Journal of Magnetism and Magnetic Materials 194 (1999) 126—131

Chemiluminescence enzyme immunoassay usingProteinA-bacterial magnetite complex

Tadashi Matsunaga!,*, Rika Sato", Shinji Kamiya", Tsuyosi Tanaka!,Haruko Takeyama!

!Department of Biotechnology, Tokyo University of Agriculture and Technology, 2-24-16, Naka-cho, Koganei, Tokyo 184-8588, Japan"TDK Akita Laboratory Corporation, 15 Aza-Gashomen, Hirasawa, Nikaho-machi, Yuri-gun, Akita 018-0493, Japan

Abstract

Bacterial magnetic particles (BMPs) which have ProteinA expressed on their surface were constructed using magAwhich is a key gene in BMP biosynthesis in the magnetic bacterium Magnetospirillum sp. AMB-1. Homogenouschemiluminescence enzyme immunoassay using antibody bound ProteinA-BMP complexes was developed for detectionof human IgG. A good correlation between the luminescence yield and the concentration of human IgG was obtained inthe range of 1—103 ng/ml. ( 1999 Published by Elsevier Science B.V. All rights reserved.

Keywords: Bacterial magnetic particles (BMPs); ProteinA-BMP complex; Chemiluminescence enzyme immunoassay;magA gene

1. Introduction

Magnetic bacteria have been isolated from freshand marine sediments and are known to producemagnetic particles [1—4]. Bacterial magnetic par-ticles (BMPs) are small in size (50—100 nm) anddisperse very well because they are covered witha stable lipid membrane [5]. Enzymes and antibod-ies immobilized onto the surface of BMPs usingboth bifunctional reagents and glutaraldehydewere found to have higher activities than thoseimmobilized onto artificial magnetic particles [6].On the basis of these properties, BMPs have been

*Corresponding author. Tel.: #81-42-388-7020; fax:#81-42-385-7713; e-mail: tmatsuna@cc.tuat.ac.jp.

applied to fluoroimmunoassay [7—9], mRNA re-covery [10], DNA carriers [11], and chemilumines-cence enzyme immunoassay (EIA) [12].

We have developed techniques to genetically ma-nipulate magnetotactic bacterium, Magnetospiril-lum sp. AMB-1 [13] and have isolated a key gene(magA) required for BMP synthesis [13]. Intracellu-lar localization of the MagA protein was confirmedusing the firefly luciferase (Luc) gene. The MagA-Luc fusion protein was detected on the surface ofBMPs from AMB-1 transformed with the magA-lucgene fusion [14]. These results demonstrated thepossibility of displaying functional proteins on thesurface of BMPs using magA gene fusions. Func-tional BMPs are produced by a transformed mag-netic bacterium and subsequently easily isolatedfrom disrupted cells.

0304-8853/99/$ — see front matter ( 1999 Published by Elsevier Science B.V. All rights reserved.PII: S 0 3 0 4 - 8 8 5 3 ( 9 8 ) 0 0 5 7 5 - 7

In this study, we have cloned a proteinA-magAhybrid gene into magnetic bacterium strain AMB-1, and produced ProteinA-BMP complexes. Wethen developed the chemiluminescence EIA usingProteinA-BMP complexes for highly sensitiveand rapid detection of human immunoglobulinG (IgG).

2. Materials and methods

2.1. Materials

All restriction endonucleases and E. coli T4 DNAligase were purchased from Takara Shuzo (Kyoto,Japan). Lumi-Phos 530, which includes lumigenPPD, 4-methoxy-4(3-phosphatephenyl) spiro[1, 2-dioxetane-3, 2’-adamantane] disodium salt (3.3]10~4 M) as luminescence substrate for alkalinephosphatase, and PicaGene Luminescence Kit,which includes luciferin (470 lM) as luminescencesubstrate for luciferase, were obtained from WakoPure Chemical Industries, Ltd. (Osaka, Japan). Bo-vine serum albumin (BSA) was purchased fromSeikagaku Co. (Tokyo, Japan). Human IgG andalkaline phosphatase conjugated anti-human IgGantibody (polyclonal) were purchased from CosmoBio (Tokyo, Japan). Other reagents were of analyti-cal-reagent or laboratory grade. Deionized, distil-led water was used in all procedures.

2.2. Bacterial strains and growth conditions

E. coli DH5aMCR (Gibco BRL) was used asa recipient for plasmid cloning. E. coli S17-1 wasused for transconjugation of Magnetospillirum sp.AMB-1 [13]. Transformed E. coli was cultured inLuria-Bertani medium at 37°C with the appropri-ate antibiotics. Magnetospillirum sp. AMB-1 wascultured in magnetic spirillum growth medium(MSGM) at 25°C as described previously [15].

2.3. Plasmids and DNA fragments

The plasmid pRK415 (Tc3, lacZ, mob`) is a broadhost range vector for gram-negative bacteria andreplicates in Magnetospirillum sp. AMB-1 [14].The plasmid pNEP (10.9 kbp), derived from

pRK415, contains the magA promoter andribosomal binding site. The plasmid pUM5A, de-rived from E. coli clonong vector pUC19 con-taining the magA gene isolated from a wild typeAMB-1 gene bank, was used as a template forPCR amplification of the magA gene. The plasmidpEZZ18 (Pharmacia Biotech) contains gene se-quence for the Z region which is the IgG anti-body binding domain in ProteinA isolated fromStaphylococcus aureus.

