isolation of alpaca anti-hapten heavy chain single domain … · 2012. 8. 2. · isolation of...

Isolation of Alpaca Anti-Hapten Heavy Chain Single DomainAntibodies for Development of Sensitive ImmunoassayHee-Joo Kim,† Mark R. McCoy,† Zuzana Majkova,† Julie E. Dechant,‡ Shirley J. Gee,†

Sofia Tabares-da Rosa,§ Gualberto G. Gonzaĺez-Sapienza,§ and Bruce D. Hammock*,†

†Department of Entomology and UCD Cancer Center, University of California, Davis, California 95616, United States‡Department of Surgical and Radiological Sciences, School of Veterinary Medicine, University of California, Davis, California 95616,United States§Cat́edra de Inmunología, Facultad de Química, Instituto de Higiene, UDELAR, Av. A. Navarro 3051, Piso 2, Montevideo 11600,Uruguay

*S Supporting Information

ABSTRACT: Some unique subclasses of Camelidae antibod-ies are devoid of the light chain, and the antigen binding site iscomprised exclusively of the variable domain of the heavychain (VHH). Although conventional antibodies dominatecurrent assay development, recombinant VHHs have a highpotential as alternative reagents for the next generation ofimmunoassay. We expressed VHHs from an immunized alpacaand developed a VHH-based immunoassay using 3-phenox-ybenzoic acid (3-PBA), a major metabolite of pyrethroidinsecticides as a model system. A phage VHH library wasconstructed, and seven VHH clones were selected bycompetitive binding with 3-PBA. The best immunoassaydeveloped with one of these VHHs showed an IC50 of 1.4 ng/mL (limit of detection (LOD) = 0.1 ng/mL). These parameterswere further improved by using the phage borne VHH, IC50 = 0.1 ng/mL and LOD = 0.01 ng/mL. Both assays showed a similartolerance to methanol and dimethylsulfoxide up to 50% in assay buffer. The assay was highly specific to 3-PBA and its 4-hydroxylated derivative, 4-hydroxy 3-PBA, (150% cross reactivity) with negligible cross reactivity with other tested structuralanalogues, and the recovery from spiked urine sample ranged from 80 to 112%. In conclusion, a highly specific and sensitiveVHH for 3-PBA was developed using sequences from immunized alpaca and phage display technology for antibody selection.

Since the first radioimmunoassay was reported,1 countlessimmunoassays have been developed and proven to beinvaluable analytical methods for in vitro diagnostics andenvironmental monitoring for wide array of substances such asviruses, bacteria, disease biomarkers, food toxins, and environ-mental pollutants including endocrine disruptors and pesticidesand their metabolites.2−6 For an immunoassay to be applied toa real sample, it should have high sensitivity and robustness inthe matrix in which it is detected. These properties are largelydependent on the availability of antibodies with high affinityand specificity to their target analyte along with a high stabilityin the matrix. Monoclonal antibodies (MAbs) mostly derivedfrom murine hybridoma cell lines, along with polyclonalantibodies (PAbs) from sera of rabbits, goats, sheep, and otherspecies, are traditional reagents used in immunoanalyticaltechniques.Conventional antibodies (IgG subclass) have an average

molecular weight of 150 kDa, and they are composed of twoidentical heavy and light chains connected by disulfide bonds.Each antibody contains two antigen binding pockets. AlthoughPAbs can be easily obtained at a low production cost, they arefinite, which requires subsequent antibody characterization and

assay optimization because of animal to animal variation inimmune response. This can be a limiting factor for the use ofPAbs in development of an assay for large scale production orcommercialization. MAbs obtained from hybridoma cell linescan overcome the reproducibility issues of PAbs. An establishedhybridoma can produce MAb indefinitely. However mAbtechnology is expensive, and sometimes frozen hybridoma celllines are hard to recover and generation of high quality MAbsto small molecules is difficult. Because of the size, therequirement for two chains, and sophisticated post-translationalmodification, functional conventional antibodies are difficult toexpress recombinantly. Many antibody-derived proteins that arederivatives of an intact antibody molecule have been developed,including monovalent fragments such as Fab, scFv, andengineered variants including diabodies, triabodies, minibodies,and single-domain antibodies.7−9 However, these constructsoften have lower affinity and are less stable than the intact

Received: November 14, 2011Accepted: December 12, 2011Published: December 12, 2011

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© 2011 American Chemical Society 1165 dx.doi.org/10.1021/ac2030255 | Anal. Chem. 2012, 84, 1165−1171

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antibody. Production can be problematic because the limitedsolubility and aggregation of the expressed antibody fragmentscan reduce yields of expressed proteins.10,11 In addition, thediversity of phage antibody libraries from hybridoma cell linesshould be very large to increase the possibility of selecting adesirable antibody fragment due to the high level ofnonfunctional association of VHs and VLs.12

In 1993, Hamers-Casterman discovered a new subclass ofantibodies in members of the Camelidae family (i.e., camels,llamas, and alpacas) that have an antigen binding pocketcomprised solely of a variable region of the heavy chain andcompletely devoid of light chains.13,14 Recombinant expressionof these heavy chain variable domains yields single domainantibodies (VHHs). The single domain nature of VHHsprovides many advantages over the other recombinant antibodyfragments; in particular, ease of expression in various expressionsystems, high thermal stability, excellent solubility, resistance toproteolysis, and ease of genetic manipulation.15−17 Antigen-binding patterns of VHHs are very unique compared toconventional antibodies. Since the binding area of a conven-tional antibody is comprised of a heavy chain and a light chain,they tend to bind to proteins through a large surface area andthey encompass small molecules in a deep cleft formed at aninterface of a heavy and a light chain. Because heavy chainantibodies need to compensate for the missing light chain, theircomplementary determining region 3 (CDR3) is normallylonger, which allows the formation of a “finger-like” shape andsubsequent binding in a groove of the protein target.18 Thisallows for heavy chain antibodies to be used as enzymeinhibitors.19 When binding to small molecules, their bindingoccurs through a lateral recognition involving the coordinatedcontribution of the three hyper variable loops and theframework regions (FRs).20−22 It has also been observed thatthe binding of a VHH against caffeine is accompanied by ahapten-induced dimerization of the VHHs.23

