A natural carrier effect and the generation of specific antibodies to biologically active peptides

Production of specific antibodies to haptens, especially antipeptides, without interference by carrier protein, is desirable. The bradykinin-potentiating peptides (BPPs) are a family of pyroglutamyl proline-rich oligopeptides with strong
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  ANALYTICALBIOCHEMISTRY Analytical Biochemistry 353 (2006) 174–$ - see front matter ©  2006 Elsevier Inc. All rights reserved.doi:10.1016/j.ab.2006.03.018 A natural carrier e V  ect and the generation of speci W c antibodies to biologically active peptides Lilian R. Tsuruta a , Mirian A.F. Hayashi a , Katsuhiro Konno a , Denise V. Tambourgi a,b , Marina T. Assakura a , Antonio C.M. Camargo a , Osvaldo A. Sant’Anna a,b, ¤ a Center for Applied Toxinology, Instituto Butantan, São Paulo, Brazil  b Immunochemistry   Laboratory, Instituto Butantan, São Paulo, Brazil  Received 10 January 2006Available online 30 March 2006 Abstract Production of speci W c antibodies to haptens, especially antipeptides, without interference by carrier protein, is desirable. The bradyki-nin-potentiating peptides (BPPs) are a family of pyroglutamyl proline-rich oligopeptides with strong antihypertensive properties. In thiswork, the production of antibodies to BPPs by use of an e Y cient immunization protocol in mice genetically modi W ed for the high anti-body responsiveness (H III  line) is described. Although it was possible to induce antibody production by single-dose administration of freeBPPs, higher antibody titers were obtained in mice preimmunized with carrier protein before administration of peptides conjugated tothis carrier. Interestingly, both mouse groups had a higher titer of IgG 1  than IgG 2a  isotypes, regardless of prior immunization with thecarrier protein. However, a lower titer of IgG 2a  was observed in unprimed mice. A single band of about 27kDa corresponding to the BPPprecursor protein was recognized by these antibodies in the cytosol of the Bothrops jararaca  venom gland. This work proposes an e Y cientimmunization protocol based on classic studies described for the hapten–carrier e V  ect for generating speci W c antibodies against biologi-cally active peptides. ©  2006 Elsevier Inc. All rights reserved. Keywords: Antipeptide antibodies; Bradykinin-potentiating peptide; Carrier protein; High-responder mice; Bothrops jararaca Small molecules such as drugs, pesticides, and peptidesare known as haptens because they are not able to elicitantibody production itself when injected into animals.However, linkage of the hapten with an immunogenic car-rier protein induces the cooperation of lymphocytes withimmunogenic carrier and hapten–carrier-primed T cells,representing the basis of the collaborative antibodyresponse of B lymphocytes recognizing the hapten. Thus,the linkage to a carrier protein may lead to a strong anti-hapten response [1]. One problem with this procedure is thegeneration of a variable degree of anti-carrier response thatin X uences mainly the quantity and a Y nity of the antibodyproduced.Several groups have reported the generation of antibodiesto synthetic peptides of 6 to 35 amino acid residues by immu-nization with free peptides. Most of these peptides were cho-sen because they are antigenic determinants of proteins andstrong candidates for vaccine production. An advantage of free peptide immunization is that it is expected to be moree Y cient in producing speci W c antibodies, as it avoids the pro-duction of anti-carrier antibodies [2–14]. However, one con-cern is that the peptide dose used in these assays varies from50  g to 1mg, and, in most cases, the antibody titer isobtained only after multiple immunizations.Another relevant aspect that should be considered isthe in X uence of the carrier protein on production of anti-hapten antibodies. The e V  ect of preimmunization with acarrier protein, followed by immunization with the haptenconjugated to the same carrier, has been widely studied.This seems to modulate the antibody titer, which is * Corresponding author. Fax: +11 55 3726 1024. E-mail address: (O.A. Sant’Anna).  A natural carrier effect and antipeptide antibody generation / L.R. Tsuruta et al. / Anal. Biochem. 