EX2221
`Eli Lilly & Co. v. Teva Pharms. Int'l GMBH
`IPR2018-01426
`
`1
`
`

`

` I l
`
`Table 1. Binding specificity of anti—2,4D monoclonal an-
`tibody MS3BF27. (Reproduced, with permission, from
`[37]
`
`
`Compound
`Cross—reactivity
`50% Mx*
`
`
`2,4D-dichlorophen0xy acetic acid
`2,4D—methyl ester
`2,4—isopropyl ester
`Dichlorprop
`2,4,5—Trichloro acetic acid
`MCPA
`Indol—3-acetic acid
`
`15
`15
`90
`l 10
`180
`360
`>1,000
`
`>l,OOO
`2,4 Dichlorophenol
`>1,000
`2,4DB
`>l,000
`2,4DB—Butyl ester
`>l,000
`2,4DB—Isobutyl ester
`>1,000
`Dicamba
`
`Picloram >1,000
`*Amount of the compound needed (ppb) to inhibit maxi-
`mal binding *Mx) of 2,4D[3 H]-glycine by 50%.
`
`several rabbits immunized with different 2,4D-protein
`
`conjugates (Panel B). The cumulative results from our
`studies revealed that increasing titres of polyclonal an-
`tibodies and higher antibody binding affinity could be
`obtained using hapten-protein conjugates containing
`an elevated ratio of 2,4D groups per molecule of pro—
`tein carrier. Using affinity column-purified anti-2,4D
`polyclonal antibodies as well as hybridoma mono-
`clonal antibodies, we studied the specificity of the
`2,4D-antibody reaction by measuring the competiti-
`ton of 2,4D—[3H]-glycine with various compounds,
`structurally both related and not to 2,4D, for antibody
`binding sites. For example, the results reported in Ta-
`ble 1 indicated that 2,4D-methyl ester, 2,4D-isopropyl
`ester, 2,4,5-Trichloro aceticaciol or MCPA, cross react
`with 2,4D antibodies to some extent, revealing each
`time a different binding affinity, while other herbicides
`such as Dicamba or the auxin Indol-3—acetic acid or
`Picloram (a potent herbicide which is normally sold
`commercially as a mixture with 2,4D) showed almost
`negligible cross reactivity.
`Figure 3 outlines a flow chart of ELISA for 2,4D
`detection and measurement. By comparing our dif—
`ferent anti—2,4D hybridoma monoclonal antibodies in
`solid phase RIA and ELISA, we found surprisingly,
`that the ELISA method sometimes failed to detect pos-
`itive antibody binding capacity for 2,4D in samples
`that turned out to be positive with RIA performed in
`solution [37]. Only three hybridoma monoclonal anti—
`
`136
`
`in order to become immunogenic and to stimulate ap—
`propriate amounts of antibodies of sufficiently high
`affinity in the animal host. Preliminary experiments
`were performed in our laboratory, testing direct cou—
`pling of the herbicide to different proteins, or using
`peptide linkers or spacers betweeen the hapten and the
`carrier molecule. The best results were achieved by
`
`using direct coupling with either glutaraldehyde or the
`carbodiimide coating chemistry procedures [37]. The
`following protein carriers gave us excellent results:
`for the immunization, keyhole—lympet—haemocyanin
`(KLH) and bovine thyroglobulin (BTG); for antibody
`detection, bovine serum albumine (BSA).
`The following hapten-protein conjugates were
`mostly used: 2,4D25-KLH; 2,4D20—BTG and 2,4Dg—
`BTG; 2,4D18-BSA and 2,4D5-BSA (the number re—
`ports the groups of herbicide coupled to a protein
`carrier molecule). Rabbits, rats and mice received sub—
`coutaneous (s.c.) or intraperitoneous (i.p.) injections
`of antigen emulsified in complete Freund’s adjuvant,
`boosted so with antigen emulsified in incomplete
`Freund’s adjuvant [37]. Serum polyclonal antibod—
`ies with increasing titre and affinity were produced
`with successive antigen injections. However, hyperirn-
`mune sera subsequently required extensive absorption
`against the uncoated protein carrier and affinity purifi-
`cation by column chromatography on beads covalently
`linked with 2,4Dlg-BSA (i.e. a different protein car—
`rier),
`to yield limited amounts of high specificity
`anti—2,4D antibodies. The hybridoma technology cir-
`cumvented this limitation; by this technique we ob-
`tained clones of hybrid cells (from the somatic cell
`fusion between myeloma P3X63Ag8 cells with anti—
`2,4D primed spleen mononuclear lymphocytes) which
`produced virtually unlimited amounts of antibodies of
`selected hapten—specificity and affinity, which allowed
`us to measure pmol quantities of 2,4D in buffer solu-
`tion as well as in river waters, in crude plant extracts
`
`and biological fluids (urines) directly or after only par-
`tial sample purification [37], in both RIA and ELISA
`assays in a reproducible manner.
`A few examples of the results normally achieved
`are reported below. Figure 1 outlines a flow—chart of
`RIA performed in our laboratory, either on solid phase
`or in solution. 2,4D—[3 H]-glycine conjugates was pre—
`pared as in [37, 38] and used as the specific ligand
`pesticide in our assays. Figure 2 shows an example of
`typical binding curves in RIA, comparing binding of
`polyclonal anti-2,4D antibodies from serum samples
`obtained from subsequent bleedings of a single rabbit,
`before and after immunization (Panel A), and from
`
`2
`
`2
`
`

