Anal. Chem. 1992, 64, 2403-2407
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Detection of Antistreptolysin 0 Antibody: Application of an Initial Rate Method of Latex Piezoelectric Immunoassay Makoto Muratsugu. Department of Laboratory Medicine, School of Medicine, Asahikawa Medical College, Asahikawa 078, Japan
Shigeru Kurosawat and Naoki Kamo Department of Biophysics and Physical Chemistry, Faculty of Pharmaceutical Sciences, Hokkaido University, Sapporo 060, Japan
Latex plezoelectrlc Immunoassay (LPEIA) Is a new latex Immunoassay uslng a plezoekctrlcquartz crystal (Kurorawa et al. Chem. P h a m Bull. 1990,S8,1117). Thls assay requlres 110 ImmoMlIzatbnof antlgen or antbody on an electrodewrlace of a plezoelectrlc crystal, whlle the lmmoblllzatlon Is lndkpensable for ordlnary Immunoassays d n g a plezoelectrlc crystal as a microbalance. The present paper Improves a prevlow method(end-polnt analysis) by lntroduclngthe lnltlal rate method udng a batch cell; reductlon of assay volume (1.2 mL) and shortenlng of assay time (2-3 mln) were achkved. Thls assay was applled to the detectlon of antktreptolysln 0 antlbody (ASO) In serum. The frequency change was proportlonalto the AS0 concentratlonup to 1040 I U mL-l, and the method hadgood sendtlvlty for actual cllnlcal appllcatlon. The volume of serum requlredfor the assay was 0.02 mL. Twenty-four cllnlcalspecbnens were analyzed wlth thls LPEIA, and the values obtalned were compared wlth thoso obtalned wlth a turbldlmetric latex agglutlnatlonmethod. The correlatlon coefflclent between these values was 0.950 ( P < 0.01).
INTRO DUCT10N The antistreptolysin 0 (ASO) test is the most valuable serologic test for infection by group A &hemolytic streptococci. Streptolysin 0 produced by most strains of the streptococci stimulates the production of a specific antibody, ASO. The most widely used method for the determination of AS0 titer is the macrotechnique of Rantz and Randall,' which is based on the Todd procedure.2 Here, the titer is the reciprocal of the highest serum dilution preventing hemolysis of red blood cells. The titer is expressed in Todd units if the potency of the streptolysin 0 used in the test has been adjusted against the Todd standard or in international units (IU) if it has been adjusted against the international standard of the World Health Organization. In order to save time, the latex agglutinationtest, which was first developed for the detection of rheumatoid factor (RF),3 was applied to the AS0 test.4 The latex method is normally used for a qualitative screening test of ASO, i.e., to check whether the sample contains AS0 + Present address: National Chemical Laboratory for Industry, Tsukuba, Ibaraki 305,Japan. (1) Rantz, L. A.; Randall, E. R o c . SOC. E z p . Biol. Med. 1945,59,22-
-". 3R
(2) Todd, E. W. J . E z p . Med. 1932,55,267-280. (3)Singer, J. M.; Plotz, C. M. Am. J . Med. 1956,21,888-892. (4)Bach, G. L.; Wiatr, R. A.; Anderson, T. 0.;Cheatle, E. Am. J . Clin. Pathol. 1969,52,126-128.
