Detection of P ara Influenza and Influenza A V iruses ... · 1. Complement fixation tests 2. H...

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Results indicated that the sandwichscheme assay performed much betterthan the competitive scheme assay, witha low detection limit of about 5 nM forthe competitive scheme compared with8 pM for the sandwich scheme for rabbitIgG. Five milligrams of graphite powderimmunosorbent and 0.5 mM of hydro-gen peroxide were the optimum concen-trations of immunoassay for anti-rabbitIgG.

No interference from influenza Avirus to para influenza assay was found.In contrast, when immunoassay responsefor detection of para influenza usinginfluenza A immunosorbent was con-ducted, it was found that para influenzainterferes with influenza A assay.

INTRODUCTION Each year, influenza afflicts millions ofpeople and animals worldwide. A virusprimarily attacks the upper respiratorytract, such as the nose and throat, andcauses influenza. Influenza virusesbelong to a group of RNA viruses(Orthomyxoviridae family), which canbe divided into 3 groups: A, B, and C.1From here it can be broken into 2 differ-ent protein components, identified as

Vol. 7, No. 1, 2007 • The Journal of Applied Research

Detection of Para Influenza andInfluenza A Viruses UsingFlow-Injection AmperometricImmunosensorRavil A. Sitdikov, PhDEbtisam Wilkins, PhD

Department of Chemical and Nuclear Engineering, University of New Mexico, Albuquerque,New Mexico

KEY WORDS: para influenza 1 virus,influenza A virus, amperometric detection, flow-injection immunoassay,amperometric immunosensor, sandwichimmunoassay

ABSTRACT This research was focused on testing aminiature biosensor device that meas-ures amperometric response using aflow-injection immunoassay system forfast, quantitative detection of low con-centrations of influenza viruses in watersamples. The goal was to achieveextremely high selectivity and sensitivitywhile maintaining fast and user-friendlyoperation. Based on data obtained pre-viously, the authors explored a flow-injection amperometric immunoassaysystem that was developed in theUniversity of New Mexico laboratoryfor hantavirus and bacteria and wasadapted for detection of influenza Avirus and para influenza virus. Resultsindicated that both viruses in concentra-tions as low as 0.5 ng/mL can be detect-ed. The higher detection limit can be 300ng/mL for influenza A and more than104 ng/mL for para influenza.

The Journal of Applied Research • Vol. 7, No. 1, 2007 109

antigens. They are composed of spike-like features called hemagglutinin (H)and neuraminidase (N) components. Intotal, there are 15 subtypes of H and 9subtypes of N. Unfortunately, all of theH and N subtypes can infect birds, ani-mals, and humans.

In 1997, the outbreak of a new avianinfluenza-like virus, known as bird flu,affected 18 humans.2 The cases, manysevere or fatal, highlighted the chal-lenges of unique influenza viruses.Researchers struggled to track a newpotentially deadly strain of influenza,which was previously thought to onlyaffect chickens. Lessons from thisepisode helped to improve internationaland national planning for influenza pan-demics.2

A H5N1 virus infection resulted inthe death of a 3-year-old child in 1997.The child died from complications ofinfluenza-associated pneumonia, includ-ing acute respiratory distress syndrome,Reye’s syndrome, and multiorgan fail-ure.2 Although serologic evidence forinfection of humans with H5N1 influen-za virus had previously been reported,3this incident resulted in the first isola-tion of an avian virus from a humanwith severe respiratory disease.Seventeen additional cases, 5 of themfatal, were associated with avian H5N1influenza virus infections.4-6

Influenza is caused by the H typecomponent, which is an antibody thatcauses the clumping of red blood cells.Due to the limitations of the assays citedby various investigators,3-10 an enzyme-linked immunosorbent assay (ELISA)does work; however, it requires a puri-fied antigen, which was not available atthe time. The disease killed 16 people inVietnam and 8 in Thailand in 2005.2Although strains have been found in theUnited States, health officials say theyare not fatal to humans. There was a sec-ond outbreak in Texas 2004, but officialssaid they are unrelated.11,12

In this situation, the development ofrapid, sensitive, and simple methods andtechniques for the detection of influenzaviruses are very important for earlier fluepidemic control.

The modern virus detection tech-niques may differ in 2 ways13:A. Classical Techniques

1. Complement fixation tests2. H inhibition tests3. Immunofluorescence techniques4. Neutralization tests5. Single radial hemolysis

B. Newer Techniques1. Radioimmunoassay2. ELISA3. Particle agglutination4. Western blot5. Recombinant immunoblot assay6. Polymerase chain reaction technique

Several rapid influenza tests are avail-able.• BD Directigen Flu A and Directigen

Flu B, from Becton & Dickinson Co,Franklin Lakes, NJ14

• The QuickVue Influenza A+B, fromQUIDEL Company, San Diego,California15

• The BinaxNOW Influenza A & B test,from Binax, Inc., Scarborough, Maine16

These test systems are adapted onlyfor quality detection of human influenzaviruses. However, now it is very impor-tant to carry out fast influenza virus con-trol with animals (chicken, pigs, etc.),farms, and plants.