`

`137
`
`RADIOIMMUNO-ASSAY (RIA)
`
`IN SOLUTION
`
`SOLID PHASE
`96 well
`late
`
`COATING
`IMMUNE REACTION MIXTURE
`coat each well with 100 pl of Rabbit anti Mouse
`In a reaction tube containing buffer‘r
`IgG solution
`0.5mg.norma.l 300, add dropwisc and mix on
`Vortex
`[ANTIBODY AGAINST PESTICIDE+(’H)I("’I)
`PESTICIDE+I- COMPETITION STANDARD
`COMPOUND 0R SAMPLE]
`
`FIRST INCUBATION
`incubate overnight at 4°C
`
`IMMUNE COMPLEX PRECIPITATION
`add cold (NI-102804 (45% f.c.) dropwise and mix
`on vortex
`
`WASH
`
`centrifuge supernatant, discard and wash 5 times
`the pellet with cold (NH4)2SO4 (45% fie.)
`
`COUNTING
`
`FIRST INCUBATION
`incubate overnight at 4°C
`
`WASH
`wash 3 times with buffer
`
`MOUSE ANTIBODY AGAINST PESTICIDE
`add 100 [.11 antibody solution
`
`SECOND INCUBATION
`incubate for 4 h. at 25°C
`
`WASH
`w'ash 3 times with buffer
`
`[Jill-PESTICIDE +/- COMPETITION
`STANDARD COMPOUND 0R SAMPLE
`add 100 pl reaction mixture
`
`THIRD INCUBATION
`incubate for 1h. at 4°C on a shaker
`
`WASH
`wash 3 times with buffer
`
`COUNTING
`Autoradiography (whole plate)/cut and count each
`well
`
`Figure 1. Flow chart illustrating our anti—2,4D radioimmunoassay (RIA) procedures,
`
`bodies (clones MS3BF27, H4L2 and D1/7) appeared
`to bind specifically 2,4D with high affinity (higher
`than 10—7 M Kd), in both assays giving always reliable
`estimates of herbicide content in untreated environ—
`mental samples. Seventy two other different clones
`producing anti-2,4D antibodies of significantly lower
`affinity for 2,4D, sometimes gave alternative results '
`RIA or ELISA, whereas the three previous indicated
`antibodies turned out to give identical results in both
`assays, with the same reagents. Reproducibility prob—
`lems in solid phase immunoassays were noticed, par-
`ticularly when hapten-protein conjugates with a low
`ratio of hapten groups bound per protein carrier mole—
`
`cule were used as the target antigen. These results, in
`agreement with similar observations reported indepen—
`dently by others with other herbicides [36—40], sug-
`gested the contamination of unknown cross-reacting
`derivates from unrelated but structurally similar com—
`pounds in unprocessed environmental samples, affect—
`ing the reliability of solid phase immunoassays, par—
`ticularly ELISA, with certain low-affinity anti—2,4D
`hybridoma monoclonal antibodies. Similar discrepan—
`cies were observed using affinity purified polyclonal
`antibodies from early bleeding serum samples of rab-
`bits grown in farms; where a large environmental use
`of 2,4D was then reported. We intend to look carefully
`
`3
`
`