To date, most reports about biotechnological applications ofVHHs are related to protein or other macromolecularantigens,23,24 while few reports refer to small molecules.Some VHH-based assays have been developed for the detectionof small haptens but, with the exception of two VHHs againstazo-dye Reactive Red20 and ochratoxin A,26 the μM rangesensitivities of the assays are somewhat unsatisfactory forpractical application to real samples.27−32 We recentlydemonstrated that the generation of VHHs to triclocarban(TCC) is not favored in the llama immune response but thathigh-affinity anti-TCC VHHs can be isolated by competitiveselection from phage libraries.33 The VHH-based ELISAshowed an IC50 value of 3.5 ng/mL. To further test thishypothesis and evaluate the potential of VHHs for thedevelopment of practical applications, we present in this workthe isolation of alpaca VHHs to 3-phenoxybenzoic acid (3-PBA) (MW = 214) and their use in the immunodetection ofthis compound in human urine samples. 3-PBA is a humanurinary metabolite of pyrethroid insecticides and can be used asa biomarker to monitor human exposure to these pesticides.We have previously developed a PAb-based enzyme-linkedimmuosorbent assay (ELISA) for 3-PBA with IC50 values of120 ng/mL for a homologous ELISA and 1.6 ng/mL for aheterologous ELISA.34 The same antigen was used in this studyto immunize an alpaca, and the VHHs were isolated from alibrary prepared from its peripheral blood lymphocytes, leadingto the development of immunoassay to 3-PBA with equalsensitivity to that obtained with conventional PAbs and

excellent recoveries in real samples. Since VHHs can beproduced easily and without the batch-to-batch variations ofPAbs, the demonstration of their high profile performance in apractical application is an important contribution to theimmunoassay technology for small molecules.

■ EXPERIMENTAL SECTIONA 3-year old castrated male alpaca was immunized subcuta-neously with 200 μg of 3-PBA hapten-thyroglobulin conjugatemixed with Freund’s incomplete adjuvant. The immunization,homologous, and heterologous coating antigens were pre-viously synthesized in this laboratory,34 and their structures arepresented in Figure S-1 in the Supporting Information. TotalRNA was extracted from peripheral blood lymphocytes isolatedfrom alpaca blood and then used for first strand cDNAsynthesis. The phage-VHH library was constructed by cloningPCR amplified VHH genes into the pComb3X phagemidvector. Phage anti-3-PBA VHHs were selected using graduallydecreasing concentrations of 3-PBA hapten-BSA conjugate andfree 3-PBA and more stringent washing. Phage VHH cloneswhose binding to coating antigen was inhibited by free 3-PBAwere sequenced, and soluble VHH fragments were expressedfrom TOP10F′ cells of E. coli transformed with phagemidsextracted from clones with unique sequences. ELISAs withisolated phage-VHHs (PELISAs) and soluble VHHs (VELI-SAs) were used to determine assay sensitivity. The phage-VHHand VHH with the highest sensitivity were further used for theestimation of cross-reactivity and assay validation using humanurine samples. For a more detailed experimental description,see the Supporting Information.

■ RESULTSLibrary Construction and Selection of 3-PBA Specific

VHHs. The total RNA was extracted from alpaca blood withthe leukoLOCK total RNA isolation system (AppliedBiosystems, Foster City, CA) following the manufacturer’sprotocols. VHH genes were obtained by PCR using a set ofsense primers specific for the 5′ region of the heavy chainvariable genes paired with a reverse primer specific for eitherlong (IgG2) or short (IgG3) hinge regions.35 The amplifiedgenes were cloned into the phagemid vector pComb3X, whichwas then electroporated into electrocompetent ER2738 cellsfrom E. coli in order to construct the phage VHH library. Thesize of the phage VHH library was 3.4 × 107 colony formingunits (cfu)/mL.Figure 1 presents the number of phage output in each

panning. The number of phage output decreased after everyround of panning except for the second. Overall, there was a 10000-fold decrease in the phage output over 5 panning cycles.The fourth and fifth phage outputs were used to identifypositive phage VHH clones for analysis of 3-PBA. Although thenumber of phage output decreased with decreasing concen-trations of the coating antigen and 3-PBA, we observed that100% of tested clones showed different degrees of bindinginhibition by free 3-PBA (data not shown). This result indicatesthat our panning scheme was very effective in isolating positiveVHH clones. We devised a panning strategy that increased theselection pressure throughout the panning and minimized thecarryover of nonspecific binders to a next round of panning,preventing the nonspecific binders from outnumbering thespecific ones. We performed five cycles of panning withmodulation of the concentration of the 3-PBA hapten-BSA

Analytical Chemistry Article

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conjugate for capturing phage-VHHs, the washing intensity, theconcentration of 3-PBA for the competitive elution of thecaptured phage-VHHs, and the postabsorption of phage eluateto BSA. To capture high affinity phage-VHHs to the coatingantigen, the concentration of the coating antigen (0.5, 0.25, 0.1,0.02, and 0.01 μg per well over the panning) was graduallydecreased. We also decreased the concentration of 3-PBA by10-fold for each subsequent round of panning to elute phage-VHHs with high affinity to the 3-PBA. To remove nonspecificand weakly binding phage-VHHs we increased the washingintensity in each round of panning. The phage-VHH capturedwells were washed five times for the first panning and washedten times for the second panning with PBS containing 0.05%Tween 20 before the competitive elution. For the rest of thepannings, the wells were washed 10 times and then the plateswere shaken for 30 min while the wells were filled with PBScontaining 0.1% Tween 20, which was followed by 10 morewashings. Nonspecific phage-VHHS were minimized byincreasingly stringent washings and postabsorption of phageoutput to BSA.Sequence Alignment of Isolated VHHs to 3-PBA. A