The aim of this work is the develop-ment and testing of a flow-injectionamperometric immunosensor for quanti-tative detection of para influenza andinfluenza A (bird flu) viruses. As a result of preliminary research efforts, anamperometric flow immunoassay systemwas adopted for detection of some bac-teria17,18 and hantavirus19-21 in field condi-tions.

Viral diseases are the primary causeof serious disorders that do not requirehospitalization among people who

are unable to multiply outside of a hostcell (intracellular, obligate parasitism).Only one type of nucleic acid (RNA orDNA) is present in the assembled virus(virion) plus, in simple viruses, a protec-tive protein coat. The nucleic acid car-ries the genetic data needed to programthe synthetic machinery of the host cellfor viral replication. The protein coatserves 2 functions; it protects the nucleicacid from extra cellular environmentalinsults such as nucleases and permitsattachment of the virion to the mem-brane of the host cell, which otherwiseexhibits a negative charge that repels anaked nucleic acid. Once the host cellhas been infected by viral genome pene-tration, virus replication becomesdependent on that host cell for its ener-gy and synthetic needs.

The basic structure of viruses24,25

seems to permit them to be simultane-ously adaptable and selective becausethe various virion components are syn-thesized separately within the cell, afterwhich they are assembled to form proge-ny particles. Distinguishing them from allother small, obligate, intracellular para-sites, this assembly type of replication isunique to the virus. Under experimentalconditions, viral genomes are so adapt-able that once they have penetrated thecell membrane, viral replication canoccur in almost any cell. Alternatively,intact viruses are so selective that mostof them are able to infect only a limitedrange of cell types, which selectivityexists largely because penetration of thenucleic acid usually requires a specificreaction that enables the coat to attachto specific intracellular components andthe host cell membrane.23,25

Virion replication normally causeshost cell damage or death, which is whyviruses tend to establish milder infec-tions in which cell death is more of anaberration than the norm. Notableexceptions include HIV, Ebola virus,hantavirus, and rabies virus; indeed,

Vol. 7, No. 1, 2007 • The Journal of Applied Research110

reside in developed countries, accordingto epidemiologic studies.22 Amonginfants and children, they exact a heavytoll in mortality and permanent disabili-ty.22 Emerging viral diseases, such asthose brought about by human immun-odeficiency virus (HIV), Ebola virus,and hantavirus, appear regularly.22 Inaddition, although antibiotics effectivelycombat most bacterial-based infections,viral infections are not so readily con-trolled; by comparison, they pose agreater threat to people’s health.Additionally, according to some data,the broad range of established viral dis-eases known today appears to beexpanding into other serious human ail-ments including tumors, juvenile dia-betes, rheumatoid arthritis, and a varietyof neurologic and immunologic disor-ders.22

Viruses may even have had a role inthe natural selection of animal speciesbecause, like other microorganisms, theyhave the ability to infect all forms of lifeincluding bacteria, plants, protozoa,fungi, insects, fish, reptiles, birds, andmammals in a manner similar to theselective role smallpox virus played inhumans.22 An example of this involvedthe natural selection of rabbits resistantto the virulent myxoma virus, which wasdocumented during several epidemicsdeliberately induced to control theAustralian rabbit population.22

Another possible way in whichviruses may affect evolution23 is throughthe introduction of viral genetic materialinto animal cells by mechanisms similarto those that govern gene transfer bybacteriophages. For instance, when genesfrom a virulent retrovirus are integratedinto genomes of chickens or mice, theyproduce resistance to reinfection byrelated, virulent retroviruses. Reports ofhuman leukemia-causing retrovirusesindicate that the same relationship mayexist for human retroviruses.

As small, subcellular agents, viruses


some viruses are capable of establishingforms of silent infection.26

Their extreme dependence on thehost cell makes viruses distinct amongother microorganisms. A virus mustgrow within a host cell and is viewedtogether with its host in any considera-tion of pathogenesis, epidemiology, hostdefenses, or therapy. Specific conditionsfor pathogenesis are imposed by thebilateral association between the virusand its host. Rhinoviruses, for instance,require temperatures not exceeding34°C. That environment restricts theirgrowth to cells in the cool outer layer ofthe nasal mucosa, thereby preventingtheir spread to areas of higher tempera-ture in deeper cells.27

Protected from some of the host’simmune mechanisms by its intracellularlocation, at the same time, the virus isvulnerable because of its dependence onthe host cell’s synthetic machinery. Thisstate, however, may be altered by subtlephysical and chemical changes producedby the viral infection itself, includinginflammation, fever, circulatory alter-ations, and interferon.28

The virus-host association is greatlyinfluenced by the virus’s epidemiologicproperties.29 As an example, certainarthropod-borne viruses in insects multi-ply only within a narrow temperaturerange; as a result, it is only under specif-ic seasonal and geographical conditionsthat the viruses are found. Transmis-sibility of viruses in aerosols and food isdetermined by other environmental con-ditions.30

