a competitive chemiluminescence enzyme immunoassay method for β-defensin-2 detection in transgenic...

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A competitive chemiluminescence enzyme immunoassay method for β-defensin-2 detection in transgenic mice Xi Yang, a,c Tao Zhou, a,b Lei Yu, a,c Wenwen Tan, a,c Rui Zhou a,c * and Yonggang Hu a,b * ABSTRACT: A competitive chemiluminescence enzyme immunoassay (CLEIA) method for porcine β-defensin-2 (pBD-2) detec- tion in transgenic mice was established. Several factors that affect detection, including luminol, p-iodophenol and hydrogen peroxide concentrations, as well as pH, were studied and optimized. The linear range of the proposed method for pBD-2 detection under optimal conditions was 0.0580 ng/mL with a correlation coefcient of 0.9960. Eleven detections of a 30 ng/mL pBD-2 standard sample were performed. Reproducible results were obtained with a relative standard deviation of 3.94%. The limit of detection of the method for pBD-2 was 3.5 pg/mL (3σ). The proposed method was applied to determine pBD-2 expression levels in the tissues of pBD-2 transgenic mice, and compared with LC-MS/MS and quantitative real-time reverse-transcriptase polymerase chain reaction. This suggests that the CLEIA can be used as a valuable method to detect and quantify pBD-2. Copyright © 2014 John Wiley & Sons, Ltd. Additional supporting information may be found in the online version of this article at the publishers web site. Keywords: chemiluminescence enzyme immunoassay; porcine β-defensins-2; transgenic mice Introduction Given recent developments in transgenic technology, the genera- tion of genetically modied organisms (GMOs) with transgenic resistance and improved production rates has received signicant research attention. Target-gene expression analysis in GMOs is mainly based on reverse-transcriptase polymerase chain reaction (RT-PCR). RT-PCR is a classical approach used to determine genetic expression by synthesis of specic mRNA species as a proxy for estimating functional differences at the protein level (1,2). However, inconsistencies among RT-PCR results suggest that gene expression at the transcriptional level is not tightly coupled to that at the translational level (35). In addition, RT-PCR is associated with the risk of amplied nucleic acid release into the environ- ment, which can contaminate subsequent analysis. As such, an alternative immunoassay method that can directly measure the concentration of specic proteins or peptides, especially in GMOs, must be developed. Traditional immunoassay methods, such as enzyme-linked immunosorbent assay, have limited applications in GMO analysis because of their lack of sensitivity. Therefore, a rapid, cost-effective, high throughput, sensitive and specic immunoassay method must be developed for expression analysis and risk assessment of transgenic organisms. Defensins, a family of small (3.54.5 kDa) cationic antimicro- bial peptides with three to four intramolecular cysteine disulde bonds, are found in mammals, insects and plants (6). Defensins and their synthetic analogues have been extensively studied because of their microbiocidal and immunoenhancing proper- ties (7,8). Porcine β-defensin-2 (pBD-2) is a recently discovered mammalian porcine defensin produced in most porcine tissues (9,10). Previous studies have demonstrated that pBD-2 has broad antimicrobial activity against Gram-negative and Gram-positive bacteria (11,12). Overexpression of pBD-2 can improve the disease resistance of transgenic animals (10,13). Thus, a reliable and simple method by which to measure protein levels of pBD-2 is highly important and necessary, especially in transgenic studies. Unfortunately, pBD-2 is a short peptide (37-mer) that is difcult to detect using traditional sandwich immunoassay methods because of steric hindrance of the identied pairs of antibodies (14). pBD-2 levels in different organisms are currently mainly measured by RT-PCR (11,15). Chemiluminescence (CL) is widely used in various elds because of its extremely high sensitivity, simplicity, wide calibration range and suitability for miniaturization in analytical chemistry (16,17). This study develops a competitive chemiluminescence enzyme immunoassay (CLEIA) method to determine pBD-2 concentrations * Correspondence to: Rui Zhou, Division of Animal Infectious Diseases in the State Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University, Shizishan Street 1, Wuhan 430070, China. Tel.: +86-2787281878; Fax: +86-2787282608. E-mail: [email protected] * Yonggang Hu, State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Shizishan Street 1, Wuhan 430070, China. Tel.: +86- 2787282608; Fax: +86-2787282608. E-mail: [email protected] a State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan 430070, China b College of Life Science and Technology, Huazhong Agricultural University, Wuhan 430070, China c Division of Animal Infectious Diseases in the State Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070, China Luminescence 2014 Copyright © 2014 John Wiley & Sons, Ltd. Research article Received: 17 December 2013, Revised: 13 April 2014, Accepted: 8 May 2014 Published online in Wiley Online Library (wileyonlinelibrary.com) DOI 10.1002/bio.2718

