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Enhancing immunoelectrochemiluminescence (IECL) for sensitive bacterial detection
JOURNAL OF IMMUWOU)GICAL METHODS ELSEVIER
Journal of Immunological
Methods
I Y2 ( I YY6! 63-7
I
Enhancing immunoelectrochemiluminescence sensitive bacterial detection Hao Yu
Received
17 November
(
IECL) for
*
1995: revised 24 January 1996: accepted 21 January 1996
Abstract Immunoelectrochemiluminescence (IECL) as an alternative method versus immunochemiluminescent and immunofluorescent methods can be used for versatile applications in biological agent detection. Although IECL offered high reproducibility and sensitive detection capability for soluble antigens and nucleic acids in aqueous phase. the IECL assays are not optimal and many factors which can affect the IECL performance still remain unclear. Further IECL kinetic studies, improvement of antibody biotinylation, magnetic particle selection and reducing non-specific binding have shown that the enhanced IECL sensitivities (signal to background noise ratios) can be potentially increased at least ten-fold compared to the sensitivities with general IECL assay procedures for bacterial detection. Kr.word.c:
bandwich
Immunoelectrochemiluminescence: immunoassay
Kinetics: Sensitive bacterial detection: Magnetic particle: Biotin/protein
1. Introduction
Electrogenerated chemiluminescence (ECL) as a highly sensitive detection technology has received considerable attention in chemical analysis and clini-
Abbreviations: IECL, immunoelectrochemiluminescence; b/p. biotin/protein; biotin-Ah. biotin-antibody; Tag-Ab. Ru(bpy)f+antibody: S/B. signal to background (negative control): Ru(bpy):+. wthenium (II) tristbipyridyl): SA. streptavidin; TPA. tripropylamine. * Tel.: (310) 671-5569: Fax: (410) 671-5807: e-mail: [email protected]. Elsevier Science B.V. PI/ SOO??- 1759(96)00036-
I
molar ratios: Direct
cal diagnostics (Norffsinger and Danielson. 1987; Danielson et al.. 1989: Uchikura and Kirisawa, 199 1: Blackburn et al.. 1991). Applications of immunoand nucleic acid-based ECL assays for virulent biological agent detection use of commercial ECL analyzers were reported (Gatto-Menking et al., 199.5: Yu et al.. 1995, Yu and Bruno. 1996). Currently. two sister ECL analyzers are available in the market, Origen from Igen (Gaithersburg, MD) and QPCR.5000 from Perkin Elmer (Foster City. CA). The major differences between these ECL analyzers are: the materials used in the electrode (platinum in Origen and gold in QPCR5000); application capabilities: and instrument internal standard. However, the principle of these ECL analyzers remains the same. Origen ECL analyzer was selected in these studies.
The principle of ECL using ruthenium (II) tris(bipyridyl) (Ru(bpy)i’ ) and tripropylamine (TPA) as oxidation-reduction reagents has been described (Blackburn et al.. 1991). Major advantages of ECL compared to common chemiluminescent and fluorescent techniques are higher signal to background (S/B) ratios and better-controlled luminescent signal intensities. In typical ECL reactions. even in the presence of biological samples. there is no natural fluorescent interference because there is no fluorescent excitation process involved. Relative ECL photon intensities are generated under well-controlled electric potential rather than reaction time or probe concentration (Leland and Powell, 1990). Immuno-ECL (IECL) assays have shown exceptional sensitivities for soluble antigen detection in biological applications. However, the assay procedures need to be refined in order to enhance the performance. This report has focused on the improvements of IECL assays for biological agent detections. Bacillus arlthracis, Bacillus subtilis var. typhinwrhrn, Escherichia coli niger, Salmmella 0157:H7 and Yersinia pestis agents were selected in these studies. Immunochemistry of antibody-biotin conjugates (with and without spacer). non-specific of magnetic partiprotein binding, characterization cles and assay kinetics were investigated.
2. Materials
and methods
2. I. Antibody purification.
conjugation
aild antigens
Goat anti-B. anthracis (spore) antiserum, ANT578 was obtained from Antibodies (Davis, CA). Goat anti-B. subrilis var. niger polyclonal IgG antibody was obtained from U.S. Naval Medical Research Institute (NMRI). These antisera were purified using immunopure immobilized protein G (Pierce, Rockford, IL) followed by an extensive dialysis. In general, IgG antibodies purification, briefly. 6% of crosslinked agarose packed in the 10 ml disposable collum; the matrix was equilibriums with 50 mM sodium acetate buffer at pH 5.8; binding proteins in the matrix were eluted by Pierce elution buffer and neutralized with 1 M Tris buffer at
pH 10.6 under 1 ml/min flow condition; antiserum dialysis was carried at 4°C in 10 mM phosphate buffer saline (PBS), pH 7.4, overnight (without sodium azide). Final antiserum concentrations were determined by Bradford protein assay (Bio-Rad. Hercules, CA). Affinity purified polyclonal antiSalmmella sp. and E. coli 0157 antibodies were obtained from Kirkegaard Perry Lab. (KPL. Gaithersburg, MD). Affinity purified mouse antiYersinia pestis Fl positive monoclonal antibodies were obtained from BioDesign International (Kennebunk. ME). Biotin-DNP-hydroxysuccinimide (NHS) (spacer 2.24 nm. Molecular Devices, Sunnyvale, CA) and biotin-LC-NHS (sulfosuccinidyl-6-(biotinamido) hexanoate) (spacer 2.24 and 1.35 nm, Pierce, IL) were used for biotinylation on anti-B. arzthracis, anti-B. subtilis var. niger antibodies. Initial molar ratios of biotin-DNP to antibody (b/p) from 5 to 25 were selected in biotinylation experiments and the final molar incorporation were determined by the absorbance measurement of each samples at 280 nm and 362 nm according to established protocol (Molecular Device). The b/p ratios from 1.4 to 5.8 were obtained on these antibodies. Ru(bpy)g+-NHS, as ECL Tag label. purchased from Igen for Ru(bpy)i’-antibody (Tag-Ab) labels. Ru(bpy)<+NHS ester was dissolved in dimethylsulfoxide (DMSO). Final molar ratios of Tag-(Ru(bpy)i’) to polyclonal and monoclonal antibodies were determined by absorbance at 280 and 455 nm. A molar ratio of 4.3 for these Tag-Ab conjugates used in these studies. Irradiated B. anthracis (Steme strain), B. subtilis var. niger spores and Fl antigen (CO92) were obtained from US Army Medical Research Institute for infectious diseases (USAMRIID, Ft. Detrick, MD). and heat-killed Irradiated E. coli 0 l57:H7 Salrmmellcr typhirnuriurn were obtained from Jerry Crawford (US Department of Agriculture) and KPL. respectively. Cell counts were determined by a hemacytometer. Ten-fold cell dilution was made from stock solution at IO’ cells/ml. Only 10 ~1 from each dilution was used for IECL assay. The final volume for ECL assay was 280 ~1 as described (Gatto-Menking et al., 1995). In current experimental setting, detection of average 100 cells/ml is equivalent to 28 cells.
H. Yu/ Journal
of Immunological
Methods
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63-71
2.2. Magnetic particles Polystyrene spheres magnetizable streptavidin (SA)-coated magnetic particles, 2.8 and 4.8 pm in diameter (M-280 and M-450) were obtained from Dynal (Lake Success, NY). Particle size I 0.5 km of MACS colloidal super paramagnetic microbeads (481-Ol), coated with SA. were obtained from Miltenyi Biotec (Sunnyvale. CA). Organic polymer based, SA-coated BioMag paramagnetic particles (84680, size around 1 pm) purchased from Advanced Magnetics (Cambridge, MS). MPG SA-coated paramagnetic glass particle (MSTR0502) was given as a gift from MPG (Lincoln Park, NJ).
