further simplification of ultrasensitive enzyme immunoassay (immune complex transfer enzyme...

Journal of Clinical Laboratory Analysis 9:204-211 (1995)

Further Simplification of U ltrasensi tive Enzyme I mm u noassay (Immune Complex Transfer Enzyme Immunoassay) for

Anti-HTLV-I IgG Using Microplates and Fluororeader Setsuko Ishikawa,' Seiichi Hashida,' Hidetaka Nakamoto,* Satoshi Tanaka?

Masaaki K~j irna,~ and Eiji Ishikawa' 'Department of Biochemistry, Medical College of Miyazaki, Miyazaki, 'Diagnostics R& D Department,

Sumitomo Pharmaceuticals Co., Osaka, and 3Shibayagi Co., Gumma, Japan

In a previously reported ultrasensitive enzyme immunoassay (immune complex transfer enzyme immunoassay) for anti-HTLV-l IgG, polystyrene beads were handled with tweezers, and bound p-D-galactosidase activity was measured with a fluorometer. The use of tweezers was causative of false-positivity by carryover, and testing many samples was diff icult. Recently, these drawbacks have been minimized using microplates and a fluororeader. However, tweezers were still required in the initial and final steps. In the present study, the immune complex, comprising 2,4-dinitrophenyl-antigen, anti-HTLV- I IgG, and antigen-P-0-galactosidase conjugate, was formed in and trapped onto microplate wells coated with (anti-2,4- dinitrophenyl group) IgG. Subsequently, the microplate wells were incubated with&N-2,4- dinitrophenyl-L-lysine and polystyrene beads, modified by attaching to plates through cylindrical bars and coated with (an-

tihuman IgG y-chain) IgG, to transfer the immune complex from the microplate wells to the modified polystyrene beads. Alterna- tively, modified polystyrene beads coated with (anti-2,4-dinitrophenyl group) IgG and microplate wells coated with (antihuman IgG y-chain) IgG were substituted for microplate wells coated with (anti-2,4-dinitrophenyl group) IgG and modified polystyrene beads coated with (antihuman IgG y-chain) IgG, respectively. The fluorescence intensity for bound p-D-galactosidase activity was quickly measured with a fluororeader. Thus the modified polystyrene beads were transferred from wells to wells more quickly and easily without tweezers, eliminating false-positivity due to carryover, and it became easy to test many samples with high sensitivity and reliability, although the assay of bound p-D- galactosidase activity became slightly more time-consuming. o 1995 Wiley-Liss, tnc.

Key words: humanT-cell leukemia virus, adultT-cell leukemia, P-D-galactosidase, fluorornetry, carryover

INTRODUCTION

An ultrasensitive solid phase enzyme immunoassay (immune complex transfer enzyme immunoassay) for antithyroglobulin IgG (l), anti-HTLV-I IgG (1) and anti-HIV-1 IgG (2) in serum and urine has been described. Antibody IgG was reacted simultaneously with 2,4-dinitrophenylated antigen and enzyme-labeled antigen. The immune complex formed, comprising the three components, was trapped onto polystyrene beads coated with affinity-purified (anti-2,4-dinitrophenyl group) IgG. After washing, the immune complex was eluted from the polystyrene beads with excess of &N-2,4- dinitrophenyl-L-lysine and transferred onto clean polystyrene beads coated with affinity-purified (antihuman IgG y-chain) IgG. The enzyme activity bound to the last polystyrene beads was assayed by fluorometry. Low levels of antithyroglobulin IgG (1), anti-HTLV-I IgG (l), and anti-HIV-1 IgG (2) below those detectable by conventional methods have been detected.

However, polystyrene beads were transferred from test tubes to test tubes with tweezers, and tips of the tweezers had to be washed carefully after each transfer of polystyrene beads to eliminate false-positives due to carryover (3). In addition, fluorescence intensities for bound enzyme activities of many samples were measured one by one with a spectrofluorophotometer. This was tedious and time-consuming, making the assay of many samples difficult.