total of 16 clones after the fourth round of panning and 25clones from the fifth round of panning that showed aninhibition of binding to the coating antigen by free 3-PBA weresequenced. In total, 3 and 4 unique sequences were found fromthe fourth and fifth round of panning, respectively (Figure S-2in the Supporting Information). The main features of heavychain antibodies that distinguish them from the variable heavychain domains of conventional IgGs are the occurrence ofamino acids, F, E, R, and F/G in the FR2, which stabilize theVHH fragments and the extended length of CDR3 regions(15−22 amino acids). The amino acid sequences of the alpacaVHH clones in the region are in agreement with the findings.The deduced amino acid sequences of the VHH clones showthese amino acids in FR2. Clones 3P5ThC19 and 3P5ThC15have G and P in the region instead of E and G. All clones show18 amino acids in the CDR3 region with no significant aminoacid variation.VELISA for 3-PBA. The sensitivities of assays were

estimated by performing an indirect competitive ELISA withthe soluble VHH fragments (Figure 2). The VHHs wereexpressed from TOP10F′ cells transformed with each phagemidvector isolated from positive clones. The TOP10F′ is anonsuppressor strain, allowing expression of VHHs withoutthe phage pIII protein. The VHH fragments with 6xHis and

HA tags were obtained by purifying cell lysates on the Ni-NTAaffinity columns. The size of purified VHHs was determined ona 12% SDS PAGE gel as 17 kDa major bands (data not shown).Prior to the indirect ELISA, the optimization of the coatingantigen (3-PBA hapten-BSA) and VHH concentrations weredetermined by a checkerboard titration. As Figure 2 shows, thelowest IC50 value (1.4 ng/mL, 0.007 μM) was observed fromclone 3P5ThC12 which was isolated after the fifth round ofpanning. The clone 3P5ThC1 showed a higher IC50 value of22.8 ng/mL (0.1 μM). Other clones showed similar IC50 valuesthat ranged from 8.2 to 15.7 ng/mL (0.04−0.07 μM).Comparing the sensitivity of VELISA with the clone3P5ThC12 to that of our previously developed PAb-based 3-PBA ELISA, the VELISA is 100-fold more sensitive than thehomologous coating antigen-based ELISA (IC50 of 120 ng/mL)and almost identical to the best heterologous coating antigen-based ELISA (IC50 of 1.6 ng/mL). Previously, we increased thesensitivity of the PAb 3-PBA ELISA by use of the 3-PBA-BSAheterologous coating antigen by conjugation using thecarboxylic acid moiety. We tested if this approach would alsoincrease the sensitivity of the VELISA; however, the VHHshowed no recognition to the heterologous coating antigen,indicating that the acid moiety plays a critical role in thebinding of the VHH to the 3-PBA. The ELISA using the VHHfrom the 3P5ThC12 clone has at least a several hundred foldbetter sensitivity than most other camelid VHH-based ELISAsreported in the literature. Therefore, the VHHs and phage-VHHs derived from the 3P5ThC12 clone were usedthroughout the study.

Cross-Reactivity. The specificity of the assay was tested forthe clone 3P5ThC12, which exhibited the best IC50 value, usingfour known pyrethroid metabolites and five pyrethroid parentcompounds (Figure 3). The cross-reactivity was determinedusing the formula, {(IC50 of 3-PBA)/(IC50 of cross-reactingcompounds)} × 100. Overall, negligible cross reactivity wasobserved, except for 4′-hydroxy 3-PBA which showed 150%cross reactivity. Since the immunizing hapten was preparedwith the linker attached to the −OH group of the 4′-hydroxy-3-PBA, this indicates that the VHH has some interaction with thisoxygen atom.

Figure 1. Number of phage output: 10 μL of the phage output werediluted in a sterile PBS from each panning cycle and were mixed with 1mL of ER2738 cells with absorbance of 0.4 at OD600. The mixture wasincubated for 25 min at 37 °C without shaking. The mixture wasplated on a LB-plate with ampicillin. The number of colonies wascounted the following day. Figure 2. Competitive ELISA with soluble VHHs. Representative

curves showing the inhibition of VHH clones binding to 3-PBAhapten-BSA with increasing amount of the free 3-PBA. 3-PBA hapten-BSA was immobilized, and the competition mixture was added. Afterwashing, bound VHH was detected with goat anti-HA tag PAb-HRP.Data are represented as an average ± standard deviation of fourreplicates.



PELISA. The M13 bacteriophage is a filamentous virus witha diameter of 6 nm and length of 0.9 μm containing singlestranded DNA packed with around 2700 major and severalminor coat proteins. When a VHH fragment possesses a goodbinding affinity, an assay using the phage displayed-VHH canprovide advantages over a monomeric soluble expressed VHHfragment. First, the phage can improve assay sensitivity by usingit as a multiple binding element, second, the phage particle isalso adaptable to various detection methods such as immuno-PCR36,37 and numerous types of biosensors.38 Anotheradvantage of the PELISA is that phage-VHH can be easilyand inexpensively prepared in high concentrations. Phage-VHHexpressed in a culture medium can be readily isolated by simpleincubation and centrifugation, eliminating laborious proceduressuch as cell lysis and Ni-NTA purification necessary for theexpression of soluble VHH fragments. Goldman et al.39,40

evaluated the utility of phage-VHHs derived from llama forricin and botulinum A employing a sandwich ELISA andLuminex technology. They observed 1 to 2 orders of magnitudeof sensitivity improvement compared to a standard ELISA withsoluble VHHs.We also evaluated the performance of the PELISA using the

phage-displayed VHH of the clone 3P5ThC12 in an indirectcompetitive ELISA format (Figure 4). The checkerboardtitration was used to determine the optimal concentrations ofthe coating antigen and the phage-VHH using two blockingreagents (1% BSA and 3% skim milk in PBS). As shown in thefigure, when the plate was blocked with 1% BSA, the IC50 valuewas 0.6 ng/mL, showing slightly enhanced sensitivity comparedto the VELISA. However, we observed a 6-fold improvement ofthe assay sensitivity when 3% of skim milk was used forblocking with an IC50 of 0.1 ng/mL, which is 10-fold betterthan the VELISA. To generate a similar maximal signal to theVELISA, the PELISA required the use of coating antigen at 0.4μg/mL which is 8-fold higher than the concentration for theVELISA. We tested the VELISA using a plate blocked with 3%skim milk, and the assay sensitivity was similarly enhanced butsignal variations among replicates were significantly higher(data not shown). Thus, we used 1% BSA blocking for theVELISA.

