Flow immunoassay using a cation exchange column and fluorescence-labelled antibody for detection of allergen

Analytica Chimica Acta 354 (1997) 29±34

Flow immunoassay using a cation exchange column and ¯uorescence-labelled antibody for detection of allergen Tae-Kyu Lim, Noriyuki Nakamura, Tadashi Matsunaga* Department of Biotechnology, Tokyo University of Agriculture and Technology, Koganei, Tokyo 184, Japan Received 3 January 1997; received in revised form 23 June 1997; accepted 29 June 1997

Abstract An allergen detection was carried out based on a ¯uorescence immunoassay with a ¯ow injection system. Allergen and ¯uorescein isothiocyanate (FITC) conjugated IgE antibody complex was separated from free FITC conjugated IgE antibody using a cation exchange column. The ¯uorescence intensity correlated linearly with a concentration of allergen in the range of 0.01±2.0 mg mlÿ1. Reuse of free FITC conjugated IgE was possible. This simple and rapid immunoassay method can be used for the continuous detection of allergen. # 1997 Elsevier Science B.V. Keywords: Flow injection; Immunoassay; Fluorescence; Cation exchange column; Antibody; Allergen detection

1. Introduction Many immunoassay methods for detecting allergens have been performed by microtitration [1]. These methods require complicated procedures and take several hours. Flow injection immunoassay is a relatively new approach for performing continuous semior fully-automated measurements of analyte concentrations [2]. During the past several years, ¯ow injection immunoassays using small immunoaf®nity columns, referred to as immunoreactors, in which the antibody or ligand binder is covalently coupled to a rigid support, have been developed. Antibodies [3,4], protein A [5,6], protein G [7] and concanavalin A [2] have been used as ligand binders and Sepharose [8], non-porous silica [9] and Biomag 4100 beads [10] *Corresponding author. Tel.: +81 (423) 887020; fax: +81 (423) 857713; e-mail: [email protected]. 0003-2670/97/$17.00 # 1997 Elsevier Science B.V. All rights reserved. PII S0003-2670(97)00438-8

as solid phases in immunoreactors for ¯ow injection immunoassays. Flow injection immunoassay is an attractive technique because it makes possible to achieve speci®c separation to identify or isolate the antigen±antibody complex continuously. However, a ¯ow immunoassay using immunoreactors required a complicated procedure for continual use because it is dif®cult to separate antibody or antigen binding the corresponding antigen or antibody. Recently, we have developed a ¯ow immunoassay system using an ion exchange column and alkaline phosphatase-conjugated immunoglobulin E (IgE) for the detection of allergen [11]. The ion exchange column was used for separation of labelled allergen±antibody complex and free labelled antibody by difference of their isoelectric points. This method does not require immobilization of antigen (or antibody) on a solid phase. In comparison with previous solid phase immunoassay systems, this system has a

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much shorter assay time, and a minimized sample volume. However, an incubation period was necessary after the addition of luminescent substrate solution and the system could therefore not be fully automated. This paper describes a ¯ow immunoassay using ion exchange column and ¯uorescein isothiocyanatelabelled antibody (FITC-conjugated monoclonal IgE [FITC-IgE]) for simple, continuous and fully automated detection of allergen. We have also investigated the reuse of free FITC-IgE after immunoreaction. 2. Experimental 2.1. Materials Sodium 2,4-dinitrobenzenesulphonate was purchased from Tokyo Chemical Industry (Tokyo, Japan). Bovine serum albumin (BSA) and FITC were obtained from Sigma (St. Louis, MO). Monoclonal mouse immunoglobulin E (IgE, anti-DNP), puri®ed from the ascitic ¯uid of BALB/c  C57 BL-F1 mice bearing the SPE-7 hybridoma [12], was purchased from Seikagaku (Osaka, Japan). Dialysis tubing was Ê purchased from Viskase (seamless cellulose, 24 A pore size, Chicago, IL). Sephacryl S-300 column, PhastGelTM Gradient 8±25, PhastGelTM IEF 3±9, and molecular markers were purchased from Pharmacia (Uppsala, Sweden). All other chemicals used were analytical-reagent or laboratory grade. Deionized±distilled water was used in all procedures. 2.2. Preparation of FITC conjugated IgE The conjugation of FITC to IgE antibody was carried out according to the following procedure [13]. FITC (5 mg) was dissolved in 1 ml of carbonate buffer (0.5 M, pH 9.1). 100 ml of IgE antibody (1 mg mlÿ1) was added to FITC solution. The mixture was incubated for 2 h at room temperature. After incubation, the sample was dialysed for 72 h at 48C against 0.5 M carbonate buffer (pH 9.1), which was changed three times. The concentration of IgE in the solution was determined by the Bradford (Bio-Rad) method [14,15] before and after conjugation. FITC conjugated IgE (anti-DNP) obtained was homogeneous as judged by gel ®ltration using a column packed with Sephacryl S-300.