Recently, microplates and a fluororeader have been substituted for test tubes and a spectrofluorophotometer, respectively (3). Polystyrene beads were transferred quickly and easily from wells to wells by placing a microplate upside down on that containing polystyrene beads and turning the two plates

Received October 4, 1994; accepted October 10, 1994.

Address reprint requests to Dr. Eiji Ishikawa, Department of Biochemistry, Medical College of Miyazaki, Kiyotake, Miyazaki 889-16, Japan.

Q 1995 Wiley-Liss, Inc.

Enzyme lmmunoassay of Anti-HTLV-l IgG 205

Aff inity-Purification of Antibodies

IgG (anti-2,4-dinitrophenyl-bovine serum albumin) and IgG (antihuman IgG y-chain) were affinity-purified by elu- tion at pH 2.5 from columns of 2,4-dinitrophenyl-bovine serum albumin-Sepharose 4B and human IgG-Sepharose 4B, respectively (6).

together upside down. Tweezers were not used for transfer of polystyrene beads, minimizing the possibility of false-positivity due to carryover, Fluorescence intensities for bound enzyme activities were measured quickly using a fluororeader. Thus it has become easy to test many samples with high sensitivity. However, tweezers have still to be used for addition and removal of polystyrene beads in the initial and final steps.

This report describes further simplification of the immune complex transfer enzyme immunoassay for anti-HTLV-I IgG.

MATERIALS AND METHODS

Buffer

The regularly used buffer was 10 mmol/L sodium phosphate buffer, pH 7.0, containing 1.0 mmol/L MgClZ, 1.0 g/L NaN3 and 1 .O g/L bovine serum albumin (fractionV, Intergen Co., Purchase, NY) (buffer A).

Cys-Env gp46(188-224) of HTLV-I

Cys-env gp46( 188-224) of HTLV-I (H2N-Cys-Pro'**-Pro- Leu-Leu-Pro-His-Ser-Asn-Leu-Asp-His-Ile-Leu-Glu-Pro- Ser-1le-Pro-Trp-Lys-Ser-Lys-Leu-Leu-Thr-u- Thr-Leu-Gln-Ser-Thr-Asn-Tyr-Thrzz4-COOH) was obtained from Peptide Institute (Osaka, Japan).

Anti bodies

Rabbit (anti-2,4-dinitrophenyl-bovine serum albumin) serum was obtained from Shibayagi Co. (Gumma, Japan). Rabbit (antihuman IgG y-chain) IgG was obtained from Medical and Biological Laboratories Co. (Nagoya, Japan). IgG was pre- pared from serum by fractionation with Na,SOJ followed by passage through a column of DEAE-cellulose (4). The amount of IgG was calculated from the absorbance at 280 nm (4).

2,4-Dinitrophenyl-Bovine Serum Albumin

Thiol groups were introduced into bovine serum albumin molecules using N-succinimidyl-S-acetylmercaptoacetate and were reacted with maleimide groups introduced into &N-2,4- dinitrophenyl-L-lysine molecules using N-succinimidyl-6- maleimidohexanoate (5 ) . The amount of bovine serum albumin, 2,4-dinitrophenyl groups, and 2,4-dinitrophenyl- bovine serum albumin was calculated from the absorbance at 280 nm and 360 nm (5) . The average number of 2,4-dinitrophenyl groups introduced per albumin molecule was 6.0.

Protein-Sepharose 4 8

2,4-Dinitrophenyl-bovine serum albumin (10 mg) and human IgG ( 1 0 mg) were coupled to CNBr-activated Sepharose 4B (1 .O g, Pharmacia LKB BiotechnologyAB, Uppsala, Swe- den) according to the instructions of Pharmacia LKB Bio- technology.

Coating of Polystyrene Beads with Affinity- Purified (Anti-2,4-Dinitrophenyl Group) IgG and Affinity-Purified (Antihuman IgG y-Chain) IgG

Colored and white polystyrene beads (3.2 mm in diameter, Immuno Chemical, Okayama, Japan) were coated by physi- cal adsorption with affinity-purified (anti-2,4-dinitrophenyl- bovine serum albumin) IgG (0.1 g/L) and affinity-purified (antihuman IgG y-chain) IgG (0.1 g/L), respectively (7).