Solvent Effect. High tolerance to solvents is beneficialwhen developing immunoassays to be used for analysis oflipophilic analytes, because water miscible solvents arecommonly used for extraction of analyte prior to analysis.When the matrix effects for solvent extracts are negligible, lessdilution of the extracts is needed, which results in enhancedassay sensitivity. Although the camelid VHHs have been knownto be highly thermally stable, no evaluation has been done forthe tolerance of VHHs or phage-displayed VHHs to such watermiscible solvents. Hence, we estimated the effect of methanol(MeOH) and dimethylsulfoxide (DMSO) on the performanceof the VELISA and PELISA and compared to that of the PAb-based 3-PBA ELISA. Each solvent was added to the assay bufferat concentrations of 0, 10, 25, and 50% (v/v), whilemaintaining constant ionic strength. Figure 5 presents theMeOH effects on the assay performance. Typically, increasingthe concentration of MeOH in ELISAs causes an increase inboth the maximal signal and the IC50 values. All assays showedthe increase of the maximal signals likewise when the MeOHconcentrations increased with a more significant increase in thePELISA. Interestingly, the assay sensitivity also was improvedfor the VELISA and PELISA, which indicates that the additionof MeOH facilitates the binding interaction with minimal effecton the protein function. However, the MeOH showed a moresignificant effect on the sensitivity of the 3-PBA ELISA showinga small but gradual increase of IC50 values. A 4-fold increase inIC50 value was observed at 50% (v/v) of MeOH. This resultdemonstrated that both the VHH and phage-VHH were highlytolerant to MeOH.DMSO did not affect the binding of the VHH and the phage-

VHH because IC50 values changed insignificantly in thepresence of up to 50% (v/v) of DMSO (Figure 6). In contrast,DMSO had a significant effect in the PAb 3-PBA ELISA

Figure 3. Cross reactivity: the 96 well plates were coated with 3-PBAhapten-BSA conjugate at 5 ng/well. Serial dilutions of 3-PBA or testedcompounds in PBS were mixed with an equal amount of a 1/1000dilution of the VHH in PBS. A volume of 100 μL of the mixture wasadded into the wells. The bound VHH was detected with a 1/10 000dilution of goat anti-HA tag PAb-HRP.

Figure 4. Competitive binding of the phage-VHH. A 96-well plate wascoated with 3-PBA hapten-BSA conjugate at 0.4 μg/mL. Dilutions of3-PBA in PBS was mixed with a 1/2500 dilution of the phage-VHH inPBS and 100 μL of the mixture was added into wells. The boundphage-VHHs were detected by adding 100 μL of mouse anti-M13phage MAb-HRP diluted in PBS at 1/5000.



causing 50% reduction in maximal signal at 50% (v/v) DMSOwith slight increase of IC50s. The above results demonstratethat the VHH and phage-VHH are more tolerant to solventeffect than the PAb ELISA.Assay Validation. The VELISA and PELISA were validated

using a urine sample collected from a person with no knownexposure to pyrethroid insecticides. The urine was spiked with3-PBA at five different concentrations. The spiked urinesamples were diluted with PBS buffer to a final concentrationof urine of 4%. An aliquot of the diluted urine samples wasmixed with an equal volume of the VHH or phage-VHHdiluted in PBS for competition (Figure 7). The matrix effect

test revealed that the urine content in the assay buffer should beless than 2% to remove the matrix interference on the assay(data not shown). In general, both assays showed goodcorrelations between the spiked and recovered concentrationswith an average recovery of 80−110%. We also observed minorfalse positive detection from urine with no 3-PBA. It should benoted that although the dilution of urine samples can be usedfor the analysis of urine containing concentrations of 3-PBAhigher than 100 ng/mL, sample preparation by solid-phaseextraction (SPE) or a mixed-mode SPE method is necessary todetect low concentrations of 3-PBA and for more accurate

Figure 5. Effects of MeOH on the performance of the VELISA (A),PELISA (B), and 3-PBA ELISA (C). PBS buffers containing MeOH atdifferent concentrations were used to make serial dilutions of 3-PBA.The serial dilutions were mixed with an equal volume of VHH, phage-VHH, or 3-PBA PAb and then 100 μL of the mixture was added intowells. The bound immunoreagents were detected by adding 100 μL ofgoat anti-HA tag PAb, mouse anti-M13 phage MAb, and goatantirabbit PAb conjugated with HRP, respectively. Values are the mean± standard deviation of four well replicates.

Figure 6. Effects of DMSO on the performance of the VELISA (A),PELISA (B), and 3-PBA ELISA (C). PBS buffers containing DMSO atdifferent concentrations were used to make serial dilutions of 3-PBA.The serial dilutions were mixed with an equal volume of VHH, phage-VHH, or 3-PBA PAb and then 100 μL of the mixture was added intowells. The bound immunoreagents were detected by adding 100 μL ofgoat anti-HA tag PAb, mouse anti-M13 phage MAb, and goatantirabbit PAb conjugated with HRP, respectively. Values are the mean± standard deviation of four well replicates.



determination in urine samples.41 For this, the urine samplewas spiked with four low concentrations of 3-PBA (1, 5, 20, and50 ng/mL) and the analytes were extracted by the mixed-modeSPE method. The recoveries by the VELISA were compared tothe recoveries by gas chromatography-mass spectrometry. Weobserved an excellent correlation between two methods withVELISA recoveries of 82−110% (Table S-1 in the SupportingInformation).