Viruses replicate only within hostcells, primarily using many of the hostcell’s biosynthetic processes, whichmakes them difficult targets forchemotherapy. The similarity of host-directed and virus-directed processesmakes it difficult to identify antiviralagents with sufficient specificity to exerttheir effect on viral replication in infect-ed cells rather than on functions in unin-

fected host cells. Through experimenta-tion, however, the scientific communityis learning that each virus may have spe-cific steps of replication that can be usedas targets for selective, carefully aimedchemotherapeutic agents. Appropriateuse of such drugs requires a thoroughknowledge of suitable targets, based onan in-depth understanding of the viri-on’s replication mechanisms and a cor-rect diagnosis.31

Successful vaccines are based onknowledge of pathogenesis and immunedefenses, while comparable considera-tions govern treatment with interferon.Correct diagnosis, prevention of the viri-on’s spread in the environment, andeffective treatment of the disease arecomplex issues. Knowledge of the patho-genetic mechanisms by which a virusenters, spreads, and exits the body is crit-ical for antibody-containing immuno-globulin treatment and requiresknowing when a virus is susceptible toan antibody (for instance, during viremicspread), and when a virus reaches targetorgans where the antibody may be lesseffective.32 Among the most difficult anddemanding situations a physician mustaddress are viral infections. Althoughtremendous progress has been made inthe past few decades, some of theseissues still lack satisfactory solutions.More aspects of medical virology areunderstood now than previously, whileothers are being clarified gradually, andyet more are still obscure.

Successful investigation and man-agement of their pathologic processes isbased on knowledge of the properties ofviruses and the relationships they estab-lish with their hosts.33

This article concentrates on a biosen-sor for detection of virus and bacteria.

ELECTROCHEMICAL BIOSENSORSFOR DETECTION OF VIRUSES ANDBACTERIA Potentiometric biosensors are usually


based on ion-selective electrodes. Thesedevices measure the change in ion con-centration during a reaction. Generally,a simple sensor consists of an immobi-lized enzyme membrane surroundingthe probe of a pH meter where the cat-alyzed reaction will generate or absorbhydrogen ions.34-36 This leads to a changein pH which can be easily read. Threemain types of ion-selective electrodesare often used in biosensors: normalglass pH electrodes, glass pH electrodescoated with a selective gas-permeablemembrane, and solid-state electrodesconsisting of a thin membrane of a spe-cific ion conductor.37 It is also possible touse metal oxide semiconductors (MOS),which can be used to measure charge ona surface that will cause a current flowproportional to the charge. MOS devicesare small and have fast response timesdue to reduced diffusion. However, thesensitivity of these can be affected bythe ionic strength and the concentra-tions of the solutions being analyzed.

Potentiometric biosensors havebeen widely used for bacterial analyses.An example is the detection of bacterialcontamination in milk using an L-lactatebiosensor, bacterial growth, andsequence-specific biosensing of DNA.Electrochemical detection of DNAhybridization involves the monitoring ofa current under controlled potentialconditions.38 The hybridization is detect-ed via increased current of a redox indi-cator or by changes in conductivity orcapacitance.

This article, however, concentrateson the use of light-addressable potentio-metric sensors, which are proving popu-lar as a platform for detecting microbes.These are semiconductor-based systemswith an electrolyte-insulator-semicon-ductor (EIS) structure. When a currentis applied across the EIS region, a deple-tion layer is formed at the insulator-semiconductor interface.39,40 Thecapacitance of the depletion layer

changes with the surface potential,which is a function of the ion concentra-tion in the electrolyte. In order to deter-mine the capacitance, the semiconductoris illuminated by modulated light andthe current is measured. LAPS have sev-eral advantages when compared withother sensors: the surface is flat, there isno need for wires or passivation, andthey can measure pH and concentration.

Researchers at the United StatesDepartment of Agriculture have used aLAPS system in combination with animmunoligand assay to detect liveEscherichia coli O157:H7. They havereported that both live and dead bacte-ria can be detected in 30 to 45 minutes.With this system, bacteria are capturedonto a filter membrane by using specificantibodies.41 A silicone-based sensor isthen placed adjacent to the membraneand, on illumination, small changes inacidity are detected. The signal is pro-portional to the number of bacteriapresent, and it was possible to detect 710dead or 25,000 live E coli O157:H7organisms/mL.42 In a recent develop-ment, a LAPS approach was used todetect E coli in drinking water.35

An immunoassay was developedsuch that there was specificity to a par-ticular capsular protein present in thebacterium. The transducer, based on theLAPS principle, was able to detect theproduction of ammonia by urease-E coliantibody conjugate. It was claimed that10 cells/mL were detected in 1.5 hours.Generally, amperometric biosensorswork by enzymatically generating cur-rent between 2 electrodes. They havefast response times, dynamic ranges, andsensitivities similar to potentiometricbiosensors. Many amperometric biosen-sors depend on dissolved oxygen con-centration, which can pose a majorproblem. To overcome this situation,mediators are employed. These transferelectrons directly to the electrode there-by eliminating the need for the reduc-


tion of an oxygen cosubstrate.43 Themost commonly used mediators are fer-rocenes. Amperometric biosensors havebeen used for the detection of E coli inwater (screen-printed electrodes), bacte-rial vaginosis, studies of bacterial con-tamination, detection of agents ofbiological warfare (eg, anthrax), anddetection of E coli heat-labile enterotox-in and other neurotoxins.37 Ampero-metric biosensors44,45 have also beenused to study bacterial luciferase reac-tions, nano scale bacterial surface pro-teins and growth, and viability ofbacterial populations.

