immunoassay

and the high performance

liquid

ing. To measure the blank reading, the sequence of the additions of the reagents was altered as follows: the specimen or the standard was mixed with the antiserum, nephelometric blank reading (NO min) was recorded, then T-HSA was added and stirred, allowed to stand for 15 min, and nephelometric reading ) WZIS recorded. The difference (AN = N1 5 min NO min) was equal at (N 15min each theophylline concentration, though of course the N values given by the normal clear serw~ and by the turbid sera were different. Therefore the nephelometric effect of the intrinsic turbidity in the opaque specimen was compensated for when the AN value was plotted on the log&log graph to prepare the dose-response curve. Discussion In the conventional nephelometric immunoassay system, the molecule to be determined is mixed with an excess of the antibodies and forms immunoprecipitate itself. In the competitive nephelometric immunoassay system, the molecule to be determined should be mixed with an amount of the polyhaptenic molecule equivalent to added antibodies. The polyhaptenic molecule can be easily prepared by the same method that is used to prepare the immunogen. The hapten-carrier of the polyhaptenic molecule must be different from the carrier of the immunogen. We did not use T-BSA (MA was the hapten-carrier) in the assay but used T-HSA or T-RSA, because the antiserum was prepared by the immunization of the rabbit with T-BSA and the antiserum contained not only the anti-theophylline antibodies but also the anti-BSA antibodies. The anti-B&4 antibodies interfered with the assay when T-BSA was used in the assay. However, T-BSA would be used if the anti-BSA antibodies had been removed from the antiserum, though the complete and specific removal would be impossible or troublesome. The sensitivity of the nephelometric immunoassay was dependent largely on the nephelometric blank reading. In other words, the test/blank ratio was the deciding factor. The blank effect was largely due to the presence of large molecules such as lipoproteins in the specimen. Cerebrospinal fluid, saliva and urine

65

are more suitable specimens for nephelometric immunoassay than plasma because they contain much lower levels of the light scattering materials and give lower blank readings. In order to decrease the blank reading we measured the scattered light on the specially designed nephelometer with a detector situated at a forward angle (31”) from the incident light of a laser (wavelength 633 nm) [8]. We had previously measured the scattered light on a spectrofluorometer (detector angle go”), and we found that the longer the wavelength, the higher the test/blank ratio, and that the detection of the scattered light at a 31’ angle gave a higher test/blank ratio than at a 90” angle. For nephelometry of immunoprecipitate which is larger than the interfering particles, the wavelength of the incident light should be longer than 600 nm and the detector should receive the forward-scattered light?[8,9]. Such an optical system is particularly preferable if the specimen is plasma or serum. Cambiaso et al. developed the automated nephelometric inhibition immunoassay of the dinitrophenyl group and of progesterone using Technicon Autoanalyzer [ 10,111. The principle of their method is analogous to that of our method. However, the optical system of the Autoanalyzer seems to be inadequate in the wavelength and in the angle of the detector, though they did not report the result with a plasma specimen. Competitive nephelometric immunoassay seems promising for roueinc therapeutic monitoring of some drugs because the method has many txactical advantages over the other drug monitoring methods. Our method is as good as the homogeneous enzyme immunoassay method (Syva Co.) in its simplicity. The simplicity results from the fact that both methods require no separation step and that the immunoreaction can be detected early. It will be difficult to prepare the reagent of enzyme-linked drug which is suitable for the homogeneous enzyme immunoassay giving a high test/blank ratio, though the method of the reagent preparation has never been precisely reported by Syva Co. On the other hand, it will be easy and economical to prepare the reagent of polybaptenic molecule which is suitable for the competitive nephelometric immunoassay. References 3 Jenne, J.W., Wyze, E., Rood,

F.S. and MacDonald, F.M. (1972) Clin. Pharmacol. Thcr. 13.149-360 P.A. and OgiIvie, R.I. (1973) N. EngI. J. Med. 239.600-603 Kubo, H., Nishikawa, T. and Saito, M. (1978) 10th Int. Congr. Clin. Chem., Rlesico. PD. 148-148. APDO Postal 24-498, Mexico 7, D.F. Erlenger, B.F., Borek, F., Beiser, S.M. and Lieberman, S. (1975) J. Biol. Chem. 228, 71%-727 Lizana, J. and IIellsing, K. (1974) Clin. Chem. 20, 415-420 Hellsing, K. (1973) Protides Biol. Fluids, Proc. Colloq. Vol. 21. PP. 579-583 Poison, A., Potgieter. G.M., Largier, J.F., Mears, G.E.F. and Joubert, F.J. (1964) Biochim. Biophys. Acta 82.463-475 Deaton, C.D., Maxwell, K.W., Smith, R.S. and CreveIing, R.L. (1976) Ciin. Chem. 22.1465-1471 MacDonald, J.Y. (1960) Bull. Serol. Mus. 23.1-6 Cambiaso. C.L., Riccomi, H., Masson, P.L., Vaerman, J.P. and Heremans, J.F. (1973) Protides Biol. Fluids, Proc. CoIIoq. Vol. 21, pp. 585-591 Cambiaso, C.L., Riccomi, H.A., Masson, P.L. and Heremans, J.F. (1974) J. Immunol. Methods 5, 293-302

2 Mitenko,

3 4 5 6 7 8 9 10

11

i

Competitive nephelometric immunoassay of theophylline in plasma.

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