■ CONCLUSIONSIn this case, we were able to obtain a homologous VELISA andPELISA that were of equal or greater sensitivity to our bestPAb-based heterologous assay for the development of asensitive assay for 3-PBA using alpaca VHH and phage-VHHfragments. We demonstrated that the alpaca is a good sourcefor the VHH recombinant antibodies leading to smallmolecular weight compounds. The assays using the VHH andphage-VHH derived from clone 3P5ThC12 showed severalhundred times better sensitivity than other assays that havebeen developed using camelid VHHs for small molecules. Thisobservation indicates that a good panning strategy can be veryeffective in isolating a high affinity VHH for small haptensstarting with alpaca RNA. The sensitivity of the VELISA andPELISA with the homologous coating antigen was approx-imately 80- and 1200-fold better than the homologous 3-PBAELISA, respectively. The VELISA showed almost identicalsensitivity to our best heterologous ELISA, but 10-fold bettersensitivity was observed from the PELISA. Although mono-clonal type VHH showed a similar sensitivity to the 3-PBAheterologous assay, the use of heterologous antigens withlinkers attached to −OH moiety of the 3-PBA hapten mayfurther improve assay sensitivity. The VHH and phage-VHHshowed higher tolerance to MeOH and DMSO than the PAb.The size of a VHH is 1/10 of conventional antibodies, and it ismuch more robust than MAbs and PAbs with high resistance toheat and solvent effects. The size and robustness of the VHH isa significant advantage when developing assays or biosensors,because the stability and the small size allow them to beimmobilized on a miniaturized sensor chip in higher densityand leads to a longer shelf life of the assay. This feature isparticularly significant for field use diagnostics whererefrigeration is often limited, solvent-based sample preparationconditions are required, and sample cleanup procedures mustbe simple. Since the phage-VHH retain the stability and affinity

of the VHH, the phage particles are very robust and serve as amultibinding scaffold for signal enhancement, which increasedthe sensitivity as we have demonstrated. The phage-VHH waseasily obtained by simple procedures. The VELISA and PELISAboth showed good recovery of spiked samples in a urine matrixwith an average recovery of 80−110%. The dilution of the urinesample can be used for urine containing relatively highconcentrations of 3-PBA; if more sensitivity is needed sampleswould have to be extracted prior to analysis. Samplepretreatment by the simple mixed-mode SPE allowed thedetection of 3-PBA at very low concentrations. As we havedemonstrated here, camelid VHHs will be a useful tool in thefuture of small molecule immunoassays.

■ ASSOCIATED CONTENT*S Supporting InformationAdditional information as noted in text. This material isavailable free of charge via the Internet at http://pubs.acs.org.

■ AUTHOR INFORMATIONCorresponding Author*E-mail: [email protected].

■ ACKNOWLEDGMENTSThis work was supported in part by the following: TheNational Institute of Environmental Health Sciences (NIEHS)Superfund Basic Research Program (Grant P42 ES004699); theNIEHS Children’s Environmental Health Center (GrantP01ES011269); and the Fogarty International Center Grant(Grant TW05718). The support of the Western Center forAgricultural Health and Safety at the University of CaliforniaDavis (Grant PHS OH07550) is also acknowledged. B.D.H. is aGeorge and Judy Marcus Senior Follow of the AmericanAsthma Foundation. We thank Bonnie Potter for the use of thealpacas.

■ REFERENCES(1) Yalow, R. S.; Berson, S. A. J. Clin. Invest. 1960, 39, 1157−1175.(2) Iqbal, S. S.; Mayo, M. W.; Bruno, J. G.; Bronk, B. V.; Batt, C. A.;Chambers, J. P. Biosens. Bioelectron. 2000, 15, 549−578.(3) Wu, J.; Fu, Z.; Yan, F.; Ju, H. TrAC, Trends Anal. Chem. 2007, 26,679−688.(4) Li, P.; Zhang, Q.; Zhang, W. TrAC,Trends Anal. Chem. 2009, 28,1115−1126.

Figure 7. The validation of the VELISA and PELISA in a urine matrix. 3-PBA standard compound was spiked into the urine sample before it wasdiluted 25-fold with PBS buffer. The dilutions of 3-PBA spiked urine were mixed with an equal volume of VHH or phage-VHH diluted in PBS beforeanalysis.



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1

Supporting Information for: 1 2

Isolation of Alpaca Anti-Hapten Heavy Chain Single Domain 3 Antibodies for Development of Sensitive Immunoassay 4

5

Hee-Joo Kim1, Mark R. McCoy1,Zuzana Majkova1, Julie E. Dechant2, Shirley J. Gee1, 1Sofia 6 Tabares-da Rosa3, Gualberto G. González-Sapienza3 and Bruce D. Hammock1* 7

1Department of Entomology and UCD Cancer Center, University of California, Davis, California 8 95616 9

2Department of Surgical and Radiological Sciences, School of Veterinary Medicine, University 10 of California, Davis, California 95616 11

3Cátedra de Inmunología, Facultad de Química, Instituto de Higiene, UDELAR, Av. A. Navarro 12 3051, piso 2, Montevideo 11600, Uruguay 13

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Materials. 3-PBA standard, bovine serum albumin (BSA), thyroglobulin, polyethyleneglycol 1

8000 (PEG 8000), Tween 20, isopropyl-β-D-thiogalactopyranoside (IPTG), Freund’s incomplete 2

adjuvant, and 3, 3’, 5, 5’-tetramethylbenzidine (TMB) were obtained from Sigma-Aldrich (St. 3

Louis, MO). Helper phage M13KO7, T4 DNA ligase, and SfiI were purchased from New 4