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Page 1: A competitive chemiluminescence enzyme immunoassay method for β-defensin-2 detection in transgenic mice

Research article

Received: 17 December 2013, Revised: 13 April 2014, Accepted: 8 May 2014 Published online in Wiley Online Library

(wileyonlinelibrary.com) DOI 10.1002/bio.2718

A competitive chemiluminescence enzymeimmunoassay method for β-defensin-2detection in transgenic miceXi Yang,a,c Tao Zhou,a,b Lei Yu,a,c Wenwen Tan,a,c Rui Zhoua,c*and Yonggang Hua,b*

ABSTRACT: A competitive chemiluminescence enzyme immunoassay (CLEIA) method for porcine β-defensin-2 (pBD-2) detec-tion in transgenic mice was established. Several factors that affect detection, including luminol, p-iodophenol and hydrogenperoxide concentrations, as well as pH, were studied and optimized. The linear range of the proposed method for pBD-2detection under optimal conditions was 0.05–80ng/mL with a correlation coefficient of 0.9960. Eleven detections of a30ng/mL pBD-2 standard sample were performed. Reproducible results were obtained with a relative standard deviationof 3.94%. The limit of detection of the method for pBD-2 was 3.5 pg/mL (3σ). The proposed method was applied to determinepBD-2 expression levels in the tissues of pBD-2 transgenic mice, and compared with LC-MS/MS and quantitative real-timereverse-transcriptase polymerase chain reaction. This suggests that the CLEIA can be used as a valuable method to detectand quantify pBD-2. Copyright © 2014 John Wiley & Sons, Ltd.

Additional supporting information may be found in the online version of this article at the publisher’s web site.

Keywords: chemiluminescence enzyme immunoassay; porcine β-defensins-2; transgenic mice

* Correspondence to: Rui Zhou, Division of Animal Infectious Diseases inthe State Key Laboratory of Agricultural Microbiology, College ofVeterinary Medicine, Huazhong Agricultural University, Shizishan Street1, Wuhan 430070, China. Tel.: +86-2787281878; Fax: +86-2787282608.E-mail: [email protected]

* Yonggang Hu, State Key Laboratory of Agricultural Microbiology, HuazhongAgricultural University, Shizishan Street 1, Wuhan 430070, China. Tel.: +86-2787282608; Fax: +86-2787282608. E-mail: [email protected]

a State Key Laboratory of Agricultural Microbiology, Huazhong AgriculturalUniversity, Wuhan 430070, China

b College of Life Science and Technology, Huazhong Agricultural University,Wuhan 430070, China

c Division of Animal Infectious Diseases in the State Key Laboratory ofAgricultural Microbiology, College of Veterinary Medicine, HuazhongAgricultural University, Wuhan 430070, China

IntroductionGiven recent developments in transgenic technology, the genera-tion of genetically modified organisms (GMOs) with transgenicresistance and improved production rates has received significantresearch attention. Target-gene expression analysis in GMOs ismainly based on reverse-transcriptase polymerase chain reaction(RT-PCR). RT-PCR is a classical approach used to determine geneticexpression by synthesis of specific mRNA species as a proxy forestimating functional differences at the protein level (1,2).However, inconsistencies among RT-PCR results suggest that geneexpression at the transcriptional level is not tightly coupled to thatat the translational level (3–5). In addition, RT-PCR is associatedwith the risk of amplified nucleic acid release into the environ-ment, which can contaminate subsequent analysis. As such, analternative immunoassay method that can directly measure theconcentration of specific proteins or peptides, especially in GMOs,must be developed. Traditional immunoassay methods, such asenzyme-linked immunosorbent assay, have limited applicationsin GMO analysis because of their lack of sensitivity. Therefore,a rapid, cost-effective, high throughput, sensitive and specificimmunoassay method must be developed for expressionanalysis and risk assessment of transgenic organisms.