Many are based on the UIDA gene,or the !-D-glucuronidase (GLUase)enzyme for which it encodes.46 Althoughit is possible to target the UIDA genedirectly47 usually GLUase activity is usedas an enzymatic marker for the identifi-cation of Salmonella and Shigella. TheGLUase activity is detected by its abilityto cleave specific chromogenic or fluoro-genic artificial substrates added to theculture medium48 or directly to filteredcells.49 This approach has led to consid-erably faster and specific methods forthe detection of Salmonella and Shigellacontamination in water, although theanalysis time is still measured in hours.Also, it still requires either extensivemanipulation and incubation times50-53 orsophisticated equipment.49

According to the recent literature,the method of immunoassay has becomerapidly popular due to high sensitivity,decrease in the time of analysis, andreproducible data that are reliable. Theclass of amperometric biosensors hasbecome extremely successful becausethey can be used in field conditionswithout the need of skilled personnel.With these amperometric biosensors, thepathogens can be detected directly inthe original samples without any pre-enrichment of the sample. Recentlydeveloped techniques for the detectionof Salmonella and Shigella include meth-

ods based on the integration of severaltechnologies like the MEMS (micro-electromechanical systems), SAMS (self-assembled monolayer), DNAhybridi-zation, and enzyme amplifica-tion.54 Gau et al has tried to detectSalmonella and Shigella by integratingseveral of the recently developed tech-nologies like MEMS,55-57 SAMS,54 DNAhybridization, and enzyme amplification,and they have succeeded in detecting aSalmonella and Shigella concentration of1000 cells/mL. But the greatest disad-vantage of this method is that the com-bination of several technologies makes ita multistep, rigorous analysis. The prepa-ration of the MEMS detector array hasto be performed in a specially equippedlaboratory and by skilled personnel.Even though the analysis time is only 40minutes, the preparation required forMEMS and SAMS is time consuming.The analyte cannot be used for directdetection. Only samples from the culturemedia of the analyte can be used, thusreducing the chances for the developedprototype to be used for field-testing. Inaddition, the developed prototype couldonly detect Salmonella and Shigella in aconcentration of 1000 cells/mL. Thisstudy will examine the possibility of test-ing other viruses such as para influenzaand influenza A viruses.

MATERIALS AND METHODSReagents and MaterialsGraphite powder was obtained fromFisher Scientific Company (Hampton,NH). Ultrafree-MC centrifugal filterunits, used as a basis for the measuringcell (immunocolumn) construction, wereobtained from Millipore Corp. (Bedford,MA). Carbon rods used as counter elec-trodes were provided as a courtesy ofDFI Pultruded Composites Inc.(Erlanger, KY). Woodward’s reagent K(N-ethyl-5-phenyl iso-xazolium- 3!-sul-fonate), trypsin inhibitor, sodium iodide,and Tween 20 were procured from


Sigma Chemical Company (St. Louis,MO). 2-propanol (US IndustrialChemicals, New York, NY) was usedwithout further purification. All chemi-cals used were of analytical grade.

Affinity purified goat anti-rabbitIgG, conjugate of goat anti-rabbit IgGwith horseradish peroxidase (HRP), andrabbit IgG were obtained from SigmaChemical Company. Inactivated parainfluenza virus (para influenza type 1virus, strain Sendai) and influenza virustype A (H3N2, strain A/Panama/2007/99), affinity purified goat antibodies tothese viruses, and HRP-labeled affinitypurified goat antibodies to viruses (con-jugates) were procured from BiodesignInternational (Saco, ME). Other chemi-cals of analytical grade were obtainedfrom standard sources. For preparationof the aqueous solutions de-ionizedwater was used.