353 (2006) 174–180 175 independent of the isotype pattern, but dependent on spe-ci W c T cell–B cell cooperation [15]. An earlier study [16] demonstrated that preimmunization of rabbits or pigswith an optimal dose of carrier protein, followed byimmunization with antigen linked to a carrier protein,elicits a hapten-speci W c secondary response. However,some groups have reported unexpected results such as theinduction of epitopic suppression for anti-hapten anti-body production by the carrier/hapten carrier immuniza-tion protocol [17–23]. The mechanism involved in thisphenomenon and its modulation are still not fully under-stood [24]. One might suppose the carrier e V  ect describedusing the binding of chemically de W ned molecules toimmunogenic protein might be a natural, spontaneousand even an endogenous phenomenon.Bradykinin-potentiating peptides (BPPs), 1  from thevenom gland and brain of the snake Bothrops jararaca (Solenoglypha, Viperidae), are biologically active peptidescharacterized by the presence of a pyroglutamyl residue atthe N terminus and a proline residue at the C terminus [25].Besides potentiating the e V  ects of bradykinin, BPPs possessantihypertensive properties and thus might play a role inthe regulation of the blood pressure [26]. BPP precursorprotein from venom gland (27.5kDa) is formed by sevensequences of BPPs (BPP10a, BPP13a, BPP10c, BPP11b,BPP11c, and two BPP5a) and one sequence of C-type natri-uretic peptide (CNP), a well known neuropeptide [25].Recently, the expression of this BPP precursor wasobserved in regions of the snake brain correlated with neu-roendocrine functions [27].Highly speci W c antibodies constitute a promising tool inthe isolation of functionally homologous endogenous pep-tides from mammals. To obtain speci W c antipeptide anti-bodies, novel and e Y cient immunization protocols weretested. As a W rst strategy, we attempted to raise antibodiesto BPP without conjugation to a carrier, by immunizingwith the free peptides only, and using mice geneticallyselected for high antibody responsiveness (H III ) [28]. Sec-ond, to obtain more antibodies speci W c for the peptides andto reduce the response to the carrier, we preimmunizedmice with the carrier protein keyhole limpet hemocyanin(KLH) before immunization with BPP–KLH. The seraobtained were qualitatively and quantitatively analyzed,and compared with those obtained from non-preimmu-nized mice.We chose biologically active peptides as the main tar-get of our investigation due to the obvious applicationsof high-a Y nity antipeptide antibodies, such as immuno-cytochemistry, speci W c identi W cation of these antigens incell lysates or tissues, and development of potentvaccines. Materials and methods Peptide synthesis The peptides pBPP5a (<EKWAP), BPP5a (QKWAP),pBPP10c (<ENWPHPQIPP), and BPP10c (QNWPHP-QIPP) were synthesized by solid-phase synthesis using theFmoc strategy as previously described [29]. “<E” representsthe pyroglutamic acid at the N terminus. Peptides werepuri W ed by HPLC (Shimadzu Corporation, Kyoto, Japan),and the structure was con W rmed with Ettan matrix-assistedlaser desorption ionization time-of- X ight (MALDI-TOF)mass spectrometry (Amersham Biosciences UK Limited,Buckinghamshire, England). Preparation of peptides conjugated to the carrier protein The peptides were conjugated to KLH (Sigma Co., St.Louis, MO, USA) or to bovine serum albumin (BSA: Sigma)by their N termini. The W rst conjugate was used for immuni-zation, whereas the second was used for the determination of antibody titers to reduce the interference of the antibodiesagainst the carrier protein. Peptides were coupled accordingto Houen and Jensen [30]. Brie X y, 30mg of divinyl sulfonate(DVS) was added to 10mg of carrier protein dissolved in10mM sodium phosphate bu V  er (pH 7.2), and the mixturewas incubated at 4°C overnight under agitation. Then 0.4mgof BPP5a was added, and pH was adjusted to 9. The mixturewas incubated at 4°C overnight under agitation, andunbound peptides were removed through dialysis against 10mM sodium phosphate bu V  er (pH 7.2). The concentration of the conjugate was determined by the Bradford method usingthe Bio-Rad Protein Assay (Bio-Rad Laboratories, Hercules,CA, USA). The average number of peptides per BSA wasdetermined with the Ettan MALDI-TOF mass spectrometer(Amersham) using BSA as internal standard. BPP5a–BSAhad approximately 15 molecules of peptide per conjugate,whereas BPP10c–BSA had an average of 7 molecules of pep-tide per conjugate. Animals and immunization protocols The genetically selected high-antibody-producer mice(H III  mice) [31] were maintained at the animal facilities of the Immunochemistry Laboratory, Instituto Butantan, andwere caged and handled according to the InternationalAnimal Welfare Recommendations and in line with theguidelines for the use of animals in biomedical research[32]. Fifty micrograms of BPP5a–KLH emulsi W ed withincomplete Freund’s adjuvant (IFA) was injected subcuta-neously into two mice, and the anti-BPP5a antibody titerswere maintained by immunization with the free peptide.Then, novel immunization protocols were tested. Groups of four mice were immunized with 10  g of free peptides(pBPP5a, BPP5a, and pBPP10c) emulsi W ed with IFA bysubcutaneous injection. Subsequent boosters were given atthe same dose by the same route. 