England Biolabs (Ipswich, MA). Mouse anti-M13 phage MAb-horseradish peroxidase (HRP) 5

was purchased from GE Health Care (Piscataway, NJ). Goat anti-HA tag PAb-HRP was 6

purchased from Abcam (Cambridge, MA). 3-PBA immunizing hapten was previously 7

synthesized in our laboratory (Figure S-1)1. The phagemid vector pComb3X was kindly provided 8

by Dr. Carlos F. Barbas (The Scripps Research Institute, La Jolla, CA). Chemically competent 9

TOP10F’ cells, Platinum® Taq DNA Polymerase and The SuperScript III First-Strand Synthesis 10

System were obtained from Invitrogen (Carlsbad, CA). LeukoLOCK™ Total RNA Isolation Kit 11

was obtained from Applied Biosystems (Foster City, CA). Plasmid purification kit, gel 12

purification kit, PCR purification kit, and 6xHis tag purification resins were obtained from 13

Qiagen (Valencia, CA). Electrocompetent ER2738 E. coli cells were purchased from Lucigen 14

Corporation (Middleton, WI). B-PER lysis solution was purchased from Thermo Pierce 15

Scientific (Rockford, IL). 16

Immunization. A three-year old castrated male alpaca was subcutaneously immunized with 17

200 µg of 3-PBA hapten-thyroglobulin conjugate diluted in 1 mL of PBS mixed with 1 mL of 18

Freund’s incomplete adjuvant. Identical booster injections followed every 2 weeks for a total of 19

6 injections. The same hapten was conjugated to BSA and used as a coating antigen for the 20

screening of positive VHH clones and for assay development. A 5 mL serum sample was taken a 21

few days prior to each immunization for titer estimation. A week after the final injection, 10 mL 22

3

of blood was withdrawn from a catheter in the neck vein into EDTA-coated Vacutainer® blood 1

collection tubes (Becton Dickinson, Franklin Lakes, NJ). 2

Library construction. Peripheral blood lymphocytes were isolated from 10 mL of EDTA-3

treated blood (about 107 cells) using the LeukoLOCK™ Total RNA Isolation Kit following the 4

manufacturer’s instructions. The concentration of RNA was calculated by measuring the 5

absorbance at 260 nm on a NanoDrop (Thermo Scientific, Fremont, CA) and the RNA integrity 6

was confirmed by denaturing agarose gel electrophoresis. The SuperScript III First-Strand 7

Synthesis System for RT-PCR with oligo(dT)s as the primers was used to synthesize cDNA from 8

2.5 µg of RNA. DNA fragments encoding the VHH IgG variable domains were amplified by 9

PCR using the forward primer Alp-Vh-F1 SfiI: CAT GCC ATG ACT GTG GCC CAG GCG 10

GCC CAG KTG CAG CTC GTG GAG TCN GGN GG targeting the framework 1 region; and 11

reverse primers AlpVhh-R1 SfiI: CAT GCC ATG ACT CGC GGC CGG CCT GGC CTC GTG 12

GGG GTC TTC GCT GTG GTG CG and AlpVhh-R2 SfiI: CAT GCC ATG ACT CGC GGC 13

CGG CCT GGC CTC GCC TTG TGG TTT TGG TGT CTT GGG corresponding to the short 14

(IgG3) and long (IgG2) hinge region, respectively. The reverse and forward primers were based 15

on previously published alpaca sequences (Maass et al2) and also introduced two different SfiI 16

sites (underlined) that were used for subsequent cloning of the fragments into the phagemid 17

pComb3X vector. We also shortened the length of the Alp-Vh-F1 annealing region because we 18

found in our first attempt that this introduced frame shifts in some of our VHH sequences. The 19

ligated vector was electroporated into electrocompetent ER2738 cells from E. coli using a 20

MicroPulser Electroporation Apparatus (Biorad Laboratories, Hercules, CA). The resulting 21

library had an estimated size of 3.4 x 10 7 independent clones. For the amplification and isolation 22

of the phage-VHH library, the transformed cells were added into a 1 L culture flask containing 23

4

400 mL Super Broth (SB, 10g of MOPS, 30g of tryptone, 20g of yeast extract, pH 7.0) 1

containing tetracycline (20 µg/mL) and ampicillin (100 µg/mL). The flask was shaken 2

vigorously at 37 oC until O.D.600 reached 0.5. The cells were super infected by adding 2 mL of 3

helper phage M13KO7 (1x1012 c.f.u/mL) by 30 min incubation at 37 oC without shaking. The 4

flask was shaken for 1 h before kanamycin (70 µg/mL) was added into the flask. On the 5

following day, the overnight culture was centrifuged at 11,000 x g for 10 min at 4 oC. The clear 6

supernatant was transferred to clean bottles and 40 mL of 20% PEG 8000/2.5 M NaCl 7

(PEG/NaCl) was added. The bottles were incubated on ice for 2 h and centrifuged for 15 min at 8

11,000 x g. The supernatants were discarded and the phage pellets were resuspended with 100 9

mL of sterilized PBS. Twenty ml of PEG/NaCl was added into each bottle and incubated for 2 h 10

on ice. The bottles were centrifuged again at 11,000 x g for 15 min at 4 oC. The supernatants 11

were discarded and the phage pellets were resuspended with 10 mL of phage suspension buffer 12

(sterilized PBS containing 0.02% sodium azide, 0.5% BSA, and 1x protease inhibitor cocktail 13

(Roche Bioscience). The phage library solution was filtered through 0.2 µm membrane and 14

stored at -70 oC until use. 15

Selection of phage anti- 3-PBA VHH clones. Six wells of an ELISA plate (Nunc MaxiSorp, 16

Thermo Fisher) were coated with 0.5 µg/mL of 3-PBA-BSA by overnight incubation at 4 °C. 17

The plates were blocked with 3% BSA in PBS (350 µL per well) for 1 h at 37 °C. One hundred 18