Defensins, a family of small (3.5–4.5 kDa) cationic antimicro-bial peptides with three to four intramolecular cysteine disulfidebonds, are found in mammals, insects and plants (6). Defensinsand their synthetic analogues have been extensively studiedbecause of their microbiocidal and immunoenhancing proper-ties (7,8). Porcine β-defensin-2 (pBD-2) is a recently discoveredmammalian porcine defensin produced in most porcine tissues(9,10). Previous studies have demonstrated that pBD-2 has broadantimicrobial activity against Gram-negative and Gram-positive

Luminescence 2014 Copyright © 2014 John

bacteria (11,12). Overexpression of pBD-2 can improve thedisease resistance of transgenic animals (10,13). Thus, a reliableand simple method by which to measure protein levels ofpBD-2 is highly important and necessary, especially in transgenicstudies. Unfortunately, pBD-2 is a short peptide (37-mer) that isdifficult to detect using traditional sandwich immunoassaymethods because of steric hindrance of the identified pairs ofantibodies (14). pBD-2 levels in different organisms are currentlymainly measured by RT-PCR (11,15).Chemiluminescence (CL) is widely used in various fields because

of its extremely high sensitivity, simplicity, wide calibration rangeand suitability for miniaturization in analytical chemistry (16,17).This study develops a competitive chemiluminescence enzymeimmunoassay (CLEIA) method to determine pBD-2 concentrations

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X. Yang et al.

in tissue samples of transgenic mice. This CLEIA method iscompared with LC-MS/MS and RT-PCR. A schematic diagram ofCLEIA for pBD-2 detection in transgenic mice is illustrated in Fig. 1.First, the synthesized pBD-2 (37-mer) was adsorbed physically onthe surface of 96-well microplates. Next, tissue samples from miceand the pBD-2 monoclonal antibody (mAb) were incubatedtogether in an Eppendorf tube. Recombinant pBD-2 peptides werelinked to the pBD-2 antibody in this step. The tissue–antibodymixture was transferred onto a coated microplate to initiate thesecond immunorecognition event. Unbound mAbs were reactedwith the synthesized pBD-2, while the matrix, recombinant pBD-2–antibody complexes and other unbound mAbs were washedout. The detection antibody-conjugated horseradish peroxidase(HRP) was added to the plate to react with the pBD-2 antibody.The unbound detection antibody was also washed out. Finally,the detection substrates were added to the immune reactionsystem. pBD-2 expressionwas determined by detecting CL intensity.

Materials and methods

Reagents

Luminol was purchased from Alfa Aesar (USA). p-Iodophenol(PIP) was purchased from Aladdin Chemical (Shanghai, China).

Figure 1. Principle diagram for the determin

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All other reagents were of analytical grade. All solutions wereprepared with deionized water produced by a Cascada IX ultra-pure water system from Pall Co., Ltd (USA). Goat anti-(mouseIgG) conjugated with HRP (IgG–HRP) was obtained fromSouthern Biotechnology Associates Inc. (Birmingham, AL, USA).A standard mature 37-mer porcine pBD-2, with an amino acidsequence of DHYICAKKGGTCNFSPCPLFNRIEGTCYSGKAKCCIR,and an 11-mer peptide, with an amino acid sequence ofPLFNRIEGTCY between the third and fourth cysteines of themature pBD-2, were synthesized according to the known proteinsequence of mature pBD-2 by Neweast Biosciences (Wuhan,China) (9).

Carbonate–bicarbonate buffer (0.05mol/L, pH 9.6) was used asthe coating buffer. The washing solution consisted of phosphate-buffered saline (PBS) (pH7.4) with 0.1% (v/v) Tween (PBST). Theblocking solution was composed of PBS containing 1% (w/v)bovine serum albumin (BSA). Tris/HCl buffers (0.05mol/L) withpH values ranging from 6.5 to 10.5 were also used. A stock solutionof PIP (0.1mol/L) was prepared in dimethylsulfoxide (DMSO) andstored at room temperature. Luminol was dissolved in 1mol/LNaOH, diluted to 32mmol/L, stored at 4 °C, and used within threedays of preparation. A stock solution of H2O2 (0.1mol/L) wasprepared by volumetric dilution of 30% (w/w) of H2O2 1h beforeaddition of the assay solution.

ation of pBD-2 using the CLEIA method.