or not, although it has also been employedfor the quantitative determination of the AS0 titer, and this has been reported by other researchers.5 Instead of using the usual visual method for the latex agglutination, the development of a simpler, easier, and more quantitative method is desirable. At present, the assay is widely performed t u r b i d i m e t x i d ~ or nephelometrically,'*12 and particle counting immunoassay was developed.13J4 Recently, latex photometric immunoassay has been developed and applied successfully to the determination of various substances such as ASO, immunoglobulins, RF, C-reactive protein (CRP), femtin, a-fetoprotein, and µglobulin.1+18 A piezoelectric quartz crystal can be used as a microbalance: a change in the resonance frequency of the crystal is proportional to the weight on the crystal surface, and the detection limit is as small as several nanograms.19 Some investigators have applied this device to immunosensors. Shons et 81.20 observed a frequency shift which was proportional to the concentration of a specific antibody in a solution when the surface of a piezoelectric crystal was precoated with the antigen. Immunoglobulinswere detected by some workers using antibody-21.22 or protein A-coated23924 crystals. Some microbes,26-2' haptens,28+29or protein antigen30 were also detected by a similar method. This method requires the (5)Gerber, M. A,; Caparaa, L. S.; Randolph, M. F. J . Clin. Microbiol. 1990,28,413-415. (6)Sawai, M.; Sudou, T.;Enomoto, S. Japanese Unexamined Patent Application No. 53-24015,1978. (7)Hoigne, R. Helu. Med. Acta 1958,4,422-429. (8)Dezelic, N.; Dezelic, Gj. Croat. Chem. Acta 1970,42,457-466. (9)Dezelic, Gj.; Dezelic, N.; Muic, N.; Pende, B. Eur. J. Biochem. 1971,20,553-560. (10)Cohen, R. J.; Benedek, G. B. Immunochemistry 1976, 12, 349351. (11)Blume, P.; Greenberg L. J. Clin. Chem. 1975,21,1234-1237. (12)Morita, S.;Sawai, M.; Mataumoto, S.; Sudo, T. US.Patent 4,313,9291982. J.;Masson, (13)Cambiaao,C.L.;Leek,A. E.;DeSteenwinkel,F.;Billen, P. L. J . Zmmunol. Methods 1977,18,33-44. (14)Masson, P. L.;Cambiaso, C. L.; Collet-Cassart, D.; Magnusson, C. G. M.; Richards, C. B.; Sindic, C. J. M. Methods Enzymol.1981,74, 106-139. (15)Ikeda, K.; Fujii, I.; Ikawa, S.; Takahashi,Y. Jpn. J . Clin. Lab. Automation 1983,8,152-155. (16)Senju,O.;Takagi,Y.;Gomi,K.;Ishii,Y.;Mochizuki,S.;Minakami, Y.; Inoue, K. Jpn. J . Clin. Lab. Automation 1983,8,161-165. (17)Fujita, S.;Tedokon, M.; Yasuhara, M.; Arisue, K.; Kouda, K.; Hayashi, C.; Miyai, K. Jpn. J . Clin. Lab. Automation 1983,8,225-229. (18)Tsutaui, S.Jpn. J . Clin. Lab. Automation 1986,11, 210-215. (19)Sauerbrey, G. Z.Phys. 1959,155,206-222. (20)Shons, A.; Dorman, F.; Najarian, J. J . Biomed. Mater. Res. 1972, 6,565-570. (21)Roederer, J. E.;Bastiaans, G. J. Anal. Chem. 1983,55,2333-2336. (22)Thompson, M.; Arthur, C. L.; Dhaliwal, G. K. Anal. Chem. 1986, 58,1206-1209. (23)Muramatsu, H.;Dicks, J. M.; Tamiya, E.; Karube, I. Anal. Chem. 1987,59,2760-2763. (24)Davis, K.A.; Leary, T. R. A d . Chem. 1989,61,1227-1230. (25)Muramatau. H.;Kajiwara, K.: Tamiya, E.: Karube, I. Anal. Chim. Acta 1986,188, 257-261.