2,4-Dinitrophenyl-Bovine Serum Albumin-Cys-Env gp46(188-224) Conjugate and Cys-Env gp46(188- 224)-P-D-Galactosidase Conjugate

Cys-env gp46( 188-224) was reacted with maleimide groups introduced into 2,4-dinitrophenyl-bovine serum albumin molecules using N-succinimidyl-6-maleimidohexanoate and into P-D-galactosidase molecules from Escherichia coli using NJV -o-phenylenedi m alei mide (8).

Previous (Immune Complex Transfer) Enzyme lmmunoassay

In test tubes, serum samples (up to 20 pl) were mixed successively with 50 pg of inactive P-D-galactosidase (p- galactosidase-mutein: Boehringer Mannheim GmbH, Mannheim, Germany) in 80 pl of buffer A containing 0.4 mol/L NaC1, up to 20 pI of nonspecific rabbit serum, and 100 fmol each of 2,4-dinitrophenyl-bovine serum albumin-Cys-env gp46( 188-224) conjugate and Cys-eni. gp46( 188-224)-P-D-galactosidase conjugate in 50 p1 of bufferA containing 0.4 mol/L NaCl (Fig. 1) (8). The total volume of serum samples and nonspecific rabbit serum was 20 pl. Inactive P-D-galactosidase was used to eliminate interference by anti-P-D-galactosidase antibodies (9). The amount of inactive P-D-galactosidase was calculated from the absorbance at 280 nm using the extinction coef- ficient for P-D-galactosidase (4). To the reaction mixtures, colored polystyrene beads coated with affinity-purified (anti-2,4-dinitrophenyl group) IgG (two beads per assay) were added with tweezers, and the test tubes were allowed to stand overnight. After removing the reaction mixtures, the colored polystyrene beads were washed twice by addition and aspiration of 2 ml of buffer A containing 0.1 mol/L NaCl, were transferred into clean test tubes with tweezers and were incubated for 1 h with 170 p1 of buffer A containing 0.1 mol/L NaCl and 1 mmol/L &N-2,4- dinitrophenyl-L-lysine and white polystyrene beads coated

Previous i m m u noassay

- + +a3

I DNP-Ag IgG Ag-Enz

Test tube

Anti-DNP- solid phase

Anti-lgG- solid phase

O U O DN P- lysine

Present immunoassay

I II

Reaction mix t u re

Well

m J

16 J

U

Fig. 1. group, Ag: antigen, Enz: enzyme, 4MUG:4-methylumbellife1yl-~-D-galactoside.

Present enzyme immunoassays I and I1 in comparison with the previous one. DNF': 2,4-dinitrophenyl


Present (Immune Complex Transfer) Enzyme lmmunoassay I

Serum samples were mixed successively with inactive P- D-galactosidase, nonspecific rabbit serum, and the two conjugates, as described above, in microplate wells (Maxisorp, A/S Nunc) coated with affinity-purified (anti-2,4-dinitrophenyl group) IgG and allowed to stand overnight (Fig. 1). The microplate wells were washed twice with 0.3 ml of buffer A containing 0.1 molb NaC1. To the microplate wells, 170 p1 of buffer A containing 0.1 mol/L NaCl and 1 mmol/L ~N-2,4-dinitrophenyl-L-lysine and modified polystyrene beads coated with affinity-purified (antihuman IgG y- chain) IgG were added, and the reaction mixture with the modified polystyrene beads in the microplate wells were allowed to stand for 3 h. The modified polystyrene beads were washed twice with buffer A containing 0.1 mol/L NaC1, and P-D-galactosidase activity bound to the modified polystyrene beads was assayed for 2.5 h in black microplate wells (Dainippon) for fluorometry with a total volume of 170 pI using 4-methylumbelliferyl-~-D-galactoside as a substrate (4). The enzyme reaction was stopped by addition of a mixture of 50 pl of 0.1 mol/L glycine- NaOH buffer, pH 10.3, and 3 pl of 8 mol/L NaOH. The fluorescence intensity was measured using 355 nm for excitation and 460 nm for emission analysis with a fluororeader for microplates (Fluoroskan 11, Labsystems, Helsinki, Finland). All the processes were performed at room temperature.