`

`138
`
`
`
`
`
`x2.40[am-glycinebound
`
`u0
`
`
`
`10‘
`
`10'
`
`10"
`l/urum dilution
`
`10‘
`
`
`to5
`
`
`
`
`
`x2.40[am-glycinebound
`
`100
`90
`IO
`70
`60
`50
`4O
`3O
`20
`10
`
`
`
`10‘
`
`10'
`
`10°
`llama dilution
`
`104
`
`,0:
`
`Figure 2. RIA standard curves showing the binding of 2,4D[3 H]—glycine conjugate to rabbit serum polyclonal antibodies. Panel A: antisera
`were obtained from the same animal before immunization (A) and after 2 (E1) or 5 (O) boost inoculations with 2,4D25 —KLH, Panel B: binding
`curves from polyclonal antibodies anti-2,4D serum samples obtained from different rabbits hyperimmunized (5 boosts) respectively with:
`2,4D5-BTG (0); 2,4Dg—KLH (O); 2,4D20-BTG (A)’ 2,4D25—KLH (Cl )r bars show SE: (Reproduced, with permission, from [37]).
`
`:
`
`
`
`for possible 2,4D—complex formation in ground-water,
`plant extract or urines from individuals exposed to
`2,4D [37], to detect degraded herbicide products, us-
`ing our large panel of anti-2,4D antibodies as no
`attempt has yet been made to do so.
`Experiments of fine epitope mapping on simple
`molecules like herbicides are difficult to perform in
`solid phase immuno-assays, due to technical limi—
`tations. Coating of the herbicide-conjugates in solid
`phase is normally obtained by physical contact of a
`diluted antigen solution on the plastic surface (such
`as polystyrene or polyvinylchloride), taking advantage
`of the strong hydrophobic interactions which occur in
`this process. It is unpredictable what factors primarly
`influence antigen coating to plastic. 2,4D—protein con—
`jugates attached to solid surface by absorption (partic—
`ularly conjugates with a low ratio of herbicide groups
`coated per protein carrier molecule), may undergo
`
`4
`
`changes that affect hapten binding properties and per»
`haps give rise to unpredictable ligand conformational
`events. The use of a second antibody with a large en-
`zyme tracer to reveal the primary immunocomplex as
`well as the detachment of loosely bound immunocom-
`plexes from plate, may represent some further criti-
`cal events limiting these immunoassays, particularly
`when low—affinity anti-herbicide antibodies are tested.
`These problems, on the contrary, are less critical when
`the immunoassay is performed in solution.
`
`SPR for Biosensing
`Although RIA and ELISA are a highly cost- and
`handling-effective alternative to analytical laboratory
`pesticide measurements, as they require minute sam-
`ples and dispense with tedious clean ups and pre-
`purification or derivatisation, these two immunoassays
`are still quite time-consuming (hours—days) and labo—
`
`4
`
`