µL of the phage VHH library (1012 c.f.u/mL) was added into each well and the plate was 19

incubated for 2 h at 4 oC with gentle shaking. After washings five times with PBST, the bound 20

phage-VHHs were competitively eluted with 100 µL of 3-PBA diluted in PBS (1 µg/mL per 21

well) by 30 min incubation at 4 oC. The phage VHH eluates were transferred to BSA-coated 22

wells followed by 1 h incubation to remove non specific binders. Unbound phage VHHs were 23

5

collected and pooled. For amplification of the phage VHH eluates for the next round of panning, 1

500 µL of overnight culture of the ER2738 cells was added into a 125 mL flask containing 10 2

mL of SB and the cells were grown by shaking at 37 oC until O.D.600 reached 0.5-0.6. Three mL 3

of the cells were then infected with 200 µL of the phage VHH eluates at 37 oC for 30 min 4

without shaking. Five mL of pre-warmed SB containing 200 µg of ampicillin and 60 µg of 5

tetracycline was added to the infected cells and the culture was shaken for 1 h at 37 oC. Five 6

hundred µg of ampicillin was added and the culture was incubated at 37 oC for 1 h with shaking. 7

After infecting the cell culture with 1 mL of helper phage by 30 min incubation at 37 oC without 8

shaking, the culture was transferred to a 500 mL culture flask containing 91 mL SB with 9.4 mg 9

of ampicillin and 920 µg of tetracycline. After shaking 1 h at 37 oC, kanamycin was added at a 10

final concentration of 70 µg/mL. The flask was shaken overnight by vigorous shaking at 250 11

rpm. The amplified phages were prepared using the procedures described above in the section of 12

library construction. Four more rounds of panning were carried out with the concentrations of the 13

3-PBA-BSA and free 3-PBA gradually decreased. For 2nd, 3rd, 4th, and 5th panning, the plate was 14

coated with 3-PBA-BSA at 0.25, 0.1, 0.02, and 0.01 µg/well, respectively and the concentrations 15

of free 3-PBA for the elution of bound phage-VHHs were 1000, 100, 10, 1, and 0.1 ng/mL, 16

respectively. For the 2nd panning, the plates were washed ten times with PBST. For the rest of 17

panning, the plates were washed ten times and the plates were shaken for 30 min with wells 18

filled with the washing buffer, followed by ten more washings prior to elution. Post-absorption 19

of the eluted phage-VHHs was performed throughout the panning under the same condition. 20

After the final round of panning, individual clones were screened to identify positive clones by 21

performing an indirect competitive phage ELISA. 22

6

Expression and purification of soluble VHHs. The pComb3X phagemid vectors encoding 1

VHHs with inhibited binding to the coating antigen and unique DNA sequences were extracted 2

from ER2738 clones and were transformed into the TOP 10F’ cells by heat shock following the 3

manufacturer instructions. A single colony was selected and grown in 5 mL of SB overnight. 4

One ml of the overnight culture was added to a 500 mL cultural flask containing 100 mL of SB 5

with tetracycline (100 µg/mL) and ampicillin (100 µg/mL). The flask was shaken until O.D.600 6

reached 0.5. IPTG was added at final concentration of 1 mM, and the flask was shaken overnight 7

at 250 rpm at 37 oC. The cell cultures were centrifuged at 3,000 x g for 10 min and the cell 8

pellets were frozen overnight at -70 oC. The melted cell pellets were lysed by adding 6 mL of B-9

PER lysis solution containing phenylmethylsulfonyl fluoride (PMSF). The cell lysates were 10

centrifuged at 13,000 x g for 15 min at 4 oC. The clear supernatant was dialyzed against 6x His 11

tag purification washing buffer (50 mM NaH2PO4 (pH 8.0), 300 mM NaCl, and 25 mM 12

imidazole). On the following day, the dialyzed solution was centrifuged again at 13,000 x g for 13

15 min at 4 oC. The clear supernatant was added into a purification column packed with 1 mL of 14

Ni-NTA resin which had been washed with 10 mL of washing buffer. The columns were gently 15

rocked for 1 h at 4 oC. The columns were washed with 10 mL of the washing buffer. The 16

captured soluble VHHs were eluted with 6 mL of elution buffer (50 mM NaH2PO4 (pH 8.0), 300 17

mM NaCl, and 250 mM imidazole). The VHH eluate was loaded onto a PD-10 desalting column 18

for buffer exchange to PBS. The purified soluble VHHs were aliquoted and stored at -70 oC until 19

use. 20

VHH ELISA (VELISA) and phage ELISA (PELISA). One hundred µL of 3-PBA hapten-21

BSA conjugate (0.05 µg/mL for the VELISAs and 0.4 µg/mL for PELISAs) diluted in 0.1 M 22

carbonate-bicarbonate buffer (pH 9.6) was added into wells of a 96-well ELISA plate followed 23

7

by an overnight incubation at 4 oC. The plate was washed and then blocked with 350 µL of 1% 1

BSA in PBS by incubation for 1 h at 37 oC. Fifty µL of various concentrations of 3-PBA diluted 2

in PBS was mixed with an equal volume of soluble VHH (1/1000 dilution in PBS) or phage-3

displayed VHH (1/2500 dilution in PBS). One hundred µL of the mixture was added to wells and 4

the plate was incubated for 2 h at room temperature with gentle shaking. The plate was washed 5

(5 times for the VELISA and 10 for the PELISA) with PBST. One hundred µL of goat anti-HA 6

tag PAb-HRP (1/10000 dilution in PBS) or goat anti-M13 phage MAb-HRP (1/5000 dilution in 7

PBS) was added to wells, followed by 1 h incubation at room temperature. After 5 washings, 100 8

µL of HRP substrate solution was added and the reaction was stopped after 15 min by adding 50 9

µL of 2 M sulfuric acid. The plates were read with a plate reader (Molecular device, Sunnyvale, 10

CA) at absorbance of 450 with background reading at 650 nm. The obtained absorbance was 11

plotted against the concentrations of 3-PBA using the Sigma plot software version 11.0. 12