1 Abbreviations used:  BPP, bradykinin-potentiating peptide; BSA, bo-vine serum albumin; IFA, incomplete Freund’s adjuvant; KLH, keyholelimpet hemocyanin; MALDI-TOF, Matrix-assisted laser desorption ioni-zation time-of- X ight; PBS, phosphate-bu V  ered saline; TBS, Tris-bu V  eredsaline.  176 A natural carrier effect and antipeptide antibody generation / L.R. Tsuruta et al. / Anal. Biochem. 353 (2006) 174–180 The e V  ect of preimmunization was investigated byimmunizing one group of three mice with 10  g of KLHemulsi W ed with IFA, while the other group did not receivethe carrier protein. After 2 weeks, both groups received10  g of BPP5a–KLH emulsi W ed with IFA by subcutane-ous injection. To determine the antibody titer, the micewere bled, and the sera were collected after centrifugationand stored at ¡ 20°C until use. ELISA procedure Peptides conjugated to BSA were used as antigen for thedetermination of antibody titers of antisera. Initially, 50  g/ml of peptide–BSA conjugate was added to an enzymeimmunoassay/radioimmunoassay plate (Costar No. 3690,Corning, NY, USA) and incubated overnight at 4°C forantigen W xation. BSA was used as a negative control.Unbound antigen was removed, and the plate was incu-bated at 37°C for 1h with the blocking solution (3% skimmilk in PBS). Then, the blocking solution was replaced witha serial twofold dilution of serum, starting at an appropri-ate dilution for each serum. The preimmune serum was alsoadded at an appropriate dilution and used as a control of binding. Wells were washed six times with PBS to removeunbound antibodies. A 1/5000 dilution of alkaline phos-phatase-conjugated anti-mouse IgG (H+L) (Promega,Madison, WI, USA) was used as secondary antibody. Theplate was then incubated at 37°C for 1h with 1mg/ml of   p -nitrophenylphosphate (Sigma) in 1.5M NaCl/ 0.1mMTris (pH 9.8), as substrate, for color development. The reac-tion was stopped by adding 2N NaOH, and the absorbancewas read at 405nm using a Spectra Max 1900 microplatereader (Molecular Devices Corporation, Sunnyvale, CA,USA). Antibody titers were expressed as log 2  maximumantiserum dilution giving a positive reaction at which theabsorbance was equal to two times the control value of thepreimmune serum.Immunoglobulin isotypes, IgG 1  and IgG 2a , from antiseraof mice primed with carrier protein and unprimed micewere evaluated. For this purpose, a 1/2000 dilution of alka-line phosphatase-conjugated rat anti-mouse-IgG 1  or anti-mouse IgG 2a  (BD Biosciences Pharmingen, San Diego, CA,USA) was used as secondary antibody. Antibody titer wasdetermined as described earlier. Western blotting  Venom glands of Bothrops jararaca  were obtained by theLaboratory of Herpetology, Instituto Butantan. The cyto-sol was obtained by homogenization followed by ultracen-trifugation at 100,000  g   for 1h at 4°C. Cytosol proteinswere separated on 12.5% sodium dodecyl sulfate–polyacryl-amide gel by electrophoresis. The proteins were transferredfrom the gel to nitrocellulose membrane, and, the next day,the membranes were blocked with 5% skim milk in TBS/0.05% Tween 20. The membranes were washed and thenincubated with a 1/100 dilution of total serum H III  anti-BPP antibodies for at least 1h at room temperature. After awash with TBS/0.05% Tween 20, a 1/5000 dilution of alka-line phosphatase-conjugated anti-mouse IgG (H+L)(Promega) was added, and the mixture was incubated for1h at room temperature. Then the membranes were washedwith TBS/0.05% Tween 20, and the band was revealed byincubating with nitroblue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate substrate in 5M NaCl/1MTris–HCl, pH 9.5/1M MgCl 2  solution [33]. Results Production of speci   W c antibodies to peptides by immunization with free peptides In preliminary experiments, we used a conventionalimmunization protocol to obtain antibodies against thepeptides. H III  mice were immunized subcutaneously with50  g of BPP5a conjugated to the carrier KLH, and highantibody titers were obtained with a single immunization(Fig.1). However, for the booster, to obtain more speci W csera against the peptides, only free peptides were adminis-tered to the animals. Surprisingly, the antibody titerremained high after more than 150 days of this procedure.Mice immunized with either 10  g of free peptide,namely, BPP5a or pBPP5a, produced antibodies with simi-lar titers against both peptides (Fig.2A). Additional immu-nizations did not signi W cantly change the responses.Similar results were also obtained with pBPP10c, whichis longer than pBPP5a and has a higher content of prolineresidues (Fig.2B), indicating that antibodies