with affinity-purified (antihuman IgG y-chain) IgG (two beads per assay). The colored polystyrene beads were removed with tweezers, and the incubation was continued for 2 h. The white polystyrene beads were washed and transferred into clean test tubes with tweezers as described above. P-D-Galactosidase activity bound to the white polystyrene beads was assayed in a total volume of 170 pl for 1.5 h by fluorometry using 4-methylumbelliferyl-j3-D- galactoside as a substrate (4). The enzyme reaction was stopped by adding 2.5 ml of 0.1 mol/L glycine-NaOH, pH 10.3. The fluorescence intensity was measured relative to that of 1 x mol/L 4-methylumbelliferone in 0.1 mol/ L glycine-NaOH buffer, pH 10.3, using 360 nm for excitation and 450 nm for emission analysis with a spectrofluorophotometer (RF-5 10, Shimadzu Seisakusho, Kyoto, Japan). All the processes were performed at room temperature.

Coating of Microplate Wells With Affinity-Purified (Anti-2,4-Dinitrophenyl Group) IgG and Affinity-Purified (Antihuman IgG YChain) IgG

An aliquot (170 111) of 0.1 mol/L sodium phosphate buffer, pH 7.0, containing affinity-purified (anti-2,4-dinitrophenyl group) IgG (25 or 50 mg/L) and affinity-purified (antihuman IgG y-chain) IgG (5 or 50 mg/L) was added to each well of transparent microplates (Maxisorp, Nunc-Immuno Module, Cat No. 468667, A/S Nunc, Roskilde, Denmark, and Falcon 3072, Becton Dickinson & Co., Lincoln Park, NJ) and black microplates for fluorometry (Dainippon Pharmaceutical Co., Osaka, Japan), respectively, and allowed to stand at 4°C overnight. The microplate wells were washed with buffer A containing 0.1 mol/L NaCl and stored at 4°C after addition of the same buffer (300 pl) to each well.

Coating of Modified Polystyrene Beads With Affinity-Purified (Antihuman IgG y-Chain) IgG and Aff inity-Purified (Anti-2,4-Dinitrophenyl Group) IgG

Polystyrene beads (5 mm in diameter, Immuno Chemical, Okayama, Japan) were modified by attaching to plates through cylindrical polystyrene bars (3.0 mm in diameter and 5.5 mm in length) with an adhesive (Quick Bond V, Kansai Polymer Research Institute, Uji, Japan) so as to be used for reaction in microplate wells (Fig. I). The modified polystyrene beads were washed with a nonionic detergent (10 g/L) (SCAT 20X- PF: polyethylene fattyalcohol ether, Dai-Ichi Kogyo Seiyaku Co., Kyoto, Japan) and immersed in 0.1 mol/L sodium phosphate buffer, pH 7.0, containing affinity-purified (antihuman IgG y-chain) IgG (50 mg/L) or affinity-purified (anti-2,4- dinitrophenyl group) IgG (5 , 25 or 50 mg/L) at 4°C overnight (7). The modified polystyrene beads were washed with buffer A containing 0.1 mol/L NaCl and stored in the same buffer at 4°C.

Present (Immune Complex Transfer) Enzyme lmmunoassay II

In conventional microplate wells (Falcon 3072, Becton Dickinson and Company, Lincoln Park, NJ), serum samples were mixed successively with inactive P-D-galactosidase, nonspecific rabbit serum, the two conjugates as described above (Fig. 1). To the reaction mixtures (170 pl), modified polystyrene beads coated with affinity-purified (anti-2,4-dinitrophenyl group) IgG were added, and the reaction mixtures with the modified polystyrene beads in the microplate wells were allowed to stand overnight. The modified polystyrene beads were washed twice with buffer A containing 0.1 mol/L NaCl and incubated for 1 h with 170 p1 of buffer A containing 0.1 mol/L NaCl and 1 mmol/L ~N-2,4-dinitrophenyl-L-lysine in black microplate wells for fluorometry (Dainippon), which had been coated with affinity-purified (antihuman IgG y-chain) IgG. The modified polystyrene beads were removed, and the incubation was continued for 2 h. The black microplate wells were washed twice with 0.3 ml of buffer A containing 0.1 mol/L NaCI. p-D-Galactosidase activity bound to the black microplate wells was assayed for 2 h as described in the present immunoassay 1. All the processes were performed at room temperature.