2.3. Immuno-ECL, -fluorescence -flow cytometry assays
microscopy,
and
Immunoassays described in this report are using the direct sandwich immunoassay format, that is, both biotin- and Tag-conjugated antibodies from the same animal species are used for primary antigen capture. In order to concentrate antibody-antigen complex from the sample solution onto the electrode surface(to achieve ECL reaction), SA-biotin based paramagnetic particles are employed in the IECL assays. Under the flow stream (flow rate 2 ml/min) with assay buffer (pH 8.5, Igen) containing excess of tripropylamine (TPA) reagent, these magnetic particles along with antigen-antibody complex are collected on the electrode surface by a permanent magnet. In ECL reaction, photon emission at 620 nm generated by electric field can be measured by a photomultiplier tube and the intensity of this emission is proportional to the amount of antigens captured by SA-biotin antibody based beads. General assay protocol was described in previous reports (Gatto-Menking et al., 1995; Yu and Bruno, 1996). Fluorescence microscopic studies were performed on an Olympus BH-2 microscope. Three sets of excitation/emission filters (380/420 nm, 490/525 nm, 545/570 nm) were used in these studies. BioRad Bryte-HS flow cytometer (Microscience, England) was used for flow cytometry studies. Forward light scattering and fluorescent data were generated at a flow rate of 1.5 pl/min and the pressure of 70 kBar. Double distilled water was used in this flow
14
I8
2.4
58
4,
B/P Ratios of Biotin-Antibody Conjugates Fig. I. IECL results of B. subtilis var. nigrr spore concentration. A: 5X 10’: B: 5 X IO”: C: 5X IO’ (cells/ml) at different b/p molar incorporation. B. fhuringiensis spores at IO’ cells/ml were used as negative controlled antigen (NJ under the same IECL assay condition. The ECL intensities were not significantly increased a, b/p ratios increase at low antigen concentration. A linear ECL response between b/p molar ratios of 1.8 and 3.2 was obtained. Greater than 4.2 (b/p molar ratio) resulted in a aaturation of IECL intensities.
cytometer. sample.
The assay
time was about
2 min per
3. Results 3.1. Determination ratios
of optimal biotin/protein
molur
Polyclonal anti-B. subtilis var. niger antibody was coupled with biotin-NHS ester. The final b/p molar ratios of 1.4, 1.8, 2.4, 4.2 and 5.8 determined as described in Material and methods were used. The IECL results at different b/p molar ratios were shown in Fig. 1. Results indicated that the ECL intensities increased as the b/p molar ratios increased. Saturation of ECL intensities occurred when the b/p molar ratios increased to 4.2 or greater in these experiments. Negative control ECL intensities also increased as b/p molar ratio increased. 3.2. Fluorescence microscop!, studies ef magnetic particles
and flow
qtometv
Four types of SA-coated magnetic particles were examined by fluorescence microscopy and flow cy-
‘Iable
1
Experimental
conditions
paramagnetic
particles
and physical
SA-CPG ?k.
biotin-Ab
(“kvtJ) Tag-Ah Antigen>
(rig//****
( @I)IOh
features
SA-BioMag
1
1
of three types of
SA-Dynal
2.8
1
8
x
x
10
I0
IO
30
30
30
cells/ml Particles ( jog) Density (g/cm’)
3.4
2.5
I .s
# of particles
IO’
5X10X
Surface areas
60
100
Fluorescence
_
-
Non-specificity
++
-
++ _
Not
Not
Uniform
uniform
uniform
1.8 (pm)
IOY 1-J
G/g)
3.3. Non-sprc$i’fic binding studies of r~arious irnmunomagnetic particles
binding Distribution particle size Where:
in
SA = streptavidin;
Cont. = concentration:
particles, their capturing efficiency is also dependant upon their magnetization. Both fluorescence microscopy and flow cytometry studies on particle wash-off and suspended solutions have shown that less than 100%~of magnetic particles are captured by magnet even for a period of capturing time (up to several hours). which indicates that some particles will remain free in solution and can be washed away in flow condition. Therefore, the total number of particles in an immunoassay and the actual number of particles captured should be taken into account in quantitative immunoassays.
at
Ah = antibody.
tometry. Some features of these magnetic particles were summarized in Table 1 except the MACS superparamagnetic particles because the density and size of these particles were not in the same order as others. Results of fluorescence microscopy and flow cytometry have revealed that Dynal magnetic beads (M-2.8 and M-4.5) have broader green fluorescent emission at 525 nm. In most cases, the use of Dynal beads will interfere with the FITC assays at the wavelength between 490 and 525 nm. BioMag particles, MACS superparamagnetic particles. and MPG beads, on the other hand, are good candidates for fluorescent assays because of the dark background. Forward scattering results of particle size distribution in flow cytometry studies showed that the sizes of MACS, BioMag and MPG paramagnetic particles were not uniform. Results from Dynal M-280 and M-450 bead studies, however, revealed a very sharp peak which indicated those Dynal beads were uniform in size distribution. For the purpose of ECL assay, stronger magnetization and spherical shaped particles are necessary for rapid capturing and easy wash-off from the surface of the electrode. Therefore, Dynal magnetic particles are the best choice over others in ECL application. Natural fluorescence from magnetic particles is not concerned in ECL assays, because there is no natural fluorescence involved in ECL assays. Besides the physical properties of the magnetic
In SA-biotin based immunomagnetic assay, sensitive detections depend not only upon the affinity of antibody-antigen interaction, but also upon the chemistry of SA linking to those magnetic particles and non-specific binding of antibody conjugates on the surface of the particles. In this IECL assay, freshmade PBS buffer containing 2.5% goat serum is used to block the non-specific binding sites on the surface of the particles and to minimize the non-specific binding. Several SA-coated paramagnetic particles were tested at low (4 ng/pl) and high (20 ng/pl) antibody (goat anti-E. cwli antibody) concentrations. ECL results (Fig. 2) showed that the SA-MPG partcles had greater non-specific protein binding potential (higher background and lower S/B ratios) than the Bio-Mag and Dynal magnetic particles at the same experimental conditions. 3.4. ECL kinetic ckwal antibodies format
studies of polyclonal and monoin direct sandwich immunoassay
Polyclonal anti-E. coli and Salmonella antibodies and monoclonal anti-Yersillia pestis (plague) Fl pasitive antibodies were selected for ECL kinetic studies. Both antibodies (biotin-Ab and Tag-Ab) from the same species were used for antigen capturing. Results (Fig. 3) showed that the ECL intensities were proportional to the reaction time when polyclonal Tag-Ab was added into pre-incubated biotin-Ab and antigen complex. However, when both capturing antibodies are monoclonal, the ECL intensities were saturated after 15-30 min reaction time. Upon the
H. Yu /Journal
of fmmunologicul
Methods 192 i I9961 O-71
67
after Tag-Ab added to pre-captured antibody-antigen complex is, the better the S/B ratios are when the polyclonal antibodies are used in direct sandwich immunoassay. Reaction of between 15-30 min will be optimal, when the monoclonal antibodies are used in direct sandwich immunoassay. 3.5. Bacterial detection by enhanced IECL ussays O! Neg Control
Ind
1X10'
1X10' MO4
MO5
E. eoli in Buffer (ceus/ml) Fig. 2. Non-specific antibody binding to three types of SA-coated magnetic particles was studied at low (4 ng/ yl) and high (20 ng/ ~1) antibody concentration conditions. Both results at different antibody concentrations showed the similar trends of ECL intensities as antigen increased and the result onIy from low protein concentration was presented. In this experiment, 25 ~1 of biotin-conjugated antibody (100 ng) was used with 20 pg of each SA-coated paramagnetic particles, MPG (H 1, BioMag (01 and Dynal (A) at various antigen concentrations. Finally, 25 ~1 of Ru(bpy)i+-conjugated antibody (200 ng) was applied to each sample prior to the IECL assay.