2.3. Preparation of dinitrophenylated bovine serum albumin (DNP-BSA) A DNP-BSA conjugate (6.2 molecules of DNP) was prepared using sodium 2,4-dinitrobenzenesulphonate and BSA, as described by Little and Eisen [16]. DNPBSA was employed as a model allergen. 2.4. Fluorescence immunoassay procedure using a cation exchange column The ¯ow immunoassay system is shown in Fig. 1(A). The ¯ow of eluent or carrier stream was produced by a pump (P-500, Pharmacia, Uppsala, Sweden). Injections of solutions were effected with a MV-7 injection valve (Pharmacia) ®tted with a 200 ml loop. The pump and injection valve were operated with a controller (LCC-501, Pharmacia). TEFZEL tube (0.5 mm i.d., Pharmacia, Uppsala, Sweden) was used. The typical procedure for the assay was as follows. At ®rst, DNP-BSA (100 ml) was mixed with 100 ml of 1.8 mg mlÿ1 FITC-IgE. The mixture was loaded into an injection loop (200 ml), incubated for 20 min, and then passed into the cation exchange column packed with Sepharose Fast Flow (sulfopropyl type, 16 mm i.d.25 mm; Pharmacia), with phosphate buffer (pH 6.0) as a binding buffer at a ¯ow rate of 1.0 ml minÿ1. A linear gradient of sodium chloride from 0 to 500 mM in phosphate buffer (pH 6.0) was applied for washing the column. The ¯uorescence intensity of FITC-IgE in the eluent was measured using a ¯uorescence spectrophotometer (F-1200, Hitachi, Tokyo, Japan), which was combined with a ¯ow cell unit at room temperature, with the excitation wavelength set at 490 nm and the emission wavelength at 520 nm. Results were recorded on a chart recorder (SP-J5C, Riken Denshi, Tokyo, Japan). 2.5. Flow injection analysis A cation exchange column packed with Sepharose Fast Flow was equilibrated with 50 mM malonate buffer (pH 5.0) at a ¯ow rate of 1.0 ml minÿ1. Then, 100 ml of DNP-BSA and 100 ml of FITC-IgE (1.8 mg mlÿ1) were loaded into a 200 ml injection loop, incubated for 20 min and passed into the cation exchange column for separation with malonate buffer

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Fig. 1. (A) Schematic diagram of flow immunoassay system and (B) principle of flow immunoassay. The reaction mixture consists of DNPBSA (allergen), FITC-IgE and allergen±antibody complex. The mixture is passed through a column that selectively traps FITC-IgE. The eluted species pass through the fluorescence spectrophotometer, and the fluorescence peak associated with the allergen±antibody complex is monitored.

(pH 5.0). When the allergen±antibody complex peak had returned to the baseline, another sample was passed into column for assay. The binding and elution ¯ow rates were maintained at 1 ml minÿ1. 2.6. Reuse of free FITC-IgE bound to the cation exchange resin After the measurement of the allergen±antibody complex, the free conjugated IgE antibody bound to the cation exchange resin was eluted with malonate buffer (pH 5.0) containing 0.5 M NaCl. A desalting column is connected in series after a cation exchange column to adjust the pH of the eluted sample, and mixed with 100 ml of DNP-BSA. The mixture passed into the column for measurement of the complex. Such a procedure was repeated with successive eluted free FITC-IgE. 2.7. Identification of allergen±antibody complex and free FITC-IgE antibody After the elution, the allergen±antibody complex and free conjugated IgE antibody were subjected to native polyacrylamide gel electrophoresis with PhastTM Gel Gradient 8±25 using PhastGel native buffer

strips. Molecular markers used were thyroglobulin (669 kDa), ferritin (440 kDa), catalase (232 kDa), lactate dehydrogenase (140 kDa), and BSA (67 kDa). Electrophoresis, focusing and silver staining of the gels were performed using the Pharmacia PhastsystemTM, according to the manufacturer's instructions. 3. Results and discussion 3.1. Preparation of FITC conjugated IgE The pH-dependence of conjugation reaction between IgE and FITC was studied at various pH values in 0.5 M phosphate (pH 6.5±8.0) and carbonate (pH 8.5±10.0) buffer at 248C. FITC (5 mg) were used for 100 ml of IgE (1 mg mlÿ1) in 1 ml of buffer solution. The conjugation reaction of IgE with FITC reached an equilibrium at 0.09 mg mlÿ1 IgE, when more than 5 mg mlÿ1 of FITC was added. The amount of FITC conjugated with IgE was calculated from the ¯uorescence intensity calibration curve of FITC and molecular weight (185 000) of IgE [17]. The result shows that approximately 106 molecules of FITC bind to a single IgE molecule.