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β-Defensin-2 detection in mice

Apparatus

A TECAN Infinite F200 multimode microplate reader was pur-chased from Tecan Trading Co., Ltd (Shanghai, China). The pHof all buffer solutions was measured using an Orion pH meter(United Initiators Co., Ltd, USA). The incubation procedures wereconducted using a heating platform (Jing Hong LaboratoryInstrument Co., Ltd., Shanghai, China).

Preparation of the anti-pBD-2 mAb

The 11-mer peptide coupled with keyhole limpet hemocyaninwas used as an immunogen to immunize Balb/C mice. Thesynthesized 37-mer pBD-2 was used as the coating antigen forscreening pBD-2 antibodies. Spleen cells from immunized micewith high antibody levels were fused with SP2/0 myeloma cells,and positive clones were selected. After two cycles of subcloningby limiting dilution, hybridomas secreting high levels of themAbs with favorable specificity were obtained.

Construction of the pBD-2 transgenic mice

The pBD-2 expression construct was microinjected into thepronuclei of fertilized mouse oocytes that were allowed todevelop to term. The oocytes were then transferred to pseudo-pregnant mice. Positive transgenics were selected and bred togenerate offspring.

Coating of pBD-2 on the microplate

Polystyrene 96-well microplates were coated with 100μL ofdiluted synthesis 37-mer mature pBD-2 peptide in coating buffer(pH 9.6) per well. After maintaining at 4 °C for 16 h, the plate waswashed three times with 250μL of PBST per well and gentlytapped against absorbent paper to remove all of the fluids.Subsequently, 100μL of 1% BSA in PBS was added to each well.The plate was placed at 37 °C for 1 h to block the active sites. Thesolution in the well was then removed and the plate was againwashed with PBST. Finally, the plate was wrapped and storedat –20 °C until use.

Immunoassay procedures

Briefly, 50μL of pBD-2 mAb diluted with 1% BSA and 50μL ofthe samples were added to an Eppendorf tube and incubatedfor 1 h at 37 °C. After incubation, the mixed liquid was addedto each well of the coated microplate. Incubation was again per-formed for 1 h at 37 °C. Unbound compounds were removed bywashing after incubation. Exactly 100μL of IgG–HRP solutionwas added to each well at 37 °C for 1 h, followed by four cyclesof washing with PBST. Finally, 100μL of the substrate solutionwas added to each well. The CL intensity was measured usinga TECAN infinite F200 multimode microplate reader.

CLEIA for the determination of pBD-2 in transgenic mice

Five 9-week-old pBD-2 transgenic mice and five wild-type (WT)mice were used for this study. The heart, liver, spleen, kidneyand lung tissues were collected, ground in liquid nitrogen anddissolved in normal saline prior to centrifugation for 10min at8000 r.p.m. The supernatants were collected and assayed usingCLEIA. Each sample was examined five times.

Luminescence 2014 Copyright © 2014 John

LC-MS/MS analysis

A high-performance liquid chromatography (HPLC) with massspectrometric (MS/MS) analysis was conducted to detect theconcentrations of pBD-2. Quantification was based on themethod by Kucukkolbasi et al. (18). Briefly, the supernatantscollected from 100mg of tissues were cleaned up with AlltechExtract-Clean C18 500mg solid-phase extraction (SPE) cartridges(GRACE, USA). The eluents were lyophilized (Freezone, Labconco,Kansas City, MO, USA) and then reconstituted with 0.1mL ofwater. Analysis was carried out on an Agilent 1260 Infinity LC(Waldbronn, Germany) coupled with an Agilent 6540 AccurateMass, Q-TOF (Waldbronn, Germany).