208 lshikawa et al.

Serum Samples

Serum samples were collected from healthy HTLV-I seropositives and seronegatives and stored at -20OC. The seropositivity was confirmed by the gelatin particle aggluti- nation test using a commercial kit (SERODTA-ATLA; Fujirebio, Tokyo, Japan) and the previous immune complex transfer enzyme immunoassay using a synthetic peptide, Cys- env gp46(188-224), as antigen (8).

RESULTS AND DISCUSSION

The previous and present (immune complex transfer) enzyme immunoassays for anti-HTLV-I IgG were performed as shown schematically in Figure 1. In the previous enzyme immunoassay, anti-HTLV-I IgG was incubated simultaneously with 2,4-dinitrophenyl-bovine serum albumin-Cys-env gp46( 188-224) conjugate and Cys-env gp46( 188-224)-P-D- galactosidase conjugate in test tubes, and the immune complex formed, comprising the three components, was trapped onto colored polystyrene beads coated with affinity-purified (anti-2,4-dinitrophenyl group) IgG (two beads per assay). After washing, the colored polystyrene beads were transferred to clean test tubes with tweezers and incubated with excess of ~N-2,4-dinitrophenyl-L-lysine and white polystyrene beads coated with affinity-purified (antihuman IgG y-chain) IgG (two beads per assay) to transfer the immune complex from the colored polystyrene beads to the white polystyrene beads. After removing the colored polystyrene beads with tweezers, the white polystyrene beads were washed and transferred to clean test tubes with tweezers to measure bound 0-D-galactosidase activities one by one using a spectrofluorophotometer.

In the present enzyme immunoassay I, the immune complex, comprising the three components, was formed in and trapped onto microplate, wells (Maxisorp, Nunc), which had been coated with affinity-purified (anti-2,4-dinitrophenyl group) IgG. The microplate wells were washed and incubated with EN-2,4-dinitrophenyl-L-lysine and polystyrene beads, which had been modified by attaching to plates through cylindrical bars and coated with affinity-purified (antihuman IgG y-chain) IgG to transfer the immune complex from the microplate wells to the modified polystyrene beads. The modfied polystyrene beads were washed and placed in black

microplate wells for fluorometry containing the substrate for P-D-galactosidase, The fluorescence intensity for P-D-galactosidase activity bound to the modified polystyrene beads was quickly read by a fluororeader.

Alternatively, in the present enzyme immunoassay 11, the immune complex, comprising the three components, was formed in conventional microplate wells and trapped onto modified polystyrene beads coated with affinity-purified (anti- 2,4-dinitrophenyl group) IgG. Subsequently, the modified polystyrene beads were washed, placed in black microplate wells for fluorometry, which had been coated with affinity- purified (antihuman IgG y-chain) IgG, and incubated with EN-2,4-dinitrophenyl-L-lysine to transfer the immune complex from the modified polystyrene beads to the black microplate wells. After removing the modified polystyrene beads and washing the black microplate wells, P-D-galactosidase activities bound to the black microplate wells were assayed by fluorometry. The fluorescence intensity was quickly measured with a fluororeader.

Condition for Coating Microplate Wells and Modified Polystyrene Beads With Aff inky-Purified (Anti-2,4-Dinitrophenyl Group) IgG and (Antihuman IgG y-Chain) IgG, Respectively

Microplate wells (Maxisorp, Nunc) were coated with two different concentrations of affinity-purified (anti-2,4- dinitrophenyl group) IgG (25 and 50 mg/L) and substituted for polystyrene beads coated with the same antibody IgG in the previous enzyme immunoassay. No difference in the signal was observed between the two conditions, and the ratios of signals for seropositives to that for a seronegative were slightly (9.0-9.4%) higher with the microplates than in the previous enzyme immunoassay (Table 1). With microplates (Falcon 3072), the ratios were - 10-fold lower. Therefore, microplates (Maxisorp, Nunc) coated with 25 mg/L were used in the subsequent experiments. Modfied polystyrene beads were coated with 50 m a of affinity-purified (antihuman IgG y-chain) IgG.