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3.2. Separation of FITC-IgE and allergen±FITC-IgE complex by cation exchange column Before the elution, DNP-BSA (allergen), FITC conjugated IgE and allergen±antibody complex were subjected to isoelectric gel electrophoresis with PhastGelTM IEF 3±9. Electrophoresis, isoelectric focusing and silver staining of the gels were performed by the Pharmacia PhastsystemTM. FITC-IgE and allergen± FITC-IgE complex was separated by the cation exchange column with a linear gradient of sodium chloride in phosphate buffer (pH 6.0), since they have isoelectric points at 4.8 (allergen), 6.7 (FITC-IgE) and 5.0 (allergen±antibody complex), respectively. When FITC-IgE was injected alone into the cation exchange column, only one peak at 5.0 min was observed (Fig. 2(A)). When the reaction mixture of 1.8 mg mlÿ1 FITC-IgE and 2 mg mlÿ1 DNP-BSA was injected, two peaks were obtained at 2.6 min and 5 min, respectively (Fig. 2(B)). The ®rst peak was the allergen± FITC-IgE complex. The ¯uorescence intensity of this complex increased with increasing DNP-BSA concentration. On the other hand, the ¯uorescence intensity of free FITC-IgE antibody decreased. Therefore, the allergen±antibody complex concentration was

Fig. 2. Separation of FITC-IgE and DNP-BSA±FITC-IgE complex by cation exchange column. (A) 1.8 mg mlÿ1 FITC-IgE and (B) reaction mixture of 1.8 mg mlÿ1 FITC-IgE and 2.0 mg mlÿ1 DNPBSA were applied to the cation exchange column respectively when a linear gradient of sodium chloride in phosphate buffer (pH 6.0) was employed. (C) Reaction mixture of 1.8 mg mlÿ1 FITC-IgE and 2.0 mg mlÿ1 DNP-BSA was applied when malonate buffer (pH 5.0) was employed.

estimated from both the increase in ¯uorescence intensity of allergen±antibody complex and decrease of FITC-IgE. However, the selective elution of allergen±antibody complex is required for continuous detection of allergen. We investigated the elution of allergen±antibody complex by keeping free FITC-IgE bound to cation exchange resin. When only free FITC-IgE was injected into the cation exchange column with 50 mM malonate buffer (pH 5.0), no peaks were observed. The reaction mixture containing the allergen±antibody complex and the free DNP-BSA was eluted with malonate buffer. The chromatogram of the reaction mixture showed one peak at 2.6 min after it was passed into the column (Fig. 2(C)). To identify this peak, the eluted solution was concentrated to onetwentieth of its original volume and analyzed by native polyacrylamide gel electrophoresis. The two bands were observed at 69 and 250 kDa (data not shown). An allergen±antibody complex band was detected at 250 kDa, and a free DNP-BSA band at 69 kDa. DNP-BSA was not monitored by ¯uorescence that was derived from FITC at an emission wavelength of 520 nm. Therefore, this chromatographic peak turned out to be the allergen±antibody complex. The ¯uorescence intensity of this complex increased with increasing DNP-BSA concentration. Furthermore, the allergen±antibody complex concentration was estimated from the increase in ¯uorescence intensity. Time course for formation of immunocomplex was examined using various concentrations of DNP-BSA and 1.8 mg mlÿ1 FITC-IgE in the reaction mixture. After immunocomplex formation, the reaction mixture was applied to the cation exchange column. The immunoreaction reached equilibrium within 20 min when 1.8 mg mlÿ1 FITC-IgE was used (Fig. 3). The ¯uorescence intensity of the allergen±antibody complex increased with increasing DNP-BSA concentration in the mixture for 1 min incubation. 3.3. Flow injection analysis and calibration for DNP-BSA concentration Using the ¯ow injection equipment described in the experimental section, various concentrations of allergen were injected into the system with malonate buffer as eluent under optimal ¯ow injection analysis conditions. The response obtained from a series of

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Fig. 3. Time course of FITC-IgE and DNP-BSA complex formation. The experiments were carried out using 1.8 mg mlÿ1 FITC-conjugated IgE (anti DNP) and various concentrations of DNP-BSA: (*) 0.6, (*) 1.0, (
) 1.3 and (&) 2.0 mg mlÿ1.