0003-2700/92/0364-2483$03.00/0 0 1992 American Chemical Society
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immobilization of antigens or antibody/specific-bindingproteins on the quartz crystal surface. On the basis of Kanazawa's treatment31-32 of the influence of solution properties on a quartz crystal, Muramatsuet al.33,34 reported a monitoring system based on the viscosity change in a solution; endotoxin or fibrinogen concentration was determined by the gelation reaction of Limulus amoebocyte lysate or fibrinogen, respectively. Kurosawa et al.35 developed a new method of immunoassay using a piezoelectric quartz crystal which does not require immobilization: the change in the oscillating frequency was observed in association with the latex agglutination. The frequency change was dosedependent and the CRP concentration was successfully determined using anti-CRP-bearinglatex. The detection limit of this method was comparable to that of latex photometric immunoassay. This new method was termed Latex piezoelectric immunoassay (LPEIA). This has an advantage in not requiring the immobilization of antibody or antigen on the quartz crystal that is necessary for the usual piezoelectric immunosensor described above, and many antibodyiantigen bearing latex particles used in clinical tests have become commercially available reagents in recent years. Piezoelectric methods for microbe detection require the immobilization of the specific antibody against the microbe on the crystal surface as described above. However, Ward et al.36developed a useful piezoelectric sensor that could detect the bacterial cells without an immobilized receptor like antibody. In a previous paper,35 a flow cell was used; the assay volume was, however, too large for clinical application, and a reduction of assay time was also required. In the present paper, we modify the original LPEIA using initial rate analysis to detect AS0 in sera using smaller assay times and sample volumes. The present LPEIA is sensitive enough to detect AS0 in the sera of various patients, and the values estimated wee in good correlation with those measured by the usual turbidimetry.
EXPERIMENTAL SECTION Apparatus and Materials. Streptolysin 0 coated latex (SERATESTAM ASO-E) (referred to as latex-SO), anti-CRP coated latex (SERATESTAM CRP-E) (referred to as latex-& CRP), AS0 standard serum (SERATESTAM S ASO, 204 IU mL-l), CRP standard serum (SERATESTAM S CRP, 5.3 mg dL-l), and the serum of high AS0 titer (1040 IU mL-l) were gifts from Hitachi Chemical Co., Ltd., Japan. Streptolysin 0 and anti-CRP were physically adsorbed on latex particles (the component was polystyrene and the size ca. 0.1 pm). Human albumin (abbreviated as HSA) (No. A-8763)and bovine albumin (abbreviated as BSA) (No. A-7030) were purchased from Sigma Chemical Co. Other chemicals used were certified reagents purchased from Wako Pure Chemical Co., Japan, and Nakarai Tesque, Inc., Japan. Water was prepared with a NANOpure I1 (26)Muramatau, H.; Watanabe, Y.; Hikuma, M.; Ataka, T.; Kubo, I.; Tamiya, E.; Karube, I. Anal. Lett. 1989,22,2155-2166. (27)Prusak-Sochaczewski, E.;Luong, J. H. T.; Guilbault, G. G. Enzyme Microb. Technol. 1990,12, 173-177. (28)Ngeh-Ngwainbi, J.;Foley, P. H.; Kuan, S. S.; Guilbault, G. G. J. Am. Chem. SOC.1986,108,5444-5447. (29)Rajakovic, L.;Ghaemmaghami, V.; Thompson, M. Anal. Chim. Acta 1897,217, 111-121. (30)Prusak-Sochaczewski, E.;Luong, J. H. T. Anal. Lett. 1990, 23, 401-409. (31)Kanazawa, K. K.; Gordon, J. G. 11. Anal. Chem. 1985,57,17711772. (32)Kanazawa, K. K.;Gordon, J. G., 11. Anal. Chim. Acta 1985,175,
atsu, H.; Tamiya, E.; Suzuki, M.; Karube, I . Anal. Chim. Acta 1988,215,91-98. (34)Muramatsu, H.; Tamiya, E.; Suzuki, M.; Karube, I. Anal. Chim. , 321-326. va,S.;Tawara,E.;Kamo,N.;Ohta,F.;Hosokawa,T. Chem. .117-1120. Foss, R. P.; Ward, M. D. BioiTechnology 1991, 9,45c-454.
, ...............
--:--'-
Bath
...............,
a
Computer Magnetic S t i r r e r
Figure 1. Schematic diagram of experimental setup. The latex suspension (1.2 mL) was stirred with a magnetic stlrrer. The sample was applied with a microplpette. The electrlc circuit for measuringthe quartz oscillatlon frequency was as descrlbed prevl0usly,3~using a universal counter controlled by a mlcrocomputer. TTL: translstortransistor logic circuit.