reaction time increase, the negative control intensities were aiso increased but the changes were not significant in these experiments; however this phenomenon should be taken into account. Kinetic ECL results suggest that the longer the reaction time of
B. anrhracis spore detection Werne strain) has been reported using an ECL assay with the sensitivity of 100 cells/ml (S/B = 3) (Gatto-Menking et al., 1995). Extended spacer arm of biotin-LC-NHS is conjugated with an anti-B. anthracis antibody (ANT578) in this application. Current ECL assays are performed at b/p molar ratio of 2.4. The pre-incubated SA-coated Dynal magnetic particles with these biotin-LC-Ab conjugates in the presence of 5% serum buffer at ice cold conditions for 10 min prior to adding Tag-Ab is necessary. Approximately 50 min reaction time was used after adding Tag-Ab to pre-captured antibody-antigen complex before ECL assay in this experiment. The intensity of IECL assay for B. anthracis detection (Fig. 4) is substantially increased after the enhancement (judged only by S/B ratios 2 31. Results in Fig. 4 (after enhancement) indicated that the detection sensitivity is about 10 cells/ml (S/B = 3.3). More than ten-fold en-
+
0
After
o
0 3
12
25
45
60
90
17.0
150
240
320
Reaction Time of Tag-Ah to Ab/Ag Complex Minutes)
Fig. 3. Kinetic studies of IECL assays are performed with polyclonal (Salmon& sp. at 1000 and 2000 cells/ml. E. coli 500 and 1000 cells/ml) and monoclonal (plague Fl positive) antibodies in sandwich immunoassay format. Negative control (without antigens in sandwich immunoassay) indicated as (0).
Fig. 4. Results of an enhanced IECL assay (after) for B. onthracis spore (Steme strain) detection compared to the IECL assay without enhancement (before). Anti-B. anthracis ANT-578 antibody was used in this study. The detection sensitivity is approximately 10 cells/ml with S/B ratio of 3.3 compared to the negative control (without B. nnthracis antigens).
68
cells/ml (S/B = 3.28) after the enhancement compared to before enhancement (ten cells/ml with S/B = 1.5, or 100 cells/ml with S/B = 3) as shown in Fig. 6.
4. Discussion
0 Neg.ca”ml
5x,o1
5X10”
5X10?
5xd
5x1us 5xd
Spores of B. Subtilis var. niger (cells/ml) Fig. 5. Results of before and after enhanced IECL assays for B. var. niger spore detection. The detection sensitivity is approximately 5000 cells/ml with S/B ratio of 4.1 compared to the negative control (without B. suhfilis var. &grr spores).
ruhtilis
hancement in assay sensitivity could be obtained compared to the previous results (ten cells/ml at S/B = 1.8 or 100 cells/ml at S/B = 3 before enhancement). Detection sensitivity of B. subtilis var. rziger also indicated about ten-fold increase after enhancement (at 5000 cells/ml with S/B = 4.1) compared to the result before enhancement (at 5000 cells/ml with S/B = 2.3 or at 50000 cells/ml with S/B = 4.9) and as results showed in Fig. 5. The comparative studies for Gram negative bacterial E. coli cell detection also revealed about ten-fold ECL intensity increase with the detection sensitivity at ten
E. eoli 0157SI7 Antigens (ceUslml) Fig. 6. Results of before and after enhanced IECL assays for E co/i cells detection. The detection sensitivity is approximately ten cells/ml with S/B ratio of 3.28 compared to the negative control (without E. co/i).
In a typical sandwich immunoassay, antibodies from different species are usually selected for antigen capturing. This allows the antibodies to interact with different epitopes of the target antigens. Unlike the typical sandwich immunoassay, in this report the same antibodies (either goat IgG polyclonal or mouse IgG monoclonal antibodies) are used in direct sandwich immunoassay format for antigen capturing. Theoretically, this format may not work if the antigens are haptens. That is because the same antigen binding sites can be occupied by one of the primary antibodies (biotin-Ab or Tag-Ab). Therefore, the detectable IECL signals will be minimized. In current IECL assays for pm-size spores and bacterial cells capturing, a biotin-Ab is allowed to interact with antigens prior to adding the Tag-Ab. A competitive reaction between Tag-Ab and biotin-Ab on the same antigen binding sites will begin after adding Tag-Ab. Besides the competitive reaction, polyclonal biotinand Tag-Ab could also bind to different binding sites (multiple sites or epitopes) on the surface of those antigens. IECL results have demonstrated that the direct sandwich immunoassay format can be used fol bacterial cell capturing (including spores, Salmonellcz and plague antigens) using either polyclonal or monoclonal antibodies. Because it is difficult to obtain more than one antibody with high affinity, the detection sensitivities for antigens in the typical sandwich immunoassay using two antibodies (either from different species or use of polyclonal and monoclonal antibodies combination) is limited. The advantage of using direct sandwich immunoassay format is to allow us to perform the optimal immunoassay with only one antibody which has the best affinity constant. In addition, the binding sites are potentially available to both biotin-Ab and Tag-Ab since no covalent bonds are formed between the antibody and antigen. The on/off rate of the free antigens to antibodies (biotin-Abs and Tag-Abs) depends upon their equi-
H. Yu / Joumal of Immunological
librium constant. This constant will be changed in respect of the antibody modifications by probe labels (biotin or Ru(bpy)z+) (Bredehorst et al., 1991) even if the antibody molecular structures are not changed by the modifications. The b/p molar ratio seems to play an important role regarding the assay sensitivity. Gretch and coworkers reported that molar ratios of biotin to antibody which were equal or greater than 8 had the maximum antigen recovery potential on SA-conjugated agarose matrix (Gretch et al., 1987). Even higher b/p molar ratios also were reported in the application of flow cytometry by labelling growth factor (De Jong et al., 1995). The ECL results have demonstrated that the selections of b/p molar ratios between 2 and 4 are optimal in current IECL assays with the higher S/B ratios. The b/p molar ratios greater than 4 did not significantly enhance the ECL sensitivities. However, the high molar ratios of biotin-NHS to antibody could potentially disrupt the antibody binding capability to antigens (Savage et al., 19921. The optical microscope also revealed possible particle-antibody-antigen complex aggregation when excess b/p ratios were used (data not shown). Since biotin-NHS esters covalently attach to amine groups on the side chain of amino acids, an excess of b/p molar ratios also means few amine groups available on polypeptide (only five amino acids containing -NH, on side chain). NH, groups usually play an important role in hydrogen bond formation with OH groups (donor or acceptor relations). Higher b/p molar ratios used in antibody conjugations could cause fewer hydrogen bond formations. As a result. weaker antibody-antigen binding is expected. On the other hand, if biotin molecules are randomly labelled on the Fab domain rather than the Fc region of an IgG antibody, the chances for antibody to capture antigens are limited. Besides the biotinylation effect, the steric molecular structures also could limit the antibody-antigen binding. Extending the spacer arm from 1.35 to 2.24 nm on biotinylated antibodies substantially enhanced the IECL assay sensitivities due to the minimization of steric hindrance of the antibodies. Higher concentration of Tag-[Ru(bpy)f’] labels in IECL assays normally results in increasing ECL intensity (Lee and Neiman, 1995). However, the
Methods 1% (19%) 63-71
69