Immobilization TechniquesWoodward’s reagent K immobilization is

a technique for obtaining covalent link-age of the proteins to the surface of thecarbon materials (covalently linkedimunoagent-solid phase conjugates).First, an activation of the carboxylicgroup of the graphite solid support isperformed. Second, coupling of the pro-teins to the activated solid supportoccurs. The pH of the solution withWoodward’s reagent K (20 mg/mL) inwater was adjusted to 4.5 using dilutedNaOH solution, followed by suspensionof 100 mg of graphite powder (size ofthe graphite particles was <45 µm) in 10mL of Woodward’s reagent K solution.This was followed by incubation at roomtemperature for 2 hours with shaking.The suspension was later washed 5 timeswith distilled water by repeated centrifu-gation and removal of the supernatant.Graphite particles thus treated withWoodward’s reagent K were suspendedin 5 mL of a solution of anti-analyteantibodies in 0.02 M Na-phosphatebuffer solution (PBS) pH 7.8, containing

Figure 1. Calibration curve for the competitive rabbit IgG assay obtained by the flow-injectionamperometric immunosensor. Conditions of detection: flow rate was 100 µL/min; the workingelectrodes (5 mg of graphite powder immunosorbent per 0.22 mm pores filter) were poised at0.125 V versus Ag/AgCl in 0.1 M acetate BS, pH4.5 with 0.15 M NaCl, 1 mM KI, and 2 mM hydro-gen peroxide.

0.15 M NaCl. The same concentration ofantibodies of 0.5 mg/mL for eachimmunosorbent (anti-rabbit IgG, anti-para influenza, and anti-influenza A)was used. The suspension was incubatedat room temperature for 2 hours withshaking. The suspension was laterwashed 5 times with PBS by repeatedcentrifugation and removal of the super-natant. After incubation, 5 mg/mL solu-tion of trypsin inhibitor in PBS wasadded to the same suspension as a block-ing agent and incubated for an additional2 hours at room temperature with shak-ing. The suspension was finally washed 5times with PBST (PBS with 0.1% Tween20) by repeated centrifugation (5 min-utes each) and removal of the super-natant. The immunosorbent was storedin the same buffer solution at 4°C.

Sensor Design and Flow-ThroughImmunoassay TechniqueThe immunosensor design concept, pre-


viously described17,20,21 was modified andadapted into the immunoassay proto-type system for detection of parainfluenza virus and influenza virus A.The immunocolumn, which was the dis-posable biosensing element, consisted ofa plastic column (micro centrifuge fil-ters) with a 0.22-µm pore size polymericmembrane at the bottom and immuno-sorbent prepared from graphite powder(size of graphite particles was !45 µm) with immobilized antibodies.The immunosorbent was deposited onthe filter membrane by vacuum, result-ing in dispersed graphite immunosor-bent forming the measuring (working)immunoelectrode. The immunocolumnswere prepared by adding immunosor-bent suspension to the micro centrifugefilters.

Experimental Procedure A previously described technique forimmobilizing analyte related antibody

Figure 2. Calibration curve for sandwich rabbit IgG assay obtained by the flow-injection amper-ometric immunosensor. Conditions of detection: flow rate was 100 µL/min; the working elec-trodes (5 mg of graphite powder immunosorbent per 0.22 mm pores filter) were poised at 0.125V versus Ag/AgCl in 0.1 M acetate BS, pH4.5 with 0.15 M NaCl, 1 mM KI, and 1 mM hydrogenperoxide.


on carbon powder was adapted forimmunosorbent preparation.17,20,21 Theimmunosorbent was prepared in anamount needed for fabrication of aseries of 20 individual sensing elements.Sensing elements were prepared byadding 50 to 250 µL of 20 mg/mLimmunosorbent suspension to theimmunocolumn. The immunocolumnwas then vacuumed, thus depositing 1 to5 mg of immunosorbent onto the filtermembrane. The prepared sensing ele-ments were stored at 4ºC. The sensingelement of the flow sensor contained animmune modified graphite powder as aworking electrode, a graphite counterelectrode, and an Ag/AgCl referenceelectrode; these were the basis of theflow-through immunoassay technique.

Flow of the analyte viruses contain-ing solution through the immunosorbentresulted in the capture of the analyteviruses by the immobilized antibodies.The second stage of the assay employedflow of anti-analyte antibodies conjugat-

ed with peroxidase. This led to the for-mation of an anti-analyte-virus-anti-analyte antibodies-peroxidase complex.The quantification of this complex wasconducted by oxidation of iodide cat-alyzed by peroxidase.

The sandwich scheme measuringprocedure involved the followingsteps20,21:• First washing stage: Flow of PBST

(washing solution) containing 0.02 MNa-phosphate buffer (pH 7.8) con-taining 0.15 M NaCl and 0.1% Tween20 through the immunosorbent, 2minutes.

• First stage of incubation: A suspen-sion of the analyte virus created byusing the PBST (pH 7.8) was passedthrough the working electrode,through the immunosorbent, andthen through the reference electrodeand the counter electrode. Thisallowed the binding of the target ana-lyte onto the unlabeled antibodies,which were already immobilized on

Figure 3. Diagram shows the relationship between concentrations of hydrogen peroxide in sub-strate solution and specific signal-to-background ratio: (A) 2 mg and (B) 5 mg of graphite pow-der immunosorbent, with immobilized anti-rabbit IgG in each sensitive element; concentration ofrabbit IgG in the sample is 10 µg/mL.


the graphite particles, 8 minutes.• Second washing stage: Flow of a wash-

ing solution (PBST alone) through theimmunocolumn, 4 minutes.