(Barnstead Co., USA), and its specificresistance was more than 18 M 52 cm-l. AT-cut piezoelectric quartz crystals (9-MHz resonance frequency, 8 X 8 mm) were purchased from Yakumo Tsushin Co., Japan. One side of each crystal was sealed with a quartz plate and silicon sealant-45 (Shin-Etsu Kagaku, Japan); as described previ~usly,~~ this treatment stabilizes the oscillation frequency in solution. The one-side-sealed piezoelectricquartz crystal was set in a small cuvette (10 X 10 X 15 mm) made of poly(methy1 methacrylate) and was inserted in a laboratory-made TTL circuit (Figure 1). This apparatus was accommodated in a chamber maintained at 25 & 1"C. The solution in the cuvette was gently stirred with a magnetic stirrer. The oscillation signal was fed to a universal counter (ModelSC-7202,Iwatsu Electric Co., Japan), and the frequency change was stored in a microcomputer (Model PC-9801, Nippon Electronic Co., Japan). Procedure. Kanazawa and Gordon31J2or Bruckenstein and S h a derived ~ ~ ~a theoretical equation describing the oscillation frequencyin a quartz crystal in solution(eq 1or 2). The oscillation of a quartz crystal in air is taken as a reference,and the frequency difference in 10 mmol L-l phosphate buffer, pH 6.8 (formed by mixing NaZHP04 and KHzPOd solutions, referred to as PB) from that in air, AFB, is given by U B = -K(PBvB)~/' (1) the frequency difference (AFs) for sucrose in PB is given by
U s = -K(PSVS)'/~ (2) where p~ or ps are the densities of PB or sucrose in PB, respectively, and VB or 9s are the viscosities of PB or sucrose in PB, respectively. In this study, the oscillating frequency in PB was a reference; thus the frequency differences in a sucrose-PB mixture from that in PB, US+, is given by
(3)
The one-side-sealed crystal was immersed into the cuvette containing 1.2 mL of PB, and the oscillation frequency was measured. After the solution was gently aspirated from the cuvette, 1.2 mL of 5 wt % of sucrose in PB was gently added to the cuvette and the frequency change from that in PB was recorded, followed by the same procedure with a sucrose solution of 10wt % . Frequency changes for these sucrose solutionsallowed us to calculate K of the quartz crystal, and the average was taken, as described later. Then, the crystal and the cuvette were gently rinsed three times with PB, after removal of the sucrose solution, and 1.2 mL of latex suspension was added to the cuvette. The stock latex suspension (0.5%) was diluted to an appropriate concentration with PB. The concentration of suspension was (37)Kurosawa, S.; Tawara, E.; Kamo, N.; Kobatake, Y. Anal. Chim. Acta 1990,230,41-49. (38)Bruckenstein, S.;Shay, M. Electrochim. Acta 1985,30,12951300.
ANALYTICAL CHEMISTRY, VOL. 64, NO. 21, NOVEMBER 1, 1992
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. 1-
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Piezoelectric crystal response to the sucrose-PB mixtures of various concentrations. The figures on the curve represent weight percent of sucrose solutions in PB (10 mmoi L-1 phosphate buffer, pH Flgurr 2. 6.8).
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expressed in terms of A570 (Le.,absorbance at 570 nm). After the frequency had stabilized, 0.02 mL of serum was added to the latex suspension and the frequency change was measured. The specific gravities and viscosities of sucrose solutionswere measured with a pycnometer and an Ostwald viscometer, respectively, at 25 f 0.5 O C . AS0 values were turbidimetrically measured with a Hitachi 7150 Automatic Analyzer using IATROACE AS0 (IATRON, Labs., Inc., Tokyo) and were expressed in internationalunits per milliliter. The total amount of serum protein was expressed in terms of Azso (i.e., absorbance at 280 nm).