2.4. Construction of a plasmid for expressingproteinA-magA gene fusions

The plasmid pRZM (12.8 kbp), containing theproteinA-magA hybrid gene with the magA pro-moter [14], was constructed as shown in Fig. 1.A BamHI-NurI fragment (1.25 kbp) containing themagA structural gene was ligated to the 3@ end ofthe proteinA gene in pEZZ18. The proteinA-magAhybrid gene was amplified using the followingprimers:5@-GGGTTAACGTAGACAACAAAT-TCAAC-3@ and 5@-GGTCGCGACCGCACCAG-CACACGGGC-3@ (HpaI and NruI sites areunderlined). The amplified hybrid gene fragmentwas cloned into pNEP consisted of the magApromoter and pRK415. The plasmid pNEPZ(11.3 kbp) was constructed by ligating an amplifiedproteinA fragment (primers: 5@-GGGTTAACGTAGACAACAAATTCAAC-3@ and 5@-GGCTAG-TATACTTTCGGCGCCTGAGCATC-3@) at theNcoI/PstI site in pNEP.

2.5. Conjugal gene transfer of magnetic bacterium

Constructed plasmids were transferred intoa wild type strain of AMB-1 by transconjugation[13]. Magnetic bacterial cells grown up to the latelogarithmic phase (approximately 8]107 cells/ml)were mixed with freshly transformed E. coli S17-1with constructed plasmid at a cell number ratio of1 : 50 (magnetic bacteria: E. coli). The mixture wasthen spotted onto an agar plate to allow mating.After 6 h incubation, the cell mixture was collectedand magnetic bacterial transconjugants were selec-ted in MSGM medium with 2.5 lg/ml tetracycline,in which only transconjuants of magnetic bacteriacan grow.

T. Matsunaga et al. / Journal of Magnetism and Magnetic Materials 194 (1999) 126—131 127

Fig. 1. Construction of plasmids for expressing proteinA-magA hybrid gene. The plasmid pRZM (12.8 kbp), containing the proteinA-magA hybrid gene, and the plasmid pNEPZ (11.3 kbp), containing the proteinA gene were constructed.

2.6. Preparation of bacterial magnetic particles

Recombinant BMPs were separated fromAMB-1 transconjugants using the following pro-cedure. Cells were collected by centrifugation andsuspended in 5 ml of 10 mM phosphate bufferedsaline (PBS, pH 7.4) containing 0.05% Tween20 togive a concentration of 1011 cells/ml. The cells weredisrupted by ultrasonic disruption (46W, 30 s, fivetimes). BMPs were collected from the disrupted cellfraction by using a neodymium—iron—boron (Nd—Fe—B) magnet (/21]12 mm) producing an in-homogeneous magnetic field (0.4 T on the surfaceof the magnet). BMPs were collected at the bottomof the tube due to the presence of the magnet, andthe supernatant was removed. The collected BMPswere washed with PBS at least six times by repeat-

ing dispersion using ultrasonication tab (velvoclear, Tokyo, Japan; VS-30 35W) and collectionusing a Nd—Fe—B magnet. The purified BMPs werekept at 4°C in PBS containing 0.1% bovine serumalbumin (BSA) and 0.1% sodium azide before use.

2.7. Preparation of antibody-bound ProteinA-BMPcomplex

In order to prepare antibody-bound ProteinA-BMP complexes, the 120 ll of alkaline phosphataseconjugated anti-human IgG antibody (ALP-Ab;1 mg/ml) was added to 3 ml of recombinant BMPssuspension (1 mg/ml PBS). The suspension wasthen dispersed by ultrasonication and incubated for1 h at room temperature to allow antibodies tobind to ProteinA on BMPs. After the incubation,

128 T. Matsunaga et al. / Journal of Magnetism and Magnetic Materials 194 (1999) 126—131

Fig. 2. Homogenous chemiluminescence enzyme immunoassay using ProteinA-BMP complexes. Alkaline phosphatase conjugatedanti-human IgG antibody bound ProteinA-BMP complexes (ALP-Ab-ProA-BMPs) were prepared by immunoreaction of alkalinephosphatase conjugated anti-human IgG antibody and ProteinA-BMP complexes. Luminescence yields were measured when severalconcentration of human IgG were added to ALP-Ab-ProA-BMPs suspensions.

antibody bound ProteinA-BMP complexes weremagnetically separated from the reaction mixtureusing a Nd—Fe—B magnet, washed three times withPBS, and then suspended in PBS containing 0.1%BSA and sodium azide.