high Tag-Ab concentration in sandwich immunoassay also could cause a decrease an ECL sensitivity. Over long incubation, the Tag-Ab potentially could adhere to the magnetic particles. This may explain why the negative controls of ECL results increased during the longer incubation. Even though a longer incubation after adding the Tag-Ab may enhance the ECL intensity, prolonged incubation prior to the ECL assay is not recommended. However, in nucleic acid-ECL applications, using multiple Ru(bpy):+ as Tag labels on a single-stranded DNA primer instead of the current single Tag per DNA primer could significantly increase the ECL intensity. This could be very useful in hybridization experiments and in quantitative PCR for sensitive DNA detection (Yu et al.. 1995). IECL assay time is about 1 h including the extended 50 min incubation time. The actual ECL testing time is less than 2 min per sample. In addition, the reaction between SA beads and biotin-Ab is almost instant because of the high affinity constant (K, = 10’” Mm ’ > between SA and biotin. There is no need to extend this pre-incubation time in IECL assays. Non-specific binding is common in immunoassays. Antiserum likely could adhere to hydrophobic surface of the magnetic particles if the surface is not properly treated. Particle selections for immunomagnetic application are critical at this point. Nonspecific studies on three types of SA-coated paramagnetic particles indicated the MPG particles had higher non-specific protein binding potential than the others. This could be due to hydrophobicity of the glass surface. Dynal and BioMag particles are good candidates in IECL assays because of their low non-specific binding potential. Particles are typically paramagnetic magnetite (Fe,O,). Unlike fen-o-fluid or organic polymer based magnetic particles, MPG particles are made by controlled pore glass. One of the main advantages of this MPG particle is its ability to freeze. That could be potentially useful for reagent lyophilization. In biological applications, selection of the proper magnetic particles according to physical and chemical properties is crucial. Dynal beads have been widely used in immunoassays and nucleic acid assays; however, the natural fluorescence of the beads could limit their applications. According to the man-
70
H. Yu / Journal
oj’Imnwzologica1
ufacturer, India ink can be used to quench green fluorescence on the Dynal beads. Fluorescence microscopy studies using 2.5% India ink with 10 mM EDTA, in pH 8.0 solution have shown that the natural fluorescence from the Dynal beads can not be completely removed. The applications of Dynal beads in fluorescent assays are limited. In IECL assay, however, the bead fluorescence is not a problem. Other advantages, such as spherical shape, uniform size, strong magnetization and well-characterized SA-linking chemistry have made Dynal beads one of the popular magnetic particles in the application of magnetic separation. It is interesting to note that the IECL kinetics of polyclonal and monoclonal antibodies are different. When polyclonal antibodies were used. the ECL responses were proportional to the time within 5 h. The ECL responses reached a plateau within 30 min when monoclonal antibodies were used. Both results suggest that a competition process occurred between the antibodies on the antigen binding sites. The reaction quickly reached a plateau in the case of monoclonal antibodies, indicating that the mono-binding sites are occupied by either biotin-Ab or TagAb, and there are no changes of ECL intensities over the long reaction. On the other hand, multiple binding sites or epitopes of the antigens provide a suitable environment for polyclonal antibodies to associate with antigens. Proportional ECL responses vs. reaction time are expected. Therefore, longer reaction time in IECL assays using polyclonal antibodies will increase the ECL intensities. A 15 min reaction time is optimal in monoclonal antibody sandwich IECL assays. Natural fluorescence from biological samples is minimal in IECL assays. However. some chemical reagents. such as proline, oxalate, gentamicin, NADH and streptomycin could cause electron transfer duroxidation (Lee and Neiman, ing the Ru(bpy)z+ 1995). Result of this may cause false positives in IECL assays. The IECL results for bacterial cell (spore) detection in biological or environmental samples have shown similar detection sensitivities to those in non-biological samples (buffers), except some fish samples which have greater ECL signals than the controls (Yu and Bruno, 1996). Improved IECL assays showed great potential of enhancing ECL sensitivity for bacterial cell detection. These
Methods
I92
(IYMI h3- 71
could also be applied in environmental and biological samples for sensitive bacterial detection.
Acknowledgements The author would like to thank Mrs. Gatto-Menking from STC, Inc., for technical support of anti-B. anthrucis antibody work and Mr. Goode from the US Army at Berger Laboratory, ERDEC, Aberdeen Proving Ground, MD, for providing laboratory support.
References Blackbum, G.F.. Shah, H.P., Kenten, J.H., Leland, J.. Kamin, R.A., Link, J., Peterman, J.. Powell. M.J., Shah, A., Talley. D.B., Tyagi. S.K., Wilkins, E.. Wu, T. and Massey, R.J. (1991) Electrochemiluminescence detection for development of immunoassays and DNA probe assays for clinical diagnostics. Clin. Chem. 37. 1534. Bredehorst. R.. Wemhoff, G.A.. Kusterbeck. A.W., Charles, P.T.. Thompson, R.B.. Ligler. F.S. and Vogel. C.W. (1991) Novel trifunctional carrier molecule for the fluorescent labeling of haptens. Anal. Biochem. 193, 272-279. Danielson, N.D., He. L., Norffsinger, J.B. and Trelli. L. (1989) Determination of erythromycin in tablets and capsules using flow-injection analysis with chemiluminescence detection, J. Pharm. Biomed. 7, 1281-1285. De Jong, M.O., Rozemuller. H., Bauman, J.G.J. and Visser. J.W.M. (1995) Biotinylation of interleukin-2 for flow cytometric analysis of IL-2 receptor expression. J. Immunol. Methods. 184, 101-I 12. Gatto-Menking. D.L., Yu, H., Bruno. J.G.. Goode, M.T.. Miller, M. and Zulich, A.W. (1995) Sensitive detection of biotoxoids and bacterial spores using an immunomagnetic electrochemiluminescence sensor. Biosens. Bioelectron. IO. 501-507. Gretch, D.R., Suter. M. and Stinski. M.F. (1987) The use of biotinylated monoclonal antibodies and streptavidin affinity chromatography to isolate herpesvirus hydrophobic proteins or glycoproteina. Anal. Biochem. 163, 270-277. Lee, W.Y. and Neiman, T.A. (1995) Evaluation of use of tris(2,2bipyridyl) ruthenium (III) as a chemiluminescent reagent for quantitation in flowing streams. Anal. Chem. 67, 1789-1796. Leland. J.K. and Powell. M.J. (1990) Electrogenerated chemiluminescence: An oxidative reduction type ECL reaction sequence using tripropyl amine. J. Electrochem. Sot. 137. 3127-3 131. Norffsinger, J.B. and Danielson, N.D. (1987) Generation of chemiluminescence upon reaction of aliphatic amines with tris(2.2’-bipyridine)ruthenium (III). Anal. Chem. 59, 865-868. Savage, M.D. Mattson, G., Desai, S., Nielander. G.W., Morgensen. S. and Conklin. E. J. (1992) Avidin-Biotin Chemistry: A Handbook. Chapter 2. Pierce Chemical Company. Rockford. IL.
Uchikura,
K. and Kirisawa.
M. (1991) Generation
of chemilumi-
nescence upon reaction of alicycline tertiary-amines with trisQ,?‘-bipyridinejruthenium (III), using flow electrochemical reaCtOr. hd. sci. 7. 803-804. Yu. H. and Bruno, J.G. (1996) Rapid and qenaitivr immunomagnetic-electrochemiluminescence detection and quantitation method for E.vcherichia co/i 0 157 and Salnror~lla in food and
environmental
samples.
Appl. Environ.
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62. 587-
597. Yu. H., Bruno. J.G.. Cheng, T., Calomiris. J.J.. Goode. M.T. and Gatto-Menking. D.L. (1995) A comparative study of PCR product detection and quantitation by electro-chemiluminescence. chemiluminescence and fluorescence. J. Biolum. Chemilum. IO. 239-255.