• Second stage of incubation: Flow ofPBST (pH 7.8) containing the HRP-labeled antibodies (concentration ofimmunoconjugate was 1 mg/mL)against the target analyte through theimmunosorbent, allowing theimmunoconjugate to form the sand-wich complex of the unlabeled anti-body, the analyte, and the labeledantibody on the immunosorbent, 7minutes.

• Third washing stage: Flow of a 0.1 Macetate buffer solution (pH 4.5) con-taining 0.15 M NaCl and 1 mM KIthrough the immunocolumn, 4 min-utes.

• Recording of results stage: Flow of a0.1 M acetate buffer solution (pH 4.5)containing 0.15 M NaCl, 1 mM KI, andhydrogen peroxide through theimmunocolumn, 3 minutes.

The flow rate of solutions for each stage

was 100 µmL/min.

RESULTS AND DISCUSSION Methodology The graphite powder offers a large sur-face area and, at the same time, func-tions as the working electrode becauseof its conductive nature. The powderpossesses large area per unit mass, andthe particle sizes lie in the same order ofmagnitude as that of proteins. This pro-vides the basis for immobilization ofantibodies on graphite particles. Such aprocess enhances the proximity of bio-logical components with the transducer,a very essential factor for biosensordevelopment.

Graphite powder (size particleswere "45 µm) was used as animmunosorbent and working electrodesimultaneously. Current collection wasachieved through a carbon rod. The ref-erence electrode and counter electrodewere prepared by Ag/AgCl and carbon,respectively.

The graphite powder particles modi-

Figure 4. Determination of detection limits for influenza A virus electrochemical immunoassay (5mg of graphite powder immunosorbent per 0.22 µm pores filter) were poised at 0.125 V versusAg/AgCl in 0.1 M acetate BS, pH4.5 with 0.15 M NaCl, 1 mM KI, and 0.5 mM hydrogen peroxide.


fied by immobilized antibodies wereused for sandwich immunoassay of rab-bit IgG, para influenza 1, and influenzaA viruses. The immune-modifiedgraphite powder was deposited in thedisposable sensing element by vacuumof suspension through centrifugal filterunits under some vacuum pressure fromthe laboratory vacuum line. Because thegraphite particles were so small, thereagent solutions in the flow-throughtechnique were mixed very well, andwith a powder as a working electrode,the rate of immuno-interaction wasincreased, causing enzymatic and elec-trochemical reactions that produced thesignal.

HRP attached as an enzyme label tothe specific antibodies catalyzed the oxi-dation of iodide into iodine. Electro-chemical reduction of iodide formed thebasis for determination of the activity ofHRP and quantification of the enzymelabel. More details of immunosensordesign and flow-injection amperometric

system were described in earlierwork.17,19,20

Comparison of ImmunosensorPerformance Using Competitive andSandwich Techniques Rabbit IgG immunoassay using ampero-metric detection was used as a model forthe flow-injection procedure. Optimalscheme of assay using competitive orsandwich immunoassays of rabbit IgGwas used. For carrying out the competi-tive assay, the preliminary 15-minuteincubation of testing solutions, contain-ing both analyte IgG and IgG-HRP con-jugate (1.0 µg/mL), was used. Thecalibration curves for competitive andsandwich techniques of IgG assay areshown in Figures 1 and 2.

Figure 1 shows a typical calibrationcurve for competitive reaction where thesignal decreased as the concentrationincreased due to the nature of the reac-tion. The lower detection limit was 5 nMof IgG. The higher concentration level

Figure 5. Determination of detection limit for para influenza virus electrochemical immunoassay(5 mg of graphite powder immunosorbent per 0.22 µm pores filter) were poised at 0.125 V versusAg/AgCl in 0.1 M acetate BS, pH4.5 with 0.15 M NaCl, 1 mM KI, and 0.5 mM hydrogen peroxide.


that could be detected with a reasonableamperometric signal was around 150 nMof IgG. However, the results shown inFigure 2 using the sandwich scheme ofIgG assay show the low detection limitwas 8 pM, which was approximately athousand times more sensitive than thecompetitive technique. Also the highdetection limit of the sandwich schemeassay was extended to more than 104 pM(107 nM) in comparison with the com-petitive scheme of high detection limit.The high detection limit of the sandwichscheme was higher than 150 nM of thecompetitive scheme. Therefore, from thispoint forward, the experiment used thesandwich assay as the main experimen-tal technique.

OPTIMIZATION OF IMMUNOSENSORPERFORMANCEThe major analytical parameters of the

amperometric immunosensor are detec-tion limit, analytical range, and signal-to-background ratio related with theamount of bounded conjugate and theconditions of its detection. The results ofoptimization of graphite powderimmunosorbent amount and composi-tion of substrate solution are presentedin Figure 3.

Several concentrations of graphitepowder as immunosorbent were usedwith an immobilized anti-rabbit IgGconcentration of antibodies of 0.5mg/mL from stock solution; the concen-tration of rabbit IgG in the sample was10 µg/mL.