RESULTS AND DISCUSSION Proportionality Coefficient,K. As described previously,36,37 sealing one side of the crystal alters the value of proportionality coefficient,K, so that the value of frequency change was different, when different crystals were used. It was, however, found that if the frequency change was expressed in terms of -AFIK (here, -M is divided by K including no units in order to reduce the value of K to 1cm2 g-1 s-1/2),the variation of -AF could be compensated (namely, the variation coefficient of -AFIK was smaller than that of -AF). Therefore, in this study, data are expressed in terms of -AF/K. Since LPEIA in this study was performed using PB as a solvent, the determination of K in the PB solution was desirable. Figure 2 shows a typical recording of frequency changes by application of 2-60 wt % sucrose in PB. The oscillation frequency in PB was taken as a reference value. After the measurement of sucrose, measurement in PB restored the original oscillation frequency. Figure 3 shows a plot of the frequency change against ( p l q ~ ) l / ~( P O ~ O ) ' / ~ using sucrose-PB mixtures with different concentrations of sucrose (see the caption of Figure 3 for the meaning of suffix 0 and 1). The values of K have so far been determined in
150
0
50
100
TIME/ MIN Flgurr 4. Typical frequency changes on the addition of serum to latex suspension. (A) a, addition of CRP serum (5.3 mg dL-I) to latex-SO (A570 = 1.3), K = 72 354 cm2g-l s-ll2;b, addition of A S 0 serum (1040 IU mL-l) to IatexdCRP (A570= 1.1), K = 80 282 cm2 g-1 s-I/*; c, addition of A S 0 serum (1040 IU mL-') to latex-SO (AsT0= 1.3), K = 72 176 cm2g-1s - ~ / ~Each . serum (0.02 mL) was added to 1.2 mL of each latex suspension at the arrow. The ordinate represents the values of frequency change (AF) divided by proportionality coefficient, K. (6) Expanded trace of c in A. Change 1, an abrupt frequency change; change 2, a gradual frequency change.
sucrose-water mixtures using a flow cell,35,3' but in this study the values were determined in sucrose-PB mixtures using the batch cell. For comparison, K values were determined in sucrose-water mixtures using the batch cell (Figure 3). Evidently the frequency change linearly depends on ( p ~ q ~ ) l / ~ - (poq0)1/2, as long as the sucrose concentration is low, whichever solvent is used; the linearity holds at lower than 30 or 40 wt 5% for PB or water, respectively. At higher concentrations, the frequency change deviates from linearity. The frequency change for 60 wt % sucrose in PB or water was slightly unstable. The deviation from linearity confirms the previously reported results in high-viscosity glycerol-water mixtures,3S4lbut the reason for this phenomenon has not yet been explained. In addition, our data shows that the deviation was larger for sucrose-PB mixtures than for sucrose-water mixtures. The reason why the discrepancy occurs is not clear at present. Frequency changes in electrolyte solutions do not follow eq 1 (or 2), the reason is obscure,3' and this may relate to some extent to the difference in deviation. Therefore, the following LPEIA was performed only within the linear frequency change limits. The value of K could be easily determined with 5 and 10 wt % sucrose solutions. The values of & and Klo were calculated for 5 and 10wt % sucrose in PB, respectively, and here an arithmetic average of K5and Klo is taken as K (mean f SD = 68 350 f 6662 cm2 g-1 s-ll2, CV = 9.75%, n = 58). Frequency Changeon Latex Agglutination. Figure 4A shows the frequency changes of one-side-sealed crystals in protein-coated latex suspensions to which each serum was added. The addition of the serum containing antistreptolysin 0 (1040 IU mL-l) to the latex-SO suspension induced the frequency change of the crystal; an abrupt frequency change appeared following a gradual change. This biphasic response is clearly illustrated as the expanded trace in Figure 4B. On the other hand, when the serum containing AS0 (1040IU mL-l) or CRP (5.3 mg dL-1) was added to latex-aCRP or latex-SO suspension, respectively, no gradual frequency change occurred. The addition of AS0 standard serum (204 IU mL-1) to latex-aCRP also changed the abrupt frequency of the crystal, which was followed by no gradual frequency change (data not shown). These results indicated that the gradual change depended on the agglutination of latex particles; in fact, the agglutination could be visually observed. (39)Muramatau, H.; Tamiya, E.; Karube, I. Anal. Chem. 1988, 60, 2142-2146. (40)Charlesworth, J. M. Anal. Chem. 1990.62, 76-81. (41)Kurosawa, S.;Kitajima, H.; Ogawa, Y.; Muratsugu, M.; Nemoto, E.; Kamo,N., unpublished work.