2.8. Homogenous immunoassay using ProteinA-BMPcomplex

Homogenous immunoassay using ProteinA-BMP complexes is described in Fig. 2. Alkalinephosphatase conjugated anti-human IgG antibodybound ProteinA-BMP complexes (ALP-Ab-ProA-BMPs, 40 lg/150 ll) was mixed with various con-centrations of human IgG solution (150 ll) in thetest tube and incubated for 1 h at room temper-ature. Luminescence substrate (100 ll, Lumi-Phos530) was added to 20 ll of this suspension.Luminescence intensity was measured for 5 minusing chemiluminescence detector CLD100(Tohoku densi sangyo Co., Ltd., Sendai, Japan).Each determination was conducted in triplicate,and the results averaged.

3. Results

3.1. Expression of ProteinA on BMPs and particlesize distribution of ProteinA-BMP complex

In order to confirm whether ProteinA was ex-pressed on BMPs in an active form, the binding

Table 1Luminescence yield of BMPs from transconjugant AMB-1.Unit: kcounts/lgBMP. Activity of alkaline phosphatase con-jugated with anti-human IgG was determined from the integ-rated luminescenece yield for 5 min

pNEPZ pRZM(proteinA) (megA-proteinA)

2.3 46.4

Fig. 3. Particle size distribution of BMP; (h) BMP from wildtype AMB-1 and (j) ProteinA-BMP complex from transcon-jugant of AMB-1.

T. Matsunaga et al. / Journal of Magnetism and Magnetic Materials 194 (1999) 126—131 129

activity of ProteinA on the BMPs to the antibodywas evaluated by measuring enzyme activity ofalkaline phosphatase conjugated to antibody. Asshown in Table 1, the MagA-ProteinA hybrid pro-tein expressed on the BMPs (pRZM) shows higherantibody binding activity than that of BMPs iso-lated from AMB-1 cells with the proteinA gene(pNEPZ). This result shows that ProteinA could beexpressed on the BMPs using proteinA-magA genefusions and ProteinA could not be expressed on theBMPs by the protein A gene only. Furthermore, thisresult reveals that the non-specific binding of anti-body to BMPs was not significant. Fig. 3 showsparticle size distribution of BMPs from wild typeand transconjugant of AMB-1. There is no differ-ence in the size of BMP and ProteinA-BMP com-plexes.

3.2. Measurement of human IgG concentration usinghomogenous immunoassay

Homogenous chemiluminescence EIA usingALP-Ab-ProA-BMPs has been developed for meas-uring human IgG concentration. Luminescenceyields were measured when several concentrationof human IgG were added to ALP-Ab-ProA-BMPssuspensions. Fig. 4 shows the relationship bet-ween the luminescence intensity and human IgG

Fig. 4. Correlation between luminescence and human IgG con-centration using ALP-Ab-ProA-BMPs in homogenous chemi-luminescence EIA. The experiments were performed undervarious human IgG concentration using 60 lg of BMP com-plexes.

concentration. The luminescence yield decreasedwith increasing human IgG concentration in therange of 1—103 ng/ml. The maximum detectableconcentration of IgG was of 1000 ng/ml, becausethe immunological reaction of anti-human IgGantibody was saturated. The minimum detectableconcentration of IgG was 1 ng/ml.

The mean particles size of BMPs after im-munoreaction was increased with increasing hu-man IgG concentration, indicating aggregation ofBMP complexes (data not shown).

4. Discussion

A magnetic particle-based immunoassay hasbeen developed due to their use in separatingbound and free analyte by application of a mag-netic field. Antibodies are covalently coupled toa magnetic particle solid phase, preventing the de-sorption of antibody during the assay. Because theparticles are dispersed evenly throughout the reac-tion mixture, they allow rapid reaction kineticswithout continuously mixing or shaking, providefor the precise addition of antibody, and are easy touse. The magnetic particles serve as the solid sup-port and provide a mean of separation. Amine-terminated magnetic particles (approximately 1 lmdiameter) developed by Advanced Magnetics, Inc.(Cambridge, MA) are commercially available andhave been used for solid phase immunoassays[16—18]. Enzyme immunoassay using specific at-razine antiserum covalently coupled to the mag-netic particle solid phase provided accurate directdetection of atrazine at 15 ppt in a 250 ll watersample [19].

In this study, BMPs expressing ProteinA wereshown to be the same size as BMPs produced bywild type AMB-1 and to have the ability to bindantibody. These results suggest that the immunoas-say system using the antibody-bound ProteinA-BMP complex would be feasible. Decrease ofluminescence intensity in homogenous chemi-luminescence EIA are attributed to aggregation ofBMPs by the immunoreaction. Aggregation ofBMPs causes inhibition of enzyme activity presum-ably by restricting substrate permeability, and/orabsorption of luminescence.

130 T. Matsunaga et al. / Journal of Magnetism and Magnetic Materials 194 (1999) 126—131

In order to utilize a greater range of lumines-cence intensity, the amount of ProteinA expressedon the BMPs should be increased. We are con-structing stronger expression systems to increasethe amount of expressed target proteins onBMPs by employing stronger promoters and/orhigh copy number plasmids. The development ofthese genetic techniques will increase the applica-tions of recombinant BMPs such as recoveringsystems for nucleoic acid, antigen, and specific cellsused in diagnostic and environmental researchfields.

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