Expression analysis by quantitative real-time RT-PCR

Total RNA was extracted from each tissue sample using an SVTotal RNA isolation system (Promega, Madison, WI, USA) accord-ing to the manufacturer’s instructions. RT-PCR was performed on1μg of RNA using a first-strand cDNA synthesis kit (Fermentas,Lithuania). Real-time PCR was performed on an ABI 7500 fastreal-time PCR system (Applied Biosystems, USA). Relative levelsof transcript abundance of PBD-2 in different tissues were mea-sured using the method by Chen et al. (10). Statistical analysiswas performed using SPSS 13.0 for Windows. All data wereanalyzed by one-way ANOVA. Significant differences weredefined as p< 0.01.

Results and discussion

Optimization of the working conditions

Chessboard titration was applied to optimize the coatingconcentration of pBD-2 and dilution ratios of the anti-(pBD-2)antibody (19). The CL intensity increased with increasing coatingconcentration of pBD-2 and decreased with increasing dilutionratio of the anti-(pBD-2) antibody (Fig. 2A). Smaller amounts ofantibody often result in higher sensitivity in a competitiveimmunoassay. Considering the sensitivity, reliability and avail-able quantity of the antigen and antibody, the optimal coatingconcentration of pBD-2 and dilution ratio of the anti-(pBD-2)antibody were found to be 320 ng/mL and 1 : 320, respectively.The influence of luminol on the CL intensity was investigated

at concentrations between 0.1 and 0.8mmol/L. Figure 2(B)shows that the CL intensity increased rapidly with increasingconcentration of luminol. The CL intensity increased significantlyand reached a plateau at a luminol concentration of 0.5mmol/L.Further addition of luminol did not enhance the CL intensity.Consequently, a luminol concentration of 0.5mmol/L waschosen for our experiments.PIP was dissolved in DMSO and diluted with Tris/HCl buffer to

different concentrations prior to CL detection. The effect ofdifferent concentrations of PIP on the CL intensity of the systemwas studied. The CL intensity increased rapidly with increasingconcentration of PIP and peaked at a PIP concentration of0.4mmol/L (Fig. 2C). Further increases in PIP concentrationreduced the CL intensity. The effect of DMSO without PIP onCL intensity was investigated, because the DMSO concentrationswere increased with the increase of PIP concentrations duringour experiments. We found that the CL signals were significantlyinhibited with the increase of DMSO. This is accorded withthat the high concentration of hydrophilic organic solvent

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Figure 2. (A) Effect of the coating concentration of pBD-2 and the dilution ratios of anti-(pBD-2) antibody. (B) Effect of luminol concentration on the CL intensity.[PIP] = 0.2mmol/L, pH 8.5, [H2O2] = 5mmol/L. The concentration range of luminol is from 0.1 to 0.8mmol/L. Error bars indicate ± SD, n= 6. (C) Effect of PIP concentrationon the CL intensity. [Luminol] = 0.5mmol/L, pH 8.5, [H2O2] = 5mmol/L. The concentration range of PIP is from 0.05 to 1mmol/L. Error bars indicate ± SD, n= 6. (D) Effectof H2O2 concentration on the CL intensity. [Luminol] = 0.5mmol/L, pH 8.5, [PIP] = 0.4mmol/L. The concentration range of H2O2 is from 1 to 6mmol/L. Error barsindicate ± SD, n= 6. (E) The effect of buffer pH on the CL intensity. [Luminol] = 0.5mmol/L, [H2O2] = 4mmol/L, [PIP] = 0.4mmol/L. The concentration range of pH is from7.8 to 8.8. Error bars indicate ± SD, n= 6.

X. Yang et al.

introduced to the CL system could generally decrease the CLsignal (20). In addition, with a constant concentration of DMSO,the effect of PIP for the CL signals was also investigated. Thequenching effect of the CL signals was also observed at a PIPconcentration> 0.4mmol/L. This is related to the quenching effectof the polar reagent, PIP, as an acidic compound and may be

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protonated the excited state of 3-aminophthalate as the speciesresponsible for light production (data not shown). The quenchingof the CL intensity in our experiments was not only due to theincrease of DMSO, but also related to the quenching effect ofthe increase of PIP. Thus, a PIP concentration of 0.4mmol/L wasselected as the baseline condition for the enhancer concentration.