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I 10
20
30
TIME / MIN Flgure 5. Frequency changes on the addition of BSA to latex-SO suspension (a)or to PB solutlon (b). BSA solution (0.02 mL) was added to 1.2 mL of each solution at the arrow. BSA was dissolved in physloioglcai saline, and the of the solution was 174. (a) K = 69 478 cm2 g-I s - ~ / ~(b); K = 88 813 cm2 g-1 s-lI2.
In addition, the dose-dependency of the initial rate of this change as described later supports the idea that the agglutination of latex particles is responsible for the gradual frequency change. The abrupt frequency change may be associated with a nonspecific adsorption of proteins in serum to the crystal, since the change also occurred even in the absence of antibody-antigen reaction (the addition of the ASO- or CRP-serum to latex-aCRP or latex-SO suspension, respectively). Here, we simply call the gradual frequency change a frequency change, except where otherwise stated. The dependency of the frequency change on the latex concentration was investigated. When the serum containing AS0 was added to the latex-SO suspensions of varying concentrations, the decrease in latex concentration did not strongly affect the change of frequency (data not shown). Even when ASO-serum was added to PB containing no latex particles, the oscillating frequency changed abruptly, obviously indicating the presence of nonspecific adsorption of serum protein to the crystal. From these results, it is supposed that the presence of latex prevents the adsorption of proteins to the crystal; most of the proteins added is adsorbed to the latex particles. This assumption was confirmed by the followingexperimental results. The addition of BSA solution to the cuvette, the concentration of which had the same AZW as the high AS0 value serum used, excluding latex particles, caused a large abrupt change of frequency of the crystal, but not a gradual one. On the other hand, when BSA of the same amount was added to the latex-SO suspension (A570 = 1.31, no gradual frequency change occurred, though a small abrupt frequency change was observed (Figure 5). Using HSA gave the same result (data not shown). Therefore, the f o l l o ~ n g experiments were performed under the condition that the absorbance of latex-SO suspension was 1.3 at 570 nm, the concentration of which could almost suppress the nonspecific adsorption of serum proteins to the crystal. The above results also support the idea that the agglutination of latex owing to the specific antigen-antibody reaction results in the gradual frequency change. In a previous paper,35 CRP could be detected with LPEIA using a flow cell. The sample volume was at least 9 mL, which is large for actual clinical application. In this study, a smaller batch cell (1.2 mL) was used and the amount of sample serum required for assay was 0.02 mL, a reasonable volume for actual clinical application. An increase in the magnitude of frequency change was necessary for CRP detection, but measurement of AS0 did not require this improvement, because a sufficient magnitude of frequency change was available in this system. Initial Rate of Frequency Change. The gradual frequency change is due to the specific latex agglutination, as described above. For the sake of quantification of the frequency change depending on the degree of agglutination, the following procedure was introduced. The data points
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.
.
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Flgure 6. Determlnation of the initial rate of frequency change. (A) Smoothing of a part of the data of the trace c in Figure 4. The arrow indlcates the additlon of serum to the latex suspension. The slope of the exhibitedline Isan lnitlalrate ( V). (B)First derivatlvesof the smoothed data in A.