Sensitivity of the Present Enzyme lmmunoassay I

Three different levels of anti-HTLV-I IgG in seropositive sera were tested by the previous enzyme immunoassay and

TABLE 1. Ratio of Seropositive to Seronegative Signals by Substituting Microplate Wells Coated With Affinity-Purified (Anti-2,4-Dinitrophenyl Group) IgG for Polystyrene Beads Coated With the Same Antibody IgG in the Previous Enzyme Immunoassaya

Previous enzyme irnmunoassay using microplates (Maxisorp, Nunc) coated with affinity-purified (anti-2,4-dinitrophenyl group) IgG

Serum Previous enzyme immunoassay with test tubes 25 m@ SO mg/L

sample Signal Ratio Signal Ratio Signal Ratio

Seropositive 355 * 22 86.6 454 + 13 94.6 456 k 10 95.0

aBound P-u-galactosidase activity was assayed for 1 .S h, and the fluorescence intensity was measured with a spectrofluorophotometer. Signals are expressed as mean * SD (n =3-8).

Seronegative 4.1 ? 0.1 1 .0 4.8 If: 0.3 I .o 4.8 * 0.3 1.0


TABLE 2. Ratio of Seropositive to Seronegative Signals by the Previous Enzyme Immunoassay and the Present Enzyme Immunoassay I'

Previous enzyme immunoassay Present enzyme immunoassay I

Serum sample Signal Ratio Signal Ratio

Seronegative 5.0 f 0.1 1 .o 0.71 t 0.04 1.0 Seropositive

1 19.3 k 0.7 3.9 2.90 * 0.04 4.1 2 153 f 5 30.6 21.1 f 0.3 30.1 3 1,064 + 36 213 143 t 2 201

'Bound P-u-galactosidase activity was assayed for 1.5 h and 2.5 h in the previous enzyme immunoassay and the present enzyme immunoassay 1, respectively. Signals are expressed as mean & SD (n=3).

the present enzyme immunoassay I, and the sensitivities were compared by calculating the ratios of signals for seropositives to that for a seronegative. Typical results are shown in Table 2. The present enzyme immunoassay I was as sensitive as the previous enzyme immunoassay, when bound P-D-galactosidase activity was assayed for a longer time in the present enzyme immunoassay I (2.5 h) than in the previous enzyme immunoassay (1.5 h).

Condition for Coating Black Microplate Wells With Affinity-Purified (Antihuman IgG yChain) IgG

Black microplate wells for fluorometry were coated with two different concentrations of affinity-purified (antihuman IgG y-chain) IgG (5 and 50 m a ) and substituted for polystyrene beads coated with the same antibody IgG in the previous enzyme immunoassay. The fluorescence intensity for P-D-galactosidase activity bound to the black microplate wells was measured by a fluororeader as in the present enzyme immunoassay. The ratios of signals for seropositives to that for a seronegative were not different between the two conditions (5 and 50 mg/L) and considerably (56.3-72.6%) higher than those by the previous enzyme immunoassay (Table 3). Therefore, the lower concentration ( 5 mg/L) was used in the subsequent experiments.

Condition for Coating Modified Polystyrene Beads With Affinity-Purified (Anti-2,4- Dinitrophenyl Group) IgG

Modified polystyrene beads were coated with three different concentrations of affini ty-purified (anti-2,4-dinitrophenyl group) IgG (5, 25, and 50 mgL) and tested by the present enzyme immunoassay I1 (Table 4). The use of modified polystyrene beads coated with 25 mg/L of the antibody IgG pro- vided slightly higher signals than that of modified polystyrene beads coated with 5 mg/L and 50 mgL of the antibody IgG. Therefore, polystyrene beads were coated with 25 mg/L of the antibody IgG in the subsequent experiments.