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Fig. 5. Calibration for DNP-BSA concentration.

precision (<2%) than the conventional ELISA using microtiter plates. Despite the use of puri®ed major allergens, only 21±85% of the variation in skin test results can be accounted for by speci®c IgE level [18]. This might suggest that skin test and the radioallergosorbent test (RAST) are imprecise. The reproducibility of DNP-BSA measurement was examined using ®ve different concentrations (0.02, 0.05, 0.2, 1.0 and 2.0 mg mlÿ1). The coef®cients of variation were within the range of 1.1±2.0%. The data obtained using this system showed good correlation (Rˆ0.990, nˆ10). Fig. 4. Continual flow injection analysis. (A) 2.0, (B) 1.3, (C) 1.0, (D) 0.6 and (E) 0 mg mlÿ1 DNP-BSA were reacted with 1.8 mg mlÿ1 FITC-IgE, and injected into the system. Arrows indicate the sample injection.

injections of the FITC-IgE and DNP-BSA complexes is shown in Fig. 4. The ¯uorescence intensity of this complex increased with increasing DNP-BSA concentration. Furthermore, reversible, reproducible and sensitive responses were obtained, indicating that the liquid-phase immunological sensing system appeared to be working. Injections of FITC-IgE alone gave no signal to indicate that the antibody was bound on cation exchange resin. A typical calibration curve for DNP-BSA is shown in Fig. 5. A linear relationship was obtained between the ¯uorescence intensity and DNP-BSA concentration in the range of 0.01±2.0 mg mlÿ1. The correlation coef®cient was 0.994 within this range. This method was faster (10 min) and simple to use, and gives higher

3.4. Reuse of the free FITC-IgE bound to the cation exchange resin When malonate buffer containing 0.5 M NaCl was used to elute free FITC-IgE antibody after continual assay of allergen±antibody complex, only one peak was observed after 5 min. The extract was analyzed by native polyacrylamide gel electrophoresis. A band was identi®ed that was equal in size to that of FITC-IgE (data not shown). The ¯uorescence intensity of this free FITC-IgE decreased with increasing DNP-BSA concentration. A linear relationship between the ¯uorescence intensity increase of the allergen±antibody complex and the ¯uorescence intensity decrease of free FITC-IgE was therefore obtained (unpublished data, Rˆ0.987, DNP-BSA concentration; 0.01± 2.0 mg mlÿ1). However, the concentration of eluted free FITC-IgE gradually decreased upon repeated use. We investigated to ¯uorescence intensity of the allergen±antibody complex using different concentrations

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of FITC-IgE. As a result, the ¯uorescence intensity of allergen±antibody complex were approximately equal in the range of 1.0±1.8 mg mlÿ1 of FITC-IgE concentration. Therefore, eluted free FITC-IgE can be reused when free FITC-IgE concentration decreased below 1.0 mg mlÿ1. Then 2.0 mg mlÿ1 of DNP-BSA (100 ml) and 100 ml of free FITC-IgE were loaded into a 200 ml injection loop, and passed into the cation exchange column. The results indicate that ¯uorescence intensity of allergen±antibody complex using free FITCIgE was approximately equal after using it seven times, without antibody activity decrease allowed (Fig. 6). The data obtained using this system was reliable, and ef®cient reuse of antibody. 3.5. Specificity of flow immunoassay system The speci®city of the system was investigated by injecting various proteins and FITC-IgE (anti DNP) (Fig. 7). The ¯uorescence intensity only increased signi®cantly in the presence of DNP-BSA. 4. Conclusion Many immunoassay methods for detecting allergens have been developed. However, these tests are time-consuming and require complicated procedures. Therefore, a simple, safe and rapid method for the detection of allergen is still required. In particular, the proportions of analyte which give a series of signals using this method are unknown. Ion-exchange chromatography could separate the FITC-IgE/DNP-BSA complex from free FITC-IgE by difference of their isoelectric points. This system allowed optimization

Fig. 6. Reuse of free FITC-IgE bound to the cation exchange resin.

Fig. 7. Specificity of flow immunoassay system.

of reaction conditions, including antibody concentration compared with ordinary solid-phase immunoassay systems, assay time is reduced. Reuse of free FITCIgE is also possible with this system. The data show that this is accurate and reproducible measurements.

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