measured intrinsically have more or less somewhat random errors. In order to avoid this problem and to evaluate first derivatives at each data point, the frequency data were analyzed on a computer by the method of least squares using convolution of the data p0inta.~2 Here, a 5-point quadratic smoothing procedure is applied to the data of Figure 4 trace c; this procedure does not excessively smooth the raw data, and therefore does not result in altering the important information in the raw data (the biphasic response in frequency changes). The smoothed data and the first derivatives calculated are presented in parts A and B of Figure 6, respectively. After the addition of serum, the first derivative changed substantially and then became almost constant, as shown in Figure 6B. Therefore, we could draw the line shown in Figure 6A and call the slope of the line the initial rate of gradual frequency change (V = (-AF/K)/time). The value of V was 49.7 X 104 (Hzs-l) in the case of Figure 4B. The frequency change for the calculation of V could be obtained within 2-3 min after the addition of serum. The magnitude of V decreases as the AS0 amount in the serum decreases, as described later. Muramatsu et al.33 also introduced a different analysis on the basis of first derivatives of frequency change for a gelation reaction. The frequency change was monitored, and the corresponding differential response was computed. The differential value increased as the gelation progressed, reached a maximum value, and decreased. They determined how long it took for the differential value to decrease to 10% of the maximum value after the beginning of gelation and considered the time as an index. The detection of CRP was used with an end-point method in a previous report35so that the assay time took about 60 min, a reduction of which was required. Since the batch cell was used here, the sample could be directly injected into the cell. This procedure allows us to measure the initial velocity of frequency change, decreasing the assay time. Effect of Serum Dilution on Linearity. Figure 7 shows the effect of dilution of serum on the linearity of the initial rate. As described above, the nonspecific adsorption effect on the change of frequency was still slightly apparent even in the presence of the latex suspension, so that if the AS0 positive serum was diluted only with PB, this effect would be artificially reduced. In order to compensate for the decrease of proteins in a serum depending on dilution, a serum of high A S 0 value (1040 IU mL-I) was diluted with physi(42) Savitzky, A.; Golay, M. Anal. Chem. 1964, 36,1627-1639.
ANALYTICAL CHEMISTRY, VOL. 64, NO. 21, NOVEMBER 1, lQ92 2487
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ological saline containing BSA (PSB), representing the same Am as the ASO-positive serum. When each serum diluted with PSB was added to the latex-SO suspensions, the frequency changes were measured. As shown in Figure 7, the Vof the frequency change increased linearly as AS0 amounts increased. The dilution of 0.125correspondedto 130IU mL-’. Since the cut-off value of AS0 is 200 or 300 IW mL-1 for adulta or children, respectively,’3 the LPEIA for AS0 can be expected for clinical use at the point of sensitivity.
Correlation of V with AS0 Value Measured with Turbidimetry. In order to investigate the possibility of application to the clinical area, clinical specimens were supplied for measurement with the LPEIA. When each serum from the 24individualsnegative or positive in AS0 was added to the latax-SO suspension, the V of the frequency change was measured according to the above described procedure. The V values were compared with AS0 values (IU mL-9 measured with the turbidimetric latex agglutination method (Figure 8). The correlation coefficient between V and the AS0 was 0.950(F< 0.01),indicatingthat the LPEIA for AS0 correlated with the usual turbidimetric method. AS0 in a serum could be detected with LPEIA, indicating that LPEIA can be used for the detection of not only antigens (43) Ohkuni, M.Nipponrinsho 1990, Suppl., 848-850.
400
800
1‘200
ASO/IU rnl-’ Flgure 8. Correlation of V with AS0 value measured with the turbidimetry. The correlation coefficient ( r ) was 0.950 (P < 0.01, n = 24).
like CRP, but also antibodies. As mentioned above, since many protein-coated latex particles have become available reagents in recent years, LPEIA requires no immobilization of antigen, antibody, etc. on the surface of piezoelectric quartz crystal, in contrast to other immunosensors using a crystal.~~*25-30 In addition, this method can be easily automated for clinical use, similar to photometric methods.6J”18 However, it ia still unknown what factorscause the frequency change of the quartz crystal owing to the latex agglutination, and this is a further important issue. Furthermore, other combinations of antigen and latex-antibody or antibody and latex-antigen should be examined.
ACKNOWLEDGMENT We are grateful to T. Hosokawa and F. Ohta, Ibaraki Research Laboratory, Hitachi Chemical, Co., Ltd., for providing the reagents and the serum with high AS0 titer; T. Fukui, Byotai-Seiri Laboratory, Tokyo, for providing ASOnegative or -positive sera and the turbidimetric measurement of A S 0 Y. Kunito, Toyo Communication Equipment Co., Ltd.,for useful advice; and the Shimadzu Science Foundation for the financial support. RECEIVED for review February 14,1992. Accepted August
6,1992.