Sensitivity of the Present Enzyme lmmunoassay II

Three different levels of anti-HTLV-I IgG in seropositive sera were tested by the previouslenzyme immunoassay and the present enzyme immunoassay 11, and the sensitivities were compared by calculating the ratios of signals for seropositives to that for a seronegative. Typical results are shown in Table 4. The signal for a seronegative (the nonspecific signal) de- creased significantly as compared with that in the present enzyme immunoassay 1 ('Tables 2 and 4). As a result, the present enzyme immunoassay I1 was as sensitive as the previous enzyme immunoassay, when bound P-D-galactosidase activity

TABLE 3. Ratio of Seropositive to Seronegative Signals by Substituting Black Microplate Wells (for Fluorometry) Coated With Affinity-Purified (Antihuman IgG y-Chain) IgG for Polystyrene Beads Coated With the Same Antibody IgG in the Previous Enzyme lmmunoassaya

Previous enzyme immunoassay using black microplates coated with affinity-purified (anti-human IgG y-chain) IgG

Previous enzyme immunoassay 5 mg/L 50 mg/L Serum sample Signal Ratio Signal Ratio Signal Ratio

Seronegative 3.7 1 .o 0.44 1 .o 0.44 1 .o Seropositive

1 391 106 75.6 172 80.5 183 2 1,507 407 286 650 280 636

aBound P-D-galactosidase activity was assayed for 1.5 h, and the fluorescence intensity was measured with a fluororeader in the previous enzyme immunoassay using black microplates.

210 lshikawa et al.

TABLE 4. Ratio of Seropositive to Seronegative Signals by the Previous Enzyme Immunoassay and the Present Enzyme Immunoassay 11"

Present enzvme immunoassav I1

Conccntration of (anti-2,4-dinitrophenyl group) IgG for coating modified polystyrene beads Previous enzyme

immunoassay 5 mg/L 25 mg/L 50 m u ,

Serum sample Signal Ratio Signal Ratio Signal Ratio Signal Ratio

Seronegative 4.7 f 0.1 1 .o 0.48 + 0.01 1 .o Seropositive

1 19.3 ? 0.4 4.1 .~ - 2.00 k 0.04 4.2 1.73 ? 0.06 3.6 2 168 f I 35.7 - - 16.9 k 0.4 35.2 12.8 + 0.1 26.7 3 1184 f 55 252 88.9 + 7.1 185 109 f 5 221 177 85.1 + 2.7

"Bound P-~-galactosidase activity was assayed for 1.5 h and 2.0 h in the previous enzyme immunoassay and the present enzyme immunoassay II, respectively. Signals are expressed as mean k SD (n=3).

was assayed for an only slightly longer time in the present enzyme immunoassay I1 (2 h) than in the previous enzyme immunoassay (1.5 h).

Advantages and Disadvantages of the Present Enzyme I m mu noassays

In the previous enzyme immunoassay using test tubes, polystyrene beads were transferred from test tubes to test tubes with tweezers. This was not only tedious but also causative of false-positivity by carryover, unless tips of the tweezers were washed carefully after each transfer of polystyrene beads (3). In addition, it took a long time to measure fluorescence intensities of bound P-D-galactosidase activities for many samples one by one with a spectrofluorophotometer. Thus testing many samples was difficult.

In the present enzyme immunoassays, polystyrene beads attached to plates through cylindrical bars were transferred quickly and easily from wells to wells. No tweezers were required at all, eliminating false-positivity due to carryover. Fluorescence intensities of bound P-D-galactosidase activities for 96 samples could be measured within 1 min and 40 sec by a fluororeader. Thus many samples could be tested much more easily with high sensitivity and higher reliability. And the modified solid phase will be commercially available (Immuno Chemical, Okayama, Japan).