to pBPP10ccould also be successfully produced. Interestingly, immu-nization with this longer peptide led to a secondaryresponse, and the antibody titer increased after the secondimmunization.The speci W city of the anti-BPPs for the B. jararaca  BPPprecursor protein was evaluated by Western blot analysis Fig.1. Anti-BPP5a antibody titer of H III  strain mice immunized initiallywith 50  g of BPP5a conjugated to KLH and then with 10  g of BPP5a.Each preparation was emulsi W ed with IFA and injected subcutaneouslyinto H III  mice. After immunization, animals were bled to analyze the anti-body titer. Appropriate dilutions of sera were added to the plate coatedwith BPP5a–BSA. The antibody titer was obtained by comparison withthe binding level of preimmune serum ( 6 log 2 ). The W rst immunizationcorresponds on Day 0, and broken lines represent the days on which otherboosters were administered. 0 80 160 240051015 Days    H    I   I   I   -  a  n   t   i  -   B   P   P   5  a   T   i   t  e  r   (   l  o  g    2    )  A natural carrier effect and antipeptide antibody generation / L.R. Tsuruta et al. / Anal. Biochem. 353 (2006) 174–180 177 using venom gland cytosol (Fig.3). Fig.3 illustrates the results obtained for the membrane probed with a pool of anti-BPP5a antisera. The antiserum of immunized micedetected a single band of about 27kDa, whereas the serumof nonimmunized mice was unable to recognize any com-ponent of the extract (Fig.3B). The same results wereobserved for the other anti-BPP antibodies obtained byimmunization with other free peptides or with the peptideconjugated to a carrier protein (data not shown). Preimmunization with the carrier protein induced higher antibody titer against peptides To understand the e V  ect of the carrier protein on thegeneration of antibodies to peptides and haptens, weattempted to preimmunize mice with the carrier proteinbefore administration of BPP5a conjugated to the samecarrier protein. The antibody titer was compared with thatobtained from mice not subjected to preimmunizations.Overall, the immunization protocol, including preimmuni-zation with carrier protein, produced a higher titer of anti-bodies against the peptide (Fig.4). Evaluation of theantibody titer for a long period revealed that anti-BPP lev-els were still high in mice preimmunized with the carrierprotein, even 100 days after administration of BPP5a– KLH.Both IgG 1  and IgG 2a  titers were compared between thecarrier-primed and unprimed mouse groups on the 13thand 71st days after immunization with BPP5a–KLH. Theresults are summarized in Table 1. The IgG 1  level was veryhigh for both groups in all antisera, whereas the IgG 2a  levelof unprimed mice was very low. However, no signi W cantdi V  erence was observed in the IgG levels throughout theresponse period.Comparative analysis of the antibody titers obtained byimmunization with free peptides and those obtained afterpreimmunization with the carrier protein indicated that ahigher antibody titer was obtained using the second Fig.2. Production of anti-BPP5a or anti-pBPP5a antibodies (A) and anti-pBPP10c antibodies (B) by immunization of H III  mice with free peptides. Micewere immunized with 10  g of free peptides emulsi W ed with IFA by subcutaneous injection at each step. Day 0 corresponds to the W rst immunization, andbroken lines represent the days on which other boosters were administered. (A) Titers of animals immunized with pBPP5a (triangle) and BPP5a (square)on a plate coated with BPP5a-BSA. (B) Titers of a group of animals immunized with pBPP10c and analyzed by a plate coated with BPP10c-BSA. The anti-body titer was obtained comparing to the binding level of preimmune serum ( 6 log 2 ). 0 40 80 120 160051015 Days    H    I   I   I   -  a  n   t   i  -   B   P   P   5  a   T   i   t  e  r   (   l  o  g    2    ) 0 40 80 120 160051015 Days    H    I   I   I   -  a  n   t   i  -  p   B   P   P   1   0  c   T   i   t  e  r   (   l  o  g    2    ) A B Fig.3. Analysis of the speci W city of H III -anti-BPPs for the cytosol of  Bothrops jararaca  venom gland revealed the recognition of one band of approximately 27kDa. (A) Sodium dodecyl sulfate (12.5%)–polyacryl-amide gel electrophoresis of B. jararaca  venom gland cytosol (gland).MW, protein size marker (in kDa). (B) Western blot analysis of the cyto-sol of B. jararaca  venom gland using nonimmunized H III  mouse serum(control) and a pool of H III -anti-BPP5a serum (anti-BPP).Fig.4. Increase in H III  anti-BPP5a antibody titer by preimmunization withcarrier protein followed by administration of BPP5a–KLH. One group of animals (closed circles) was preimmunized with 10  g of KLH emulsi W edin IFA, and the other group (open circles) was not preimmunized. Twoweeks later, both groups received 10  g of BPP5a–KLH emulsi W ed in IFA.Appropriate dilutions of sera were added to the plate coated with BPP5a– BSA. The antibody titer was obtained by comparison with the bindinglevel of preimmune serum ( 6 log 2 ). Day 0 corresponds to the immuniza-tion with BPP5a–KLH. 0 40 80 120051015 Days    H         I        I        I   -  a  n   t   i  -   B   P   P   5  a   T   i   t  e  r   (   l  o  g         2    )  178 A natural carrier effect and antipeptide antibody generation / L.R. Tsuruta et al. / Anal. Biochem. 