`

`ENZYME-LINKED IMMUNO-SORBENT ASSAY (ELISA)
`
`SANDWICH/EPITOPE MAPPING
`
`DIRECT/COMPETITION
`
`139
`
`COATING
`coat each well with 100 pl of pesticide protein-
`carrier solution
`
`FIRST INCUBATION
`incubate overnight at 4°c
`
`WASH
`wash 3 times with buffer
`
`
`
`FIRST ANTIBODY AGAINST PESTICIDE
`add 100 [.11 antibody solution with/w.o. competition
`standard compound or sample
`
`SECOND INCUBATION
`incubate for 14 h. at 25°C (on shaker)
`
`WASH
`wash 3 times with buffer
`
`SECOND ANTIBODY REACTION
`add AP-goat anti Ig in buffer
`
`THIRD [NCUBATION
`incubate for 1 h. at 25 °C
`
`WASH
`wash 3 times with buffer
`
`DETECTION
`add substrate; stop with 1N NaOH; read at OD“),
`
`
`
`
`
`COATING
`coat each well with 100 pl of rabbit anti
`mouse lgG solution
`
`FIRST INCUBATION
`incubate overnight at 4°C
`
`WASH
`wash 3 times with buffer
`
`MOUSE MoAb AGAINST PESTICIDE
`add 100 pl antibody solution
`
`SECOND INCUBATION
`incubate for 6 h. at 25°C
`
`WASH
`wash 3 times with buffer
`
`PESTICIDE ENTRAPPING
`add 100 pl Pesticide-Protein carrier solution
`
`THIRD INCUBATION
`incubate for 4 h. at 4 “C
`
`WASH
`wash 3 times with buffer
`
`SECOND ANTIBODY SYSTEM
`add 100 pl of AP—Rabbit Polyclonal (or AP-
`2nd mouse MoAb) anti Pesticide
`
`FOURTH INCUBATION
`incubate for 1-4 h. at 25 °C
`
`WASH
`wash 3 times with buffer
`
`
`
`DETECTION
`Add substrate; stop enzyme reaction; read at
`OD”; with 1N NaOH
`
`
`
`Figure 3. Flow chart illustrating our enzyme—linked—immunosorbent assay (ELISA) procedures.
`
`rious and require a labelling or secondary molecule as
`tracer. The labelling process by itself may denature
`the haptenic molecule and consequently its antibody
`binding characteristics. Thus,
`the measured binding
`pattern may not necessarely be the same as for the
`native molecule, leading sometimes to erroneoiis con-
`clusions about the interactions. While looking for an
`
`alternative approach, we have recently become very
`interested in the optical, non-radiative SPR technol-
`ogy. SPR is a non—labelling technique for real-time
`monitoring of biomolecular interactions [40—44]. A
`surface plasmon (SP) can be regarded as a strongly
`
`localized optical wave that is driven by an external
`light source (laser or a laser diode), that propagates
`along the interface between the metal and the ambient
`medium (e.g. a buffer or a bio—fluid). This wave is
`extremely sensitive to local variations in the refractive
`index near the metal surface. The SP phenomenon can
`be observed as a dramatic drop in the reflected light
`intensity that occurs at a given angle of incidence of
`the light with respect to the metal surface, the so-called
`angle @sp (see Figure 4, Panel A). Small changes in
`the ambient medium refractive index near the sensing
`surface caused for example by binding of biomole—
`
`5
`
`

`

`140
`
`Convergent
`Light Beam
`
`Photo Diode
`To Computer
`Array fl
` Cylindrical
`Glass Prism
`
`
`Opto
`
`Intarface
`‘ “x Sensing Surface
`on a Glass Shde
`
`
`
`Thin Flow Througflx
`Sample C911
`
`
`
`‘11
`
`2
`
`131136
`
`figure 4. Schematic illustration of a SPR phenomenon using the BIACUW technology. Panel A. A baseline (")sptlit representing the sensing
`matrix with the immobilized antigen is (,lctot'mincd under contihous flow of the buffer, Panel B. After injncting the antibody over the sensing
`xuitticc,
`tho adsorbed immunocomplcx formation inciucm :1 change in the optical propcnics of the ambient medium {(5)3512} influencing the
`resonance condition and thus generating it shift of A (fish referred as the tm‘mtanrif or Si’k’ ts'iggm'tlt The final response; of the interaction is;
`ohtztinmt after rinsing with 1mm buffer A (fish : («1&th -«
`(6513(1). Patio! C. Since 815% is 21 real-time method when the change in msoimnw
`angle is followed us :1 function of time (“)spt't)‘ it, um also provide infommtion about the kimono (association, dissociation costunts) and th
`affinity of tho biomoiccttieu‘ interaction,
`
`
`
`6
`
`