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β-Defensin-2 detection in mice

The effect of H2O2 concentration on the CL intensity wasdetermined by varying the concentration of H2O2 from 1.0 to6.0mmol/L. The CL intensity increased with increasing H2O2

concentration and reached a maximum at a H2O2 concentra-tion of 4.0mmol/L (Fig. 2D). The CL intensity decreased atH2O2 concentrations higher than 4.0mmol/L because excessamounts of peroxide can affect HRP activities (21). Thus, anoptimal H2O2 concentration of 4.0mmol/L was used in allfurther experiments.

The influence of the buffer solution pH on the CL intensity wasalso investigated. Figure 2(E) shows that the CL intensityincreased significantly at pH 8.3 and decreased with furtherincreases in pH. The pH-dependence of light emission has beenattributed to the combined effects of enzyme inactivation andstability of reaction intermediates (22). A buffer pH of 8.3balances these two factors appropriately.

Performance of the immunoassay method

Figure 3 shows representative CL responses in a dilution series ofstandard pBD-2 under optimal experimental conditions. Thelinear range of the calibration curve was 0.05–80 ng/mL andthe coefficient of determination was 0.9960, which indicates thatthe proposed method has good linearity. The reproducibilitywas expressed as the relative standard deviation (RSD) of 11

Figure 3. Calibration curve of chemiluminescence response versus dilution seriesof standard pBD-2. [Luminol] = 0.5mmol/L, [H2O2] = 4mmol/L, pH= 8.3, [PIP] = 0.4mmol/L. Error bars indicate ± SD, n= 6.

Table 1. Parameters of regression equations for the three-tissue

Tissuelysatedilution(1 : 10)

Linear equation Determinationcoefficient (r2)

Added 5 ng/m

Detected(ng/mL)

Re

Heart y= –2151.7x+ 543,806 0.9865 4.86 ± 0.41Liver y= –234.78x+ 238,057 0.9915 5.43 ± 0.31 1Spleen y= –781.05x+ 165,220 0.9878 5.26 ± 0.33 1Lung y= –944.07x+ 201,376 0.9910 4.80 ± 0.31Kidney y= –986.04x+ 219,636 0.9841 5.65 ± 0.19 1

Luminescence 2014 Copyright © 2014 John

replicate determinations of the standard solution (30 ng/mLpBD-2); the RSD obtained was 3.94%. The limit of detection(LOD) was calculated as the pBD-2 concentration correspond-ing to the blank (1% BSA) average minus three standard de-viations (3σ); the LOD of the proposed method was found tobe 3.5 pg/mL.A series concentration of systhesis pBD-2 was added to the 1 :

10 tissue lysate dilution (heart, jejunum and liver) for thestandard curve, and different amounts of pBD-2 standard weremixed with the 1 : 10 tissue lysate dilution for the recoveries.The recovery experiment was repeated five times. The averagerecoveries and series standard curves are shown in Table 1.The recovery results were between 94.62 and 113.60%, indi-cating that the proposed method is reliable.

Determination of pBD-2 in transgenic mice using CLEIA

The heart, liver, spleen, kidney and lung tissues were collectedfrom five transgenic mice and five WT mice for pBD-2 detection.The standard addition method was used to quantitativelydetermine the pBD-2 in each sample using the standardaddition calibration curve method. Each sample was examinedfive times, and the 250 measurement results were statisticallyanalyzed using T-test. A comparison of pBD-2 protein levelsbetween transgenic and WT mice is shown in Fig. 4(A). pBD-2levels in all of the transgenic mice tissues were significantlyhigher than those in WT mice (p< 0.01). BLAST database searchresults showed that the amino acid sequence of pBD-2 is similarto those of several other mouse endogenous proteins. Forexample, the 25 to 61 amino acid sequence of mouse β-defensin-38 is DTKKCVQRKNACHYFECPWLYYSVGTCYKGKGKCCQK, indicating38% sequence similarity with pBD-2 (http://blast.ncbi.nlm.nih.gov/). In addition, the composition can vary significantlyamong different organs of the same mouse or even in thesame organ in different mice (23). All these may be the possi-ble reasons for the high negative values (WT mice) in sometissues.The heart tissue showed the highest concentration of pBD-2