Figure 3 shows the dependence ofthe amperometric immunosensorresponse on concentration of hydrogenperoxide in substrate solution, theamount of immunosorbent, and its influ-ence on signal-to-background ratio.

Figure 6. Calibration curve for sandwich para influenza virus assay obtained from the flow-injec-tion amperometric immunosensor. Conditions of detection: flow rate is 100 µL/min; the workingelectrodes (5 mg of graphite powder immunosorbent per 0.22 µL/min; the working electrodes (5mg of graphite powder immunosorbent per 0.22 µm pores filter were poised at 0.125 V versusAg/AgCl in 0.1 M acetate BS, pH4.5 with 0.15 M NaCl, 1 mM KI, and 0.5 mM hydrogen peroxide.


Figure 3B shows that the specific signalwas approximately 1.5 times higher for 5mg of graphite powder immunosorbentin comparison with Figure 3A, whichshows 2 mg of graphite powderimmunosorbent. However, the conjugatesignals (nonspecific binding of conju-gate) with different concentration ofhydrogen peroxide were similar for bothcases. As shown in Figure 3, the signal-to-background ratio (illustrated in red)was 2 times higher for 3B using 0.5 mMconcentration of substrate (hydrogenperoxide). Therefore 5 mg of graphiteimmunosorbent with 0.5 mM of hydro-gen peroxide was used through theexperiments.

Flow-injection AmperometricImmunoassay of Para Influenza andInfluenza A VirusesCalibration curves for influenza A andpara influenza using 5 mg of graphite

powder immunosorbent for both experi-ments were similar (Figures 4 and 5). InFigure 4, influenza A concentration wasplotted versus amperometric signal,which illustrated a linear response up to50 ng/mL. Above 50 ng/mL, it was anexponential signal increase. The lowerdetection limit of influenza A was 0.4ng/mL with a coefficient of variation(COV) of 0.05. The upper detection limitwas as high as 300 ng/mL. Statisticallylower detection limit signals can bedetermined by multiplying 3 times theCOV of the background signal.

Similar results were obtained withpara influenza; Figure 5 describes theamperometric sensor response to differ-ent concentrations of para influenza.However, the amperometric signal ininfluenza A was smaller than andapproximately half that of the parainfluenza values. This could be due tothe differences in the outer protein shell,

Figure 7. Specific and nonspecific binding of para influenza and influenza A viruses withimmunosorbent. Signals of specific binding: for para influenza virus with anti-para influenza(curve 1) and influenza A virus with anti-influenza virus (curve 3) immunosorbents. Signals of non-specific binding for para influenza virus (curve 2) and influenza A virus (curve 4) with anti-rabbitIgG immunosorbent.


which is called capsid protein. This is arepeating proteins subunit. The architec-tural arrangement of this capsid proteinor its subunit structure is different ininfluenza A than para influenza. Thepresence of a membranous envelopesurrounding the capsid protein couldalso explain these differences. The lowerdetection limit of para influenza was 0.5ng/mL with a COV of 0.05. The upperdetection limit is illustrated in Figure 6.

Figure 6 describes the amperometricsensor response to different concentra-tions of para influenza in ng/mL. Lowerdetection limit was 0.5 ng/mL with aCOV of 0.06. The upper detection limitwas as high as 10,000 ng/mL. Above thatconcentration, the sensor reached its sat-uration limit. Once the sample wasinjected, it took 4 minutes to reach asteady state, and the total time of theassay from the beginning to the end wasabout 22 minutes. The current was pro-portional to the concentration with theupper limit of the device of 15microamps.

Investigation of Nonspecific Binding ofConjugates, Para Influenza andInfluenza A to Immunosorbents Nonspecific binding of the conjugatemolecules (IgG with HPR) and bothviruses analyte to the solid surface ofthe immunosorbent was a critical factoraffecting the sensitivity and detectionlimit of the immunoassay because it wasthe major obstacle in decreasing thelower detection limit and increasing theselectivity. The background responses(shown as dash lines in Figures 4, 5 and6) were obtained when no viruses werepresent in the sample solutions. It maybe attributed to nonspecific binding ofthe conjugates and nonenzymatic oxida-tion of substrate and adsorption of thevirus to the immunosorbent.

There were 3 possibilities for non-specific binding of conjugate moleculesthat could affect the background signal:1) nonspecific binding with immobilizedantibodies, 2) adsorption of conjugatemolecules directly on the surface ofgraphite particles, and 3) adsorption to

Figure 8. Nonspecific for influenza A virus (curve 1) and specific for para influenza virus (curve 2)binding with anti-para influenza immunosorbent.


the membrane in the filter support andto the walls of flow-through systemtubes. In this investigation, goat anti-influenza A and goat anti-para influenzaantibodies and the same goat antibodiesconjugated with HRP were used. In thiscase, the nonspecific binding of the con-jugate molecules with immobilized anti-bodies was minimized. In order to lowerthe absorption of conjugate on graphiteparticles, blocking reagent 5 mg/mLtrypsin inhibitor solution was used at thestage of immunosorbent preparationand PBST with 0.1% Tween 20 was usedfor all immunoassay stages.

Figure 7 illustrates the immunoassayresponse for detection of influenza Aand para influenza, in curves 1 and 3.Curves 1 and 3 were the specific signalsto 2 and 4, which described the nonspe-cific signal obtained by using only theimmunosorbent (anti-rabbit IgG, notanti-virus IgG). The signals from thenonspecific binding curves 2 and 4 hadsimilar signals of the background cur-rent up to a concentration less than 50ng/mL. Also nonspecific binding curve 2

(para influenza) had a higher signal thanthe nonspecific binding curve 4 (influen-za A). This may be due to higher conju-gate affinity, reaction sites, and thereaction constant.

CROSS-REACTIVITY OF THEIMMUNOASSAY SYSTEMDEVELOPED Selectivity and SpecificitySelectivity is the ability of a particularanalysis method or technology to identi-fy and quantify a particular compoundin the presence of other, potentiallyinterfering compounds.58

The term specificity is used toexpress the same general idea as selec-tivity, but to an even higher degree. Atest with a high degree of specificity willnot only resist nontarget interferences,but will also give unique responses thatpermit closely related compounds to bedistinguished from a unique target ana-lyte. Nonspecific methods are designedto give a response that integrates theindividual responses of all respondingtarget analytes present in the sample.58

Figure 9. Nonspecific for para influenza virus (curve 1) and specific for influenza A virus (curve 2)binding with anti-influenza A immunosorbent.


In order to determine the specificityand selectivity of para influenzaimmunosorbent (anti-para influenzaantibodies) in presence of both parainfluenza and influenza A viruses,influenza A virus was added to parainfluenza immunosorbent. Figure 8 illus-trates the immunoassay response fordetection of para influenza presented incurve 2. Curve 1 represented theimmunoassay response for detection ofinfluenza A using para influenzaimmunosorbent.

A low signal was obtained in curve 1similar to curve 4 in Figure 7, which rep-resented the nonspecific binding forinfluenza A. These 2 curves confirmedthat was no interference from influenzaA virus to para influenza assay. In con-trast, Figure 9 illustrates the immunoas-say response for detection of influenzaA presented in curve 2. Curve 1 repre-sented the immunoassay response fordetection of para influenza, usinginfluenza A immunosorbent. It was con-cluded from this curve that para influen-za interferes with influenza A assay.

The Main Characteristics of ParaInfluenza and Influenza AAmperometric ImmunoassayFigure 10 shows the steady state signals

for influenza A with a concentration 0ng/mL of influenza A to determine thebackground current. Additional sampleswere added with 3, 30, 100, and 300ng/mL concentrations. The signal wasstable and reproducible for the steadystate value, and it could be used to pro-gram a hand-held meter for read out ofthe concentration of the influenza Avirus. For all the concentrations ofinfluenza A virus listed above, the back-ground signal was very stable and repro-ducible using a small device such as theone used in this study.

Once the sample was injected, ittook 4 minutes to reach a steady state(Figure 10), and the total time of theassay from the beginning to end wasabout 22 minutes for 1 measurement.Similar signals were obtained with parainfluenza virus.

CONCLUSION The sandwich scheme assay performedmuch better than the competitivescheme assay. The low detection limitwas about 5 nM for the competitivescheme in comparison with 8 pM for thesandwich scheme for rabbit IgG; that isa thousand times more sensitive.However, the upper detection limit forthe sandwich scheme can exceed a thou-sand times more than the competitivescheme for rabbit IgG.

Using different concentrations ofhydrogen peroxide with different con-centrations of graphite powderimmunosorbent, it was concluded that 5mg of graphite powder immunosorbentand 0.5 mM of hydrogen peroxide werethe optimum concentrations forimmunoassay for anti-rabbit IgG. Thesame concentrations of graphite powderimmunosorbent and hydrogen peroxidewere used for both influenza A and parainfluenza.

The signal from the nonspecificbinding curves 2 and 4 in Figure 7 affect-ed the lower detection limit up to a con-

Figure 10. Sensor’s response signals for differ-ent concentration of influenza A virus, ng/mL:1) 0; 2) 3.0; 3) 30; 4) 100; 5) 300.


centration of more than 50 ng/mL. Thespecific binding curve 1 for para influen-za was higher than the specific bindingcurve 3 for influenza A. This may be dueto higher conjugate affinity, reactionsites, and the reaction constant.

Figure 8 confirmed that there wasno interference from influenza A virusto para influenza assay. In contrast,Figure 9 illustrated the immunoassayresponse for detection of influenza Arepresented in curve 2. Curve 1 repre-sented the immunoassay response fordetection of para influenza, usinginfluenza A immunosorbent. It is con-cluded from this curve that para influen-za interferes with influenza A assay.

In Figure 10, the signal response fordifferent concentrations of influenza Avirus were very stable with reproducibleand stable background currents andsteady state signals. It also showed thereal signal of the immunoassay detec-tion.

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