`

`ldl
`
`vetting the COOH groups in the tiiatris into c
`active N—hydrosysuccinitnidc esters by exposure to
`pools of Nwliydrosysuccinlinide (NHS) and Nvethyb
`N—(ditnethyiaminowpropyl)carhodiiniidc (EEC). The
`protein to link is subsequently injected over the sens-
`ing layer and free amino groups on the o‘otein {for
`example, the 2,4Dig—BSA conjugates or the Fc portion
`of the entrapping lgG molecules) react. covalently with
`the N~hydro>tysucciniinide ester groups in the matrix
`during formation of a peptide bond.
`fl“he remaining
`{unreactive) N~hydroxysuccininiidc esters groups are
`then inactivated with cthanolarnine
`
`(ii) Antibody binding to antigen immobilizer! to
`the Izya'rogel matrix, Polyclonal and liybridoina ntotr
`oclonal antinnD antibodies were assayed using the
`BiAcorel against 2,4D.g~BSA immobilized to the
`
`dextran matrix Cttnnpetition of antibody binding to
`immobilized 2,41318~8853 by“ the free 2,4}?! or other
`structurally related and unrelated compounds was also
`tested. Results (not shown in figures) confirmed those
`obtained with RIA and ELISA reported above,
`indi-
`cating different binding capacities among the panel ol’
`antibodies examined.
`in these assays,
`the biosensor
`ensured great sensitivity, precision and reproducibility
`veca se the samples were run on the some detection
`
`the sensing layer could be rapidly
`aret. Moreover;
`and completely regenerated by lowering the pH of
`he rt nning bul‘i‘er. This enables comparison of exper—
`‘men s sequentially within a few minutes, using the
`same reagents,
`
`Concerning the specificity of the 214i?) antibody
`‘eact’onsi Figure 5 illustrates a typical competition
`standard binding curve obtained alter measuring on»
`react've binding sites of antibodies pre~incubated at
`he same lined concentration, in the absence or pres
`ence of increasing concentration of free 25511:) (from
`{1.00 gig to 1.0 gig/ml) used as competitor.
`{ iii) Mir/time binding analysis: By this method it
`'s possible to perform experiments ot” cpitopc rnao
`sing, with the aim ol' verilying whether the binding
`o 214D groups of one monoclonal antiwlili} anti»
`and}: can influence the binding of a second and then
`a third monoclonal antibody injected sequentially or
`not. Alter regenerating the sensing layer the process of
`“finding can be repeated using a different antibody se—
`quence of" injection until all possible ctntiblnations are
`examined. Figure 6 schematically outlines the overall
`nultisite binding procedure and shows an example ol‘
`sensorgrants.
`
`By continuing th ’- above procedure? screening one
`ianel (3175 anti-2.4D hybridonia monoclonal antibot‘h
`
`
`
`
`
`