Journal of Immunological
Methods
I Y2 ( I YY6! 63-7
I
Enhancing immunoelectrochemiluminescence sensitive bacterial detection Hao Yu
Received
17 November
(
IECL) for
*
1995: revised 24 January 1996: accepted 21 January 1996
Abstract Immunoelectrochemiluminescence (IECL) as an alternative method versus immunochemiluminescent and immunofluorescent methods can be used for versatile applications in biological agent detection. Although IECL offered high reproducibility and sensitive detection capability for soluble antigens and nucleic acids in aqueous phase. the IECL assays are not optimal and many factors which can affect the IECL performance still remain unclear. Further IECL kinetic studies, improvement of antibody biotinylation, magnetic particle selection and reducing non-specific binding have shown that the enhanced IECL sensitivities (signal to background noise ratios) can be potentially increased at least ten-fold compared to the sensitivities with general IECL assay procedures for bacterial detection. Kr.word.c:
bandwich
Immunoelectrochemiluminescence: immunoassay
Kinetics: Sensitive bacterial detection: Magnetic particle: Biotin/protein
1. Introduction
Electrogenerated chemiluminescence (ECL) as a highly sensitive detection technology has received considerable attention in chemical analysis and clini-
Abbreviations: IECL, immunoelectrochemiluminescence; b/p. biotin/protein; biotin-Ah. biotin-antibody; Tag-Ab. Ru(bpy)f+antibody: S/B. signal to background (negative control): Ru(bpy):+. wthenium (II) tristbipyridyl): SA. streptavidin; TPA. tripropylamine. * Tel.: (310) 671-5569: Fax: (410) 671-5807: e-mail: [email protected]. Elsevier Science B.V. PI/ SOO??- 1759(96)00036-
I
molar ratios: Direct
cal diagnostics (Norffsinger and Danielson. 1987; Danielson et al.. 1989: Uchikura and Kirisawa, 199 1: Blackburn et al.. 1991). Applications of immunoand nucleic acid-based ECL assays for virulent biological agent detection use of commercial ECL analyzers were reported (Gatto-Menking et al., 199.5: Yu et al.. 1995, Yu and Bruno. 1996). Currently. two sister ECL analyzers are available in the market, Origen from Igen (Gaithersburg, MD) and QPCR.5000 from Perkin Elmer (Foster City. CA). The major differences between these ECL analyzers are: the materials used in the electrode (platinum in Origen and gold in QPCR5000); application capabilities: and instrument internal standard. However, the principle of these ECL analyzers remains the same. Origen ECL analyzer was selected in these studies.
The principle of ECL using ruthenium (II) tris(bipyridyl) (Ru(bpy)i’ ) and tripropylamine (TPA) as oxidation-reduction reagents has been described (Blackburn et al.. 1991). Major advantages of ECL compared to common chemiluminescent and fluorescent techniques are higher signal to background (S/B) ratios and better-controlled luminescent signal intensities. In typical ECL reactions. even in the presence of biological samples. there is no natural fluorescent interference because there is no fluorescent excitation process involved. Relative ECL photon intensities are generated under well-controlled electric potential rather than reaction time or probe concentration (Leland and Powell, 1990). Immuno-ECL (IECL) assays have shown exceptional sensitivities for soluble antigen detection in biological applications. However, the assay procedures need to be refined in order to enhance the performance. This report has focused on the improvements of IECL assays for biological agent detections. Bacillus arlthracis, Bacillus subtilis var. typhinwrhrn, Escherichia coli niger, Salmmella 0157:H7 and Yersinia pestis agents were selected in these studies. Immunochemistry of antibody-biotin conjugates (with and without spacer). non-specific of magnetic partiprotein binding, characterization cles and assay kinetics were investigated.
2. Materials
and methods
2. I. Antibody purification.
conjugation
aild antigens
Goat anti-B. anthracis (spore) antiserum, ANT578 was obtained from Antibodies (Davis, CA). Goat anti-B. subrilis var. niger polyclonal IgG antibody was obtained from U.S. Naval Medical Research Institute (NMRI). These antisera were purified using immunopure immobilized protein G (Pierce, Rockford, IL) followed by an extensive dialysis. In general, IgG antibodies purification, briefly. 6% of crosslinked agarose packed in the 10 ml disposable collum; the matrix was equilibriums with 50 mM sodium acetate buffer at pH 5.8; binding proteins in the matrix were eluted by Pierce elution buffer and neutralized with 1 M Tris buffer at
pH 10.6 under 1 ml/min flow condition; antiserum dialysis was carried at 4°C in 10 mM phosphate buffer saline (PBS), pH 7.4, overnight (without sodium azide). Final antiserum concentrations were determined by Bradford protein assay (Bio-Rad. Hercules, CA). Affinity purified polyclonal antiSalmmella sp. and E. coli 0157 antibodies were obtained from Kirkegaard Perry Lab. (KPL. Gaithersburg, MD). Affinity purified mouse antiYersinia pestis Fl positive monoclonal antibodies were obtained from BioDesign International (Kennebunk. ME). Biotin-DNP-hydroxysuccinimide (NHS) (spacer 2.24 nm. Molecular Devices, Sunnyvale, CA) and biotin-LC-NHS (sulfosuccinidyl-6-(biotinamido) hexanoate) (spacer 2.24 and 1.35 nm, Pierce, IL) were used for biotinylation on anti-B. arzthracis, anti-B. subtilis var. niger antibodies. Initial molar ratios of biotin-DNP to antibody (b/p) from 5 to 25 were selected in biotinylation experiments and the final molar incorporation were determined by the absorbance measurement of each samples at 280 nm and 362 nm according to established protocol (Molecular Device). The b/p ratios from 1.4 to 5.8 were obtained on these antibodies. Ru(bpy)g+-NHS, as ECL Tag label. purchased from Igen for Ru(bpy)i’-antibody (Tag-Ab) labels. Ru(bpy)<+NHS ester was dissolved in dimethylsulfoxide (DMSO). Final molar ratios of Tag-(Ru(bpy)i’) to polyclonal and monoclonal antibodies were determined by absorbance at 280 and 455 nm. A molar ratio of 4.3 for these Tag-Ab conjugates used in these studies. Irradiated B. anthracis (Steme strain), B. subtilis var. niger spores and Fl antigen (CO92) were obtained from US Army Medical Research Institute for infectious diseases (USAMRIID, Ft. Detrick, MD). and heat-killed Irradiated E. coli 0 l57:H7 Salrmmellcr typhirnuriurn were obtained from Jerry Crawford (US Department of Agriculture) and KPL. respectively. Cell counts were determined by a hemacytometer. Ten-fold cell dilution was made from stock solution at IO’ cells/ml. Only 10 ~1 from each dilution was used for IECL assay. The final volume for ECL assay was 280 ~1 as described (Gatto-Menking et al., 1995). In current experimental setting, detection of average 100 cells/ml is equivalent to 28 cells.
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Methods
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63-71
2.2. Magnetic particles Polystyrene spheres magnetizable streptavidin (SA)-coated magnetic particles, 2.8 and 4.8 pm in diameter (M-280 and M-450) were obtained from Dynal (Lake Success, NY). Particle size I 0.5 km of MACS colloidal super paramagnetic microbeads (481-Ol), coated with SA. were obtained from Miltenyi Biotec (Sunnyvale. CA). Organic polymer based, SA-coated BioMag paramagnetic particles (84680, size around 1 pm) purchased from Advanced Magnetics (Cambridge, MS). MPG SA-coated paramagnetic glass particle (MSTR0502) was given as a gift from MPG (Lincoln Park, NJ).
2.3. Immuno-ECL, -fluorescence -flow cytometry assays
microscopy,
and
Immunoassays described in this report are using the direct sandwich immunoassay format, that is, both biotin- and Tag-conjugated antibodies from the same animal species are used for primary antigen capture. In order to concentrate antibody-antigen complex from the sample solution onto the electrode surface(to achieve ECL reaction), SA-biotin based paramagnetic particles are employed in the IECL assays. Under the flow stream (flow rate 2 ml/min) with assay buffer (pH 8.5, Igen) containing excess of tripropylamine (TPA) reagent, these magnetic particles along with antigen-antibody complex are collected on the electrode surface by a permanent magnet. In ECL reaction, photon emission at 620 nm generated by electric field can be measured by a photomultiplier tube and the intensity of this emission is proportional to the amount of antigens captured by SA-biotin antibody based beads. General assay protocol was described in previous reports (Gatto-Menking et al., 1995; Yu and Bruno, 1996). Fluorescence microscopic studies were performed on an Olympus BH-2 microscope. Three sets of excitation/emission filters (380/420 nm, 490/525 nm, 545/570 nm) were used in these studies. BioRad Bryte-HS flow cytometer (Microscience, England) was used for flow cytometry studies. Forward light scattering and fluorescent data were generated at a flow rate of 1.5 pl/min and the pressure of 70 kBar. Double distilled water was used in this flow
14
I8
2.4
58
4,
B/P Ratios of Biotin-Antibody Conjugates Fig. I. IECL results of B. subtilis var. nigrr spore concentration. A: 5X 10’: B: 5 X IO”: C: 5X IO’ (cells/ml) at different b/p molar incorporation. B. fhuringiensis spores at IO’ cells/ml were used as negative controlled antigen (NJ under the same IECL assay condition. The ECL intensities were not significantly increased a, b/p ratios increase at low antigen concentration. A linear ECL response between b/p molar ratios of 1.8 and 3.2 was obtained. Greater than 4.2 (b/p molar ratio) resulted in a aaturation of IECL intensities.
cytometer. sample.
The assay
time was about
2 min per
3. Results 3.1. Determination ratios
of optimal biotin/protein
molur
Polyclonal anti-B. subtilis var. niger antibody was coupled with biotin-NHS ester. The final b/p molar ratios of 1.4, 1.8, 2.4, 4.2 and 5.8 determined as described in Material and methods were used. The IECL results at different b/p molar ratios were shown in Fig. 1. Results indicated that the ECL intensities increased as the b/p molar ratios increased. Saturation of ECL intensities occurred when the b/p molar ratios increased to 4.2 or greater in these experiments. Negative control ECL intensities also increased as b/p molar ratio increased. 3.2. Fluorescence microscop!, studies ef magnetic particles
and flow
qtometv
Four types of SA-coated magnetic particles were examined by fluorescence microscopy and flow cy-
‘Iable
1
Experimental
conditions
paramagnetic
particles
and physical
SA-CPG ?k.
biotin-Ab
(“kvtJ) Tag-Ah Antigen>
(rig//****
( @I)IOh
features
SA-BioMag
1
1
of three types of
SA-Dynal
2.8
1
8
x
x
10
I0
IO
30
30
30
cells/ml Particles ( jog) Density (g/cm’)
3.4
2.5
I .s
# of particles
IO’
5X10X
Surface areas
60
100
Fluorescence
_
-
Non-specificity
++
-
++ _
Not
Not
Uniform
uniform
uniform
1.8 (pm)
IOY 1-J
G/g)
3.3. Non-sprc$i’fic binding studies of r~arious irnmunomagnetic particles
binding Distribution particle size Where:
in
SA = streptavidin;
Cont. = concentration:
particles, their capturing efficiency is also dependant upon their magnetization. Both fluorescence microscopy and flow cytometry studies on particle wash-off and suspended solutions have shown that less than 100%~of magnetic particles are captured by magnet even for a period of capturing time (up to several hours). which indicates that some particles will remain free in solution and can be washed away in flow condition. Therefore, the total number of particles in an immunoassay and the actual number of particles captured should be taken into account in quantitative immunoassays.
at
Ah = antibody.
tometry. Some features of these magnetic particles were summarized in Table 1 except the MACS superparamagnetic particles because the density and size of these particles were not in the same order as others. Results of fluorescence microscopy and flow cytometry have revealed that Dynal magnetic beads (M-2.8 and M-4.5) have broader green fluorescent emission at 525 nm. In most cases, the use of Dynal beads will interfere with the FITC assays at the wavelength between 490 and 525 nm. BioMag particles, MACS superparamagnetic particles. and MPG beads, on the other hand, are good candidates for fluorescent assays because of the dark background. Forward scattering results of particle size distribution in flow cytometry studies showed that the sizes of MACS, BioMag and MPG paramagnetic particles were not uniform. Results from Dynal M-280 and M-450 bead studies, however, revealed a very sharp peak which indicated those Dynal beads were uniform in size distribution. For the purpose of ECL assay, stronger magnetization and spherical shaped particles are necessary for rapid capturing and easy wash-off from the surface of the electrode. Therefore, Dynal magnetic particles are the best choice over others in ECL application. Natural fluorescence from magnetic particles is not concerned in ECL assays, because there is no natural fluorescence involved in ECL assays. Besides the physical properties of the magnetic
In SA-biotin based immunomagnetic assay, sensitive detections depend not only upon the affinity of antibody-antigen interaction, but also upon the chemistry of SA linking to those magnetic particles and non-specific binding of antibody conjugates on the surface of the particles. In this IECL assay, freshmade PBS buffer containing 2.5% goat serum is used to block the non-specific binding sites on the surface of the particles and to minimize the non-specific binding. Several SA-coated paramagnetic particles were tested at low (4 ng/pl) and high (20 ng/pl) antibody (goat anti-E. cwli antibody) concentrations. ECL results (Fig. 2) showed that the SA-MPG partcles had greater non-specific protein binding potential (higher background and lower S/B ratios) than the Bio-Mag and Dynal magnetic particles at the same experimental conditions. 3.4. ECL kinetic ckwal antibodies format
studies of polyclonal and monoin direct sandwich immunoassay
Polyclonal anti-E. coli and Salmonella antibodies and monoclonal anti-Yersillia pestis (plague) Fl pasitive antibodies were selected for ECL kinetic studies. Both antibodies (biotin-Ab and Tag-Ab) from the same species were used for antigen capturing. Results (Fig. 3) showed that the ECL intensities were proportional to the reaction time when polyclonal Tag-Ab was added into pre-incubated biotin-Ab and antigen complex. However, when both capturing antibodies are monoclonal, the ECL intensities were saturated after 15-30 min reaction time. Upon the
H. Yu /Journal
of fmmunologicul
Methods 192 i I9961 O-71
67
after Tag-Ab added to pre-captured antibody-antigen complex is, the better the S/B ratios are when the polyclonal antibodies are used in direct sandwich immunoassay. Reaction of between 15-30 min will be optimal, when the monoclonal antibodies are used in direct sandwich immunoassay. 3.5. Bacterial detection by enhanced IECL ussays O! Neg Control
Ind
1X10'
1X10' MO4
MO5
E. eoli in Buffer (ceus/ml) Fig. 2. Non-specific antibody binding to three types of SA-coated magnetic particles was studied at low (4 ng/ yl) and high (20 ng/ ~1) antibody concentration conditions. Both results at different antibody concentrations showed the similar trends of ECL intensities as antigen increased and the result onIy from low protein concentration was presented. In this experiment, 25 ~1 of biotin-conjugated antibody (100 ng) was used with 20 pg of each SA-coated paramagnetic particles, MPG (H 1, BioMag (01 and Dynal (A) at various antigen concentrations. Finally, 25 ~1 of Ru(bpy)i+-conjugated antibody (200 ng) was applied to each sample prior to the IECL assay.