When microplates (Maxisorp, Nunc) coated with affinity- purified (anti-2,4-dinitrophenyl) IgG were substituted for polystyrene beads coated with the same antibody IgG in the previous enzyme immunoassay and when black microplates coated with affinity-purified (antihuman IgG y-chain) IgG were substituted for polystyrene beads coated with the same antibody IgG in the previous enzyme immunoassay, the sensitivity was improved 9.&9.4% and 56.3-72.6%, respectively, as compared with that of the previous enzyme immunoassay as described above (Tables 1 and 3). By contrast, the use of microplates (Maxisorp, Nunc) coated with affinity-purified (anti-2,4-dinitrophenyl group) IgG in combination with modified polystyrene beads coated with affinity-purified (antihu-

man IgG y-chain) IgG (the present enzyme immunoassay I) and the use of black microplates coated with affinity-purified (antihuman IgG y-chain) IgG in combination with modified polystyrene beads coated with affinity-purified (anti-2,4- dinitrophenyl group) IgG (the present enzyme immunoassay 11) required a 1.67- and 1.33-fold, respectively, longer time for the assay of bound P-D-galactosidase activity to obtain the same sensitivity as that of the previous enzyme immunoassay (Tables 2 and 4). Namely, the use of modified polystyrene beads considerably simplified the immune complex transfer enzyme immunoassay but made the assay of bound P-D-galactosidase activity more time-consuming. This draw- back remains to be overcome.

REFERENCES 1.

2.

3.

4.

5.

6.

7.

8.

Ishikawa E, Hashida S, Kohno T, Hirota K, Hashinaka K, Ishikawa S: Principle and applications of ultrasensitive enzyme immunoassay (immune complex transfer enzyme immunoassay) for antibodies in body fluids. J Clin Lab Anal 7~316-393, 1993. Hashida S, Hirota K, Hashinaka K, Saitoh A, Nakata A, Shinagawa H, Oka S, Shimada K, Mimaya J, Matsushita S, Ishikawa E: Detection of antibody IgG to HIV-I in urine by sensitive enzyme immunoassay (immune complex transfer enzyme immunoassay) using recombinant pro- teins as antigens for diagnosis of HIV-1 infection. J Clin Lab Anal

Ishikawa S, Hashida S, Kohno T. Ishikawa E: Use of microplates and fluororeader for ultrasensitive enzyme immunoassay (immune complex transfer enzyme immunoassay) of anti-HTLV-I IgG. J Clin Lab Anal

Ishikawa E, Imagawa M, Hashida S,Yoshitake S, HamaguchiY, UenoT Enzyme-labeling of antibodies and their fragments for enzyme immunoassay and immunohistochemical staining. .I Immunoassuy 4:209-327, 1983. Hashida S, Tanaka K, Yamamoto N, Uno T, Yamdguchi K, Ishikawa E: Detection of one attomole of [Arg8]-vasopressin by novel noncompeti- tive enzyme immunoassay (hetero-two-site complex transfer enzyme immunoassay). J Biochem 110:486492, 1991. Kohno T, Ishikawa E: A highly sensitive enzyme immunoassay of anti- insulin antibodies in guinea pig serum.JBiochem 100:1247-1251,1986. Ishikawa E, Kato K: Ultrasensitive enzyme immunoassay. Scand J Tm- muno18 (suppl7):43-55, 1978. Kohno T, Sakoda I, Ishikawa E Sensitive enzyme immunoassay (immune complex transfer enzyme immunoassay) for (antihumanT-cell leu-

71353-364, 1993.

8~327-331, 1994.

Enzyme lmmunoassay of Anti-HTLV-l IgG 21 1

kemia virus type I) IgG in serum using a synthetic peptide. cys-env gp46(188-224), as antigen. J Clin Lab Anal 6:105-112, 1992.

9. KohnoT, Katsumaru H, Nakamoto H, YasudaT, MitsukawaT, Matsukura S, TsunetoshiY, Ishikawa E: Use of inactive P-D-galactosidase for elimi-

nation of interference by anti-P-D-galactosidase antibodies in immune complex transfer enzyme immunoassay for antithyroglobulin IgG in serum using P-D-galactosidase from Escherichiu coli as label. J Clin Lab Anal 5: 197-205, 1991.

further simplification of ultrasensitive enzyme immunoassay (immune complex transfer enzyme...

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