`cules on the sensing St rl’acc, will change @sp and t. to
`obtained shift
`(it @sp is normally referred to as t to
`SPR or resonance signal [42~4éll (iDanel B). Nylant er
`ct 211.
`[451 and Liedhe‘g ct al. 146] dei'nonstrated tie
`nse oi“ the so~called K'ctschtnann configuration (KC)
`lilo] for sensing applications to set up an SP on tie
`sur ’ace of a thin gold l’ltn deposited onto a glass sli e
`The resonance R ((5))«curves shown in Figure ill Pat e1
`8 provide, however, only information about the initial
`an linal states, ie. be ’ore and after the interaction. A
`
`more convenient way to present the data is to plot 1 to
`angular posit'on as a "unction of time Gisptt) the so
`ctl ed ‘Sensoigi‘am’ (Figure 43 Rtnel C). This presenv
`talion enables us to t‘ol ow the interaction in real time,
`ant
`to estrac informaion about the concentration of
`
`
`
`
`
`
`
`
`
`
`the biomolec iles in the analyte, the kinetic (assocnw
`tion and dissociation constants) and the a “trinity of tie
`trio nolecular interactions [47, 48:1.
`in our latioratory, SPR measurernens are per—
`t‘ormcd on the BlAcoreTM (BlAcore AB,
`ppst 1a
`Sweden),
`21 SPR»based on 01112 tic biosensor sgstein.
`"the modifie
`gold surface (Sensor Chi), C S, by
`WAcore AB),
`covered w‘ '1
`an
`extended can
`boxymcthylated hydrogel na r'x 149]: (llows covet"
`Ecntly coupling of proteins ("or example ant'bodies)
`:tsing cotwcntional carhod‘itn’te coupling cl eniist‘y
`ts 01 at one stun
`[501 The Blincore also £10193
`’
`
`
`
`
`
`
`
`
`
`
`
`'dic cartridge 51} for
`grim and an integrated mic ‘oli
`the introduction and trans iotation of the sttnple o
`the sensing surface. The change of refractive index
`tortiiportional to the concent‘tt'on of the intet‘acti g
`molecules) causes a shift it the angle of incit once at
`which the SPR phenomenon occurs. Such shifts a‘e
`conntinously monitored auto 1 atict 11y and are shown
`as sensorgrams [471.
`
`x a
`
`Stale/”or 2,419 (Infection turd antibody binding (tiialwis,
`. few examples will now be reported. illust‘ating the
`:Eoplication of SPR for 2,413 detection using anti~2,4D
`zétilllJtXllCS.
`
`{ i) Imumbiliwtion plottedtme. Two dil‘l‘e ‘ent strate—
`
`cs were used:
`1) direct antibody binding”
`41313—133A conjugates which was prev‘ously do
`l’iilCnlly coupled to COOH groups in tlc dextrnn
`matrix by the carbodiimidc coupling method 149?
`73%)}; 2) binding ol‘ 2,4131gg—BSA conjugates to ho
`Eoogcncously oriented anti—2,41) antibod'es (polyr
`iiional/nmnoclonalj captured via specific 21 thlgtllic
`antibodies pre-inunobilizcd to the sensor chip. Dur—
`ing the immobilization step,
`the coupling 01‘ a pro
`in is dont automatically in the hiosenso by con~
`
`
`
`
`
`
`
`§ w
`
`7
`
`