reaction time increase, the negative control intensities were aiso increased but the changes were not significant in these experiments; however this phenomenon should be taken into account. Kinetic ECL results suggest that the longer the reaction time of
B. anrhracis spore detection Werne strain) has been reported using an ECL assay with the sensitivity of 100 cells/ml (S/B = 3) (Gatto-Menking et al., 1995). Extended spacer arm of biotin-LC-NHS is conjugated with an anti-B. anthracis antibody (ANT578) in this application. Current ECL assays are performed at b/p molar ratio of 2.4. The pre-incubated SA-coated Dynal magnetic particles with these biotin-LC-Ab conjugates in the presence of 5% serum buffer at ice cold conditions for 10 min prior to adding Tag-Ab is necessary. Approximately 50 min reaction time was used after adding Tag-Ab to pre-captured antibody-antigen complex before ECL assay in this experiment. The intensity of IECL assay for B. anthracis detection (Fig. 4) is substantially increased after the enhancement (judged only by S/B ratios 2 31. Results in Fig. 4 (after enhancement) indicated that the detection sensitivity is about 10 cells/ml (S/B = 3.3). More than ten-fold en-
+
0
After
o
0 3
12
25
45
60
90
17.0
150
240
320
Reaction Time of Tag-Ah to Ab/Ag Complex Minutes)
Fig. 3. Kinetic studies of IECL assays are performed with polyclonal (Salmon& sp. at 1000 and 2000 cells/ml. E. coli 500 and 1000 cells/ml) and monoclonal (plague Fl positive) antibodies in sandwich immunoassay format. Negative control (without antigens in sandwich immunoassay) indicated as (0).
Fig. 4. Results of an enhanced IECL assay (after) for B. onthracis spore (Steme strain) detection compared to the IECL assay without enhancement (before). Anti-B. anthracis ANT-578 antibody was used in this study. The detection sensitivity is approximately 10 cells/ml with S/B ratio of 3.3 compared to the negative control (without B. nnthracis antigens).
68
cells/ml (S/B = 3.28) after the enhancement compared to before enhancement (ten cells/ml with S/B = 1.5, or 100 cells/ml with S/B = 3) as shown in Fig. 6.
4. Discussion
0 Neg.ca”ml
5x,o1
5X10”
5X10?
5xd
5x1us 5xd
Spores of B. Subtilis var. niger (cells/ml) Fig. 5. Results of before and after enhanced IECL assays for B. var. niger spore detection. The detection sensitivity is approximately 5000 cells/ml with S/B ratio of 4.1 compared to the negative control (without B. suhfilis var. &grr spores).
ruhtilis
hancement in assay sensitivity could be obtained compared to the previous results (ten cells/ml at S/B = 1.8 or 100 cells/ml at S/B = 3 before enhancement). Detection sensitivity of B. subtilis var. rziger also indicated about ten-fold increase after enhancement (at 5000 cells/ml with S/B = 4.1) compared to the result before enhancement (at 5000 cells/ml with S/B = 2.3 or at 50000 cells/ml with S/B = 4.9) and as results showed in Fig. 5. The comparative studies for Gram negative bacterial E. coli cell detection also revealed about ten-fold ECL intensity increase with the detection sensitivity at ten
E. eoli 0157SI7 Antigens (ceUslml) Fig. 6. Results of before and after enhanced IECL assays for E co/i cells detection. The detection sensitivity is approximately ten cells/ml with S/B ratio of 3.28 compared to the negative control (without E. co/i).
In a typical sandwich immunoassay, antibodies from different species are usually selected for antigen capturing. This allows the antibodies to interact with different epitopes of the target antigens. Unlike the typical sandwich immunoassay, in this report the same antibodies (either goat IgG polyclonal or mouse IgG monoclonal antibodies) are used in direct sandwich immunoassay format for antigen capturing. Theoretically, this format may not work if the antigens are haptens. That is because the same antigen binding sites can be occupied by one of the primary antibodies (biotin-Ab or Tag-Ab). Therefore, the detectable IECL signals will be minimized. In current IECL assays for pm-size spores and bacterial cells capturing, a biotin-Ab is allowed to interact with antigens prior to adding the Tag-Ab. A competitive reaction between Tag-Ab and biotin-Ab on the same antigen binding sites will begin after adding Tag-Ab. Besides the competitive reaction, polyclonal biotinand Tag-Ab could also bind to different binding sites (multiple sites or epitopes) on the surface of those antigens. IECL results have demonstrated that the direct sandwich immunoassay format can be used fol bacterial cell capturing (including spores, Salmonellcz and plague antigens) using either polyclonal or monoclonal antibodies. Because it is difficult to obtain more than one antibody with high affinity, the detection sensitivities for antigens in the typical sandwich immunoassay using two antibodies (either from different species or use of polyclonal and monoclonal antibodies combination) is limited. The advantage of using direct sandwich immunoassay format is to allow us to perform the optimal immunoassay with only one antibody which has the best affinity constant. In addition, the binding sites are potentially available to both biotin-Ab and Tag-Ab since no covalent bonds are formed between the antibody and antigen. The on/off rate of the free antigens to antibodies (biotin-Abs and Tag-Abs) depends upon their equi-
H. Yu / Joumal of Immunological
librium constant. This constant will be changed in respect of the antibody modifications by probe labels (biotin or Ru(bpy)z+) (Bredehorst et al., 1991) even if the antibody molecular structures are not changed by the modifications. The b/p molar ratio seems to play an important role regarding the assay sensitivity. Gretch and coworkers reported that molar ratios of biotin to antibody which were equal or greater than 8 had the maximum antigen recovery potential on SA-conjugated agarose matrix (Gretch et al., 1987). Even higher b/p molar ratios also were reported in the application of flow cytometry by labelling growth factor (De Jong et al., 1995). The ECL results have demonstrated that the selections of b/p molar ratios between 2 and 4 are optimal in current IECL assays with the higher S/B ratios. The b/p molar ratios greater than 4 did not significantly enhance the ECL sensitivities. However, the high molar ratios of biotin-NHS to antibody could potentially disrupt the antibody binding capability to antigens (Savage et al., 19921. The optical microscope also revealed possible particle-antibody-antigen complex aggregation when excess b/p ratios were used (data not shown). Since biotin-NHS esters covalently attach to amine groups on the side chain of amino acids, an excess of b/p molar ratios also means few amine groups available on polypeptide (only five amino acids containing -NH, on side chain). NH, groups usually play an important role in hydrogen bond formation with OH groups (donor or acceptor relations). Higher b/p molar ratios used in antibody conjugations could cause fewer hydrogen bond formations. As a result. weaker antibody-antigen binding is expected. On the other hand, if biotin molecules are randomly labelled on the Fab domain rather than the Fc region of an IgG antibody, the chances for antibody to capture antigens are limited. Besides the biotinylation effect, the steric molecular structures also could limit the antibody-antigen binding. Extending the spacer arm from 1.35 to 2.24 nm on biotinylated antibodies substantially enhanced the IECL assay sensitivities due to the minimization of steric hindrance of the antibodies. Higher concentration of Tag-[Ru(bpy)f’] labels in IECL assays normally results in increasing ECL intensity (Lee and Neiman, 1995). However, the
Methods 1% (19%) 63-71
69
high Tag-Ab concentration in sandwich immunoassay also could cause a decrease an ECL sensitivity. Over long incubation, the Tag-Ab potentially could adhere to the magnetic particles. This may explain why the negative controls of ECL results increased during the longer incubation. Even though a longer incubation after adding the Tag-Ab may enhance the ECL intensity, prolonged incubation prior to the ECL assay is not recommended. However, in nucleic acid-ECL applications, using multiple Ru(bpy):+ as Tag labels on a single-stranded DNA primer instead of the current single Tag per DNA primer could significantly increase the ECL intensity. This could be very useful in hybridization experiments and in quantitative PCR for sensitive DNA detection (Yu et al.. 1995). IECL assay time is about 1 h including the extended 50 min incubation time. The actual ECL testing time is less than 2 min per sample. In addition, the reaction between SA beads and biotin-Ab is almost instant because of the high affinity constant (K, = 10’” Mm ’ > between SA and biotin. There is no need to extend this pre-incubation time in IECL assays. Non-specific binding is common in immunoassays. Antiserum likely could adhere to hydrophobic surface of the magnetic particles if the surface is not properly treated. Particle selections for immunomagnetic application are critical at this point. Nonspecific studies on three types of SA-coated paramagnetic particles indicated the MPG particles had higher non-specific protein binding potential than the others. This could be due to hydrophobicity of the glass surface. Dynal and BioMag particles are good candidates in IECL assays because of their low non-specific binding potential. Particles are typically paramagnetic magnetite (Fe,O,). Unlike fen-o-fluid or organic polymer based magnetic particles, MPG particles are made by controlled pore glass. One of the main advantages of this MPG particle is its ability to freeze. That could be potentially useful for reagent lyophilization. In biological applications, selection of the proper magnetic particles according to physical and chemical properties is crucial. Dynal beads have been widely used in immunoassays and nucleic acid assays; however, the natural fluorescence of the beads could limit their applications. According to the man-
70
H. Yu / Journal
oj’Imnwzologica1
ufacturer, India ink can be used to quench green fluorescence on the Dynal beads. Fluorescence microscopy studies using 2.5% India ink with 10 mM EDTA, in pH 8.0 solution have shown that the natural fluorescence from the Dynal beads can not be completely removed. The applications of Dynal beads in fluorescent assays are limited. In IECL assay, however, the bead fluorescence is not a problem. Other advantages, such as spherical shape, uniform size, strong magnetization and well-characterized SA-linking chemistry have made Dynal beads one of the popular magnetic particles in the application of magnetic separation. It is interesting to note that the IECL kinetics of polyclonal and monoclonal antibodies are different. When polyclonal antibodies were used. the ECL responses were proportional to the time within 5 h. The ECL responses reached a plateau within 30 min when monoclonal antibodies were used. Both results suggest that a competition process occurred between the antibodies on the antigen binding sites. The reaction quickly reached a plateau in the case of monoclonal antibodies, indicating that the mono-binding sites are occupied by either biotin-Ab or TagAb, and there are no changes of ECL intensities over the long reaction. On the other hand, multiple binding sites or epitopes of the antigens provide a suitable environment for polyclonal antibodies to associate with antigens. Proportional ECL responses vs. reaction time are expected. Therefore, longer reaction time in IECL assays using polyclonal antibodies will increase the ECL intensities. A 15 min reaction time is optimal in monoclonal antibody sandwich IECL assays. Natural fluorescence from biological samples is minimal in IECL assays. However. some chemical reagents. such as proline, oxalate, gentamicin, NADH and streptomycin could cause electron transfer duroxidation (Lee and Neiman, ing the Ru(bpy)z+ 1995). Result of this may cause false positives in IECL assays. The IECL results for bacterial cell (spore) detection in biological or environmental samples have shown similar detection sensitivities to those in non-biological samples (buffers), except some fish samples which have greater ECL signals than the controls (Yu and Bruno, 1996). Improved IECL assays showed great potential of enhancing ECL sensitivity for bacterial cell detection. These
Methods
I92
(IYMI h3- 71
could also be applied in environmental and biological samples for sensitive bacterial detection.
Acknowledgements The author would like to thank Mrs. Gatto-Menking from STC, Inc., for technical support of anti-B. anthrucis antibody work and Mr. Goode from the US Army at Berger Laboratory, ERDEC, Aberdeen Proving Ground, MD, for providing laboratory support.
References Blackbum, G.F.. Shah, H.P., Kenten, J.H., Leland, J.. Kamin, R.A., Link, J., Peterman, J.. Powell. M.J., Shah, A., Talley. D.B., Tyagi. S.K., Wilkins, E.. Wu, T. and Massey, R.J. (1991) Electrochemiluminescence detection for development of immunoassays and DNA probe assays for clinical diagnostics. Clin. Chem. 37. 1534. Bredehorst. R.. Wemhoff, G.A.. Kusterbeck. A.W., Charles, P.T.. Thompson, R.B.. Ligler. F.S. and Vogel. C.W. (1991) Novel trifunctional carrier molecule for the fluorescent labeling of haptens. Anal. Biochem. 193, 272-279. Danielson, N.D., He. L., Norffsinger, J.B. and Trelli. L. (1989) Determination of erythromycin in tablets and capsules using flow-injection analysis with chemiluminescence detection, J. Pharm. Biomed. 7, 1281-1285. De Jong, M.O., Rozemuller. H., Bauman, J.G.J. and Visser. J.W.M. (1995) Biotinylation of interleukin-2 for flow cytometric analysis of IL-2 receptor expression. J. Immunol. Methods. 184, 101-I 12. Gatto-Menking. D.L., Yu, H., Bruno. J.G.. Goode, M.T.. Miller, M. and Zulich, A.W. (1995) Sensitive detection of biotoxoids and bacterial spores using an immunomagnetic electrochemiluminescence sensor. Biosens. Bioelectron. IO. 501-507. Gretch, D.R., Suter. M. and Stinski. M.F. (1987) The use of biotinylated monoclonal antibodies and streptavidin affinity chromatography to isolate herpesvirus hydrophobic proteins or glycoproteina. Anal. Biochem. 163, 270-277. Lee, W.Y. and Neiman, T.A. (1995) Evaluation of use of tris(2,2bipyridyl) ruthenium (III) as a chemiluminescent reagent for quantitation in flowing streams. Anal. Chem. 67, 1789-1796. Leland. J.K. and Powell. M.J. (1990) Electrogenerated chemiluminescence: An oxidative reduction type ECL reaction sequence using tripropyl amine. J. Electrochem. Sot. 137. 3127-3 131. Norffsinger, J.B. and Danielson, N.D. (1987) Generation of chemiluminescence upon reaction of aliphatic amines with tris(2.2’-bipyridine)ruthenium (III). Anal. Chem. 59, 865-868. Savage, M.D. Mattson, G., Desai, S., Nielander. G.W., Morgensen. S. and Conklin. E. J. (1992) Avidin-Biotin Chemistry: A Handbook. Chapter 2. Pierce Chemical Company. Rockford. IL.
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M. (1991) Generation
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nescence upon reaction of alicycline tertiary-amines with trisQ,?‘-bipyridinejruthenium (III), using flow electrochemical reaCtOr. hd. sci. 7. 803-804. Yu. H. and Bruno, J.G. (1996) Rapid and qenaitivr immunomagnetic-electrochemiluminescence detection and quantitation method for E.vcherichia co/i 0 157 and Salnror~lla in food and
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597. Yu. H., Bruno. J.G.. Cheng, T., Calomiris. J.J.. Goode. M.T. and Gatto-Menking. D.L. (1995) A comparative study of PCR product detection and quantitation by electro-chemiluminescence. chemiluminescence and fluorescence. J. Biolum. Chemilum. IO. 239-255.