353 (2006) 174–180 immunization protocol, although the same antigen dosewas given under both conditions. Discussion The present work demonstrates that speci W c antibodiesto biologically active peptides can be successfully generatedeven by immunization with the peptides in the free form.Although the anti-BPP titers obtained by immunizationwith the peptides in their free form were lower than thoseobtained with the peptides conjugated to the carrier pro-tein, satisfactory antibody levels were generated in H III mice immunized with either peptide: BPP5a, pBPP5a, orpBPP10c (Fig.2). It is well known that the pyroglutamicacid residue at the N terminus of peptides and proteins pro-tects against degradation by N  -peptidase/proteases [34].Our results revealed similar antibody titers against bothpentapeptides (BPP5a and pBPP5a) (Fig.2A), suggestingthat anti-BPP5a antibodies were produced for both forms.It is noteworthy that determination of the antibody titer byenzyme-linked immunosorbent assay is speci W cally relatedto recognition of the C terminus of these BPPs, because thepeptides were linked to the BSA by their N termini.Lerner etal. [2] reported that production of antipeptideantibodies against hepatitis B virus was successful when thepeptides contained one or more proline residues, as theseresidues are known to play a crucial role in antigenic speci- W city [35,36]. Considering that the BPPs are proline-rich oli-gopeptides, invariably containing functionally importantproline residues at the C terminus [37–39], their structuremay have signi W cantly contributed to the antipeptideresponses.The speci W city of the antipeptide antibodies was evi-denced by Western blot analysis of the cytosol from the B. jararaca  venom gland (Fig.3). The antipeptide antibodyspeci W cally recognized a single band of about 27kDa,which corresponds to the BPP precursor protein present inthe venom gland [25].Minimizing the production of antibodies against carrierproteins is always of great interest because it allows theproduction of more speci W c anti-hapten antibodies [40–44].However, in these studies, structural modi W cations wereintroduced into the srcinal molecule. It is desirable thate Y cient antibody production against peptides is achievedfollowing minimal modi W cation of antigens. It may, how-ever, be impossible to state which immunization approachis most likely to succeed in production of the desired anti-bodies because success depends on the peptide sequence.The peptides are randomly folded and rarely occupy theirnative conformation, as within the srcinal protein; thus,they are not likely to elicit antibodies against a conforma-tion that was not maintained. However, when the peptide islinked to a carrier protein, there is a considerable loss of randomness (entropy), which may reduce the ability of thegenerated antibodies to recognize the peptide sequencewithin the proteins displaying its speci W c conformation. Alack of correlation has been observed between antipeptideand cross-reactive antiprotein responses, and it is indepen-dent of whether the peptide is injected as a conjugate or inits free form [45–47]. Generation of antibodies against pep-tides depends on the conformational stability and integrityof the immunogen and on the molecular form used for theassay, that is, free, polymerized, or carrier-bound [48]. It isalso known that antipeptide antibodies tend to reactextremely well with the denatured form of the correspond-ing protein. Thus, to analyze the cross reaction of antipep-tide antibodies with peptide and proteins, it is preferable totest the cross reaction in a variety of assay formats [49]. Inthe present work, generated anti-BPP antibodies will serveas tool for the identi W cation of endogenous correlate andmight be able to recognize various conformations of pep-tides.Analysis of the carrier function can contribute to severalcharacteristics of the hapten-speci W c immune response. Itwas veri W ed that in the case of BPPs, peptides conjugated tothe carrier protein elicited a markedly higher hapten-spe-ci W c secondary response in H III  mice when these animalswere previously immunized with carrier protein. However,several investigators failed to