`

`1500
`
`108%
`
`EGG
`
`
`
`ResonanceUnits;(RU)
`
`1
`
`,
`
`,
`
`2‘40
`
`erg/ml
`
`Figure 5. Bioscnsor nnnlysix (Toinpetititm standard curve showing the response of the binding between the immobilized 2,4l)l8~BS/-\ and a
`ttxctl conccntmtion of untold!) purified nionoelonnl antibody MS38F£7 (2t) [Lg/1111) til’tcr pt’t)~lttCttlf>ttlltm with varying amounts of free 2.413
`(from (3:001 itgiml to l fig/ml).
`
`m ‘
`
`icst we ended up by discriminating two major antibody
`classes: three antibodies which specifically recognize
`2.4l31g—BSA at the some epitope (or strongly overlap—
`ping epitopes) (clones M5338??? t'l-éllJZ and Dl/7)
`and the other antibodies partially overlapping this
`2.41? ‘core’ site. As 211] example“, Figure 6,
`)tmttl B
`shows that when clone hil53l3l7’27 antibody was; used
`to entrnp 2,4Dm—BSAA, pnrilied polyclonttl nnti~2,4D
`antibodies were Stiil reacting with the immobilized ini—
`inunoconiplex, strongly suggesting ntttltisite binding
`ettptteity.
`One possible explanation of" these results is: that
`effective hapten-protein conjugates obtained with glu»
`tttrntdehyde or the cttrbodiintit’le to couple a hnptcn via
`the primary ainino— and cnrboxylfigrottps ol the pro~
`6}“
`tein cttrrier can also rcaet with other i rottp residues,
`Smelt as sulphydril groupa phenolic liylroxyls rind the
`irnidttml of histidine reaidties, This may result in rnttlm
`tiple inter» or intrtrinolecttle reactions which may t‘tlltlt“
`epitope prexentntion ol' the relevant hnpten niolceule
`and generate eross~linl<ing, with the production ol~ tin-
`tihodies ol’ different binding capacity for the given
`httpten group l52~53l. in the present case, antiQAD
`antibody may therclore include both ttnti»2,éll.) tintir
`
`‘-
`
`
`
`hodics directed to the ‘core’ herbicide molecule and
`also anti-ct’trrier antibodies (ire. antibodies directed to
`the joining portion of the carrier protein). The lttt~
`ter may also be present, on the carrier 2,4Dig~BSA
`ctiinjugntes used its target antigen in the solid phase
`assays and therefore generate titlse positive results in
`the assay.
`
`Conclusions
`
`
`
`We have developed three imninottssnys (KIA, ELISA
`nod 21 SPR—based bioscnsor technology) for 2,41) de
`tection, using highly specific anti 2,41) polyclonal
`and monoclonal nntibodien Antibodies 01‘ certain by»
`bridotnas (for example clone MSBBISZPF) ensured great;
`sensitivity and specificity and proved to be suitable for
`detceting 2,40 in etwironinentttl samples with no or
`only partial pre‘ptiriliezttion. Other monoclonal anti
`BAD antibodies showed lower binding affinity for
`the native 2,41) molecule and were probably directed
`against overlapping epitopets) ol‘ the some herbicide or
`recognized pint of the carrier molecule on 2,41) protein
`I
`conjugates.
`
`8
`
`

`

`
`
`lmmcbiiizatim of
`filming protein
`RAMFC to COOH
`groups in the dcxtran
`matrix.
`
`The first magmas!
`antibody is bound to
`RAMfc.
`
`Antigua is bound
`m the first maximal
`antimdy.
`
`Sequmtial exposure
`to diffmt 112mm
`polyclmlal antibedim.
`
`Y
`
`Y
`
`
`
`$144
`
`
`
`0
`
`5C!)
`
`11m
`Tins
`
`1&0
`
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`
`

`

`,
`‘
`
`I
`
`144
`
`Comparison of the results obtained with the three
`immunoassays, shows the SPR-based biosensor tech-
`nique is very powerful, with several advantages over
`RIA and ELISA. It requires very low quantities of
`reagents; no labeling or a second reagent and washing
`steps; allows measurements in real time with high pre-
`cision and specificity of the assay sharpening yes/no
`results.
`
`These differences in sensitivity and specificity be-
`tween the biosensor and the other two conventional
`
`solid phase immunoassays have several explanations:
`the dextran matrix allows a much larger number of
`recognition sites per surface area than the plastic
`monolayer to be homogeneously immobilized;
`the
`carbohydrate chains are a flexible coupling matrix;
`they offer excellent diffusional accessibility in solu-
`tion, with low non-specific binding of proteins, which
`is very important since SPR is an extremely sensitive
`technique.
`In conclusion, SPR technology, using highly spe-
`cific antibodies enables quantitative pesticide determi-
`nation, identification of its active regions, discrimina-
`tion of the pesticide molecule from other structurally
`related compounds or complex products of the pesti-
`cide itself, so it is an attractive tool for environmental
`pesticide monitoring.
`
`Acknowledgements
`
`This work was supported in part by CNR.PB ‘Italy—
`Sweden’
`and the European Science Foundation—
`Programme ‘Artificial Biosensing Interfaces’.
`
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