13

8

1

2

3

4

5

6

7

8

9

10

11

12

O

O

HOO

O

HO

O OH

O

O

HO

ONH BSA

O

O

HO

ONH Thyroglobulin

Figure S-1. Structure of 3-PBA, 3-PBA hapten, homologous, and heterologous coating

antigens

3-PBA 3-PBA hapten

3-PBA hapten-thyroglobulin immunization antigen 3-PBA hapten-BSA homologous

coating antigen

O

O

HN BSA

3-PBA-BSA heterologous coating antigen

9

1

2

3

3P5ThC19 QLQLVESGGGLAQPGGSLRLSCLAS GFTLDSYG IGWFRQAPGKGREPFGC ISSFGGST NYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYC 3P5ThC15 ...........V..........T.. .A..EH.. ..........E....A. F.TLD... ..R................................... 3P5ThC1 .V.........V..........A.. .TS..Y.. ..........E..GVA. ...S..G. .I..---..............F................ 3P5ThC12 .V.........V..........A.. ..S.EY.. ..........E..GVA. ...S..R. .IRE---..........E......L.........I... 3P4ThC9 .V.........V..........A.. ..S.EY.. .........MA..GVA. ...R.... .LRE---..........E......L.........I... 3P4ThC6 ...........V..........A.. ..S.EY.. ..........E..GVA. ...G..R. .LRE---..........E......L.........I... 3P4ThC11 .V.........V..........A.. ..S.E... ..........E..GIV. ...Y.... ..R...T..........E........N...........

3P5ThC19 AAAPFYKCGTYNYSAFRS WGQGTQVTVSSEPKTPKPQD 3P5ThC15 .............N.... .................... 3P5ThC1 ....Q.R....R...... .................... 3P5ThC12 ....Q.I....R.T...T .................... 3P4ThC9 ....Q.I....Q.T...T .................... 3P4ThC6 ....K.T....Q.T...T .................... 3P4ThC11 ....Q.I....R...... ....................

FR1 CDR1 FR2 CDR2 FR3

CDR3 FR4

4

5

Figure S-2. Amino acid sequence alignment of the isolated clones. The deduced amino acid 6

sequences of the seven clones isolated from the phage VHH library are given in the single-letter 7

code. Dots are used to denote residues identical to those of clone 3P5ThC19 that is arbitrarily 8

taken as a reference sequence. Gaps were introduced to improve the alignment and are marked 9

with dashes. Solid-line boxes outline the characteristic amino acid substitutions of VHH FR2. 10

11

12

13

14

15

16

17

18

10

1

2

3

4

5

References 6

(1). Shan, G.; Huang, H.; Stoutamire, D. W.; Gee, S. J.; Leng, G.; Hammock, B. D. Chem. Res. 7

Toxicol. 2004, 17, 218-225. 8

(2). Maass, D. R.; Sepulveda, J.; Pernthaner, A.; Shoemaker, C. B. J. Immunol. Methods 2007, 9

324, 13-25. 10

11

Table S-1. Recovery of 3-PBA by GC-MS and VELISA

Fortification level (ng/mL)

GC-MS VELISA

Measured (ng/mL)

Average recovery (%)

Measured (ng/mL)

Average recovery (%)

1 1ND 1.1±0.04 110

5 3.4±0.3 68 4.9±0.2 98

20 18±0.7 87.5 18±0.4 91

50 51±1.7 101.4 41±1.0 82

1Not detected

Correction to Isolation of Alpaca Antihapten Heavy Chain SingleDomain Antibodies for Development of Sensitive ImmunoassayHee-Joo Kim, Mark R. McCoy, Zuzana Majkova, Julie E. Dechant, Shirley J. Gee, Sofia Tabares-da Rosa,Gualberto G. Gonzaĺez-Sapienza, and Bruce D. Hammock*Anal. Chem. 2012, 84 (2), 1165−1171. DOI: 10.1021/ac2030255

Incorrect primer sequences for the library construction wereincluded in the Supporting Information of the originalmanuscript (page 3, lines 10−14).Current version:DNA fragments encoding the VHH IgG variable domains

were amplified by PCR using the forward primer Alp-Vh-F1SfiI: CAT GCC ATG ACT GTG GCC CAG GCG GCC CAGKTG CAG CTC GTG GAG TCN GGN GG targeting theframework 1 region; and reverse primers AlpVhh-R1 SfiI: CATGCC ATG ACT CGC GGC CGG CCT GGC CTC GTGGGG GTC TTC GCT GTG GTG CG and AlpVhh-R2 SfiI:CAT GCC ATG ACT CGC GGC CGG CCT GGC CTCGCC TTG TGG TTT TGG TGT CTT GGG correspondingto the short (IgG3) and long (IgG2) hinge region, respectively.Corrected version:DNA fragments encoding the VHH IgG variable domains

were amplified by PCR using the forward primer Alp-Vh-F1SfiI: CAT GCC ATG ACT GTG GCC CAG GCG GCC CAGKTG CAG CTC GTG GAG TC targeting the framework 1region; and reverse primers AlpVhh-R1 SfiI: CAT GCC ATGACT CGC GGC CGG CCT GGC CAT GGG GGT CTTCGC TGT GGT GCG and AlpVhh-R2 SfiI: CAT GCC ATGACT CGC GGC CGG CCT GGC CGT CTT GTG GTTTTG GTG TCT TGG G corresponding to the short (IgG3)and long (IgG2) hinge region, respectively.The original version of the forward primer will result in

increased rates of random mutations due to the guanosine-richstretch of nucleotides at the 3′ end. The original versions of thereverse primers will result in a frame shift at the N-terminus ofthe pIII protein.

Addition/Correction

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© 2011 American Chemical Society A dx.doi.org/10.1021/ac301927h | Anal. Chem. XXXX, XXX, XXX−XXX
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isolation of alpaca anti-hapten heavy chain single domain … · 2012. 8. 2. · isolation of...

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