in transgenic mice, followed by the spleen (Fig. 4A), which issimilar to the distribution of pBD-2 mRNA determined by real-time RT-PCR (Fig. 4B). The concentration of pBD-2 in the kidneywas slightly higher than that in the lung, again similar to thereal-time RT-PCR results. However, the distributions of pBD-2and its mRNA were not identical in all tissues. Differences inrelative levels were found in the liver. The pBD-2 protein levelin the liver was only slightly over half that in the lung, but thepBD-2 mRNA was not highly enriched in the lung. These differ-ences are unlikely to have stemmed from pretreatment of the

sample and the recovery rates (n= 5)

L Added 20 ng/mL Added 50 ng/mL

covery(%)

Detected(ng/mL)

Recovery(%)

Detected(ng/mL)

Recovery(%)

97.20 19.09 ± 0.63 95.45 47.31 ± 0.95 94.6208.60 21.14 ± 0.45 105.70 53.74 ± 0.69 107.4805.20 20.41 ± 0.94 102.05 50.58 ± 0.92 101.1696.00 19.97 ± 0.73 99.85 49.48 ± 0.99 98.9613.00 21.19 ± 0.28 105.95 50.65 ± 0.57 101.30

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Figure 4. (A) The pBD-2 level (ng/g) in tissues of transgenic and wild-type mice as analyzed by CLEIA. Data are presented as the means ± SE, n= 5. **Significantly differentfrom the control group (p< 0.01). (B) Quantitation of pBD-2 mRNA expression in transgenic mice tissues. The pBD-2 PCR products were normalized to RPII as the referencegenes, and data shown are pBD-2 mRNA expression as a ratio of the lung’s expression. Data plotted represent the mean ratio value ± SE for three independent experiments.(C) Correlations between pBD-2 concentrations measured by CLEIA and LC-MS/MS. Data are presented as the means ± SE, n= 5.

X. Yang et al.

tissue samples because all the samples were prepared via thesame procedure and other factors considered, such as the useof various buffers and the buffer/tissue ratios, do not affect tis-sue distribution. Disparities between the relative tissue distribu-tions of the protein and mRNA may reflect the translational and/or post-translational regulation of pBD-2 production or cellulartransport of pBD-2 protein (24). Transcription and translationdo not always show a simple linear relationship (5). Differentmechanisms involving cis- and trans-acting mechanisms gener-ate a large repertoire of systems that enhance or repress the syn-thesis of proteins from a certain copy number of mRNAmolecules (25–27). CLEIA results showed that the distributionof pBD-2 protein is similar but not identical to that of pBD-2mRNA. LC-MS/MS was also used for pBD-2 detection in thisstudy. The results were then compared with that of CLEIA de-rived from the same samples of transgenic mice. Results in Fig. 4(C) showed that the heart expressed higher levels of pBD-2 thanthat of the spleen of transgenic mice. This is agreement withthat of CLEIA. Interestingly, the CLEIA values were higher thanthose of LC-MS/MS. Moreover, the concentrations of pBD-2 pep-tide in the transgenic liver, kidney and lung were much lowerthan those in the heart and spleen, which could be detectedby CLEIA, but could not by LC-MS/MS. This might be due thatthe recombinant pBD-2 peptide in the tissues could exist informs of pBD-2-protein complexes (28,29). These complexescould not be detected by the LC-MS/MS using the synthesizedpBD-2 molecule as standard sample, because it is difficult to pre-pare different forms of pBD-2–protein complexes as standardsamples due to the lack of reference data. Fortunately, all pBD-2-containing molecules including pBD-2–protein complexescould be detected by this developed CLEIA method becausepBD-2 antibody can bind pBD-2 and its complexes.

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ConclusionAn effective competitive CLEIA method for pBD-2 detection hasbeen developed. The proposed method is simple, rapid andcost-effective, high throughput, sensitive and specific. Moreover,it is more effective than RT-PCR and LC-MS/MS for the detectionof pBD-2 levels in the tissues of transgenic animals.

Acknowledgements

This work was supported by the National Basic Research Programof China (2012CB518802), and National Transgenic BreedingProjects (2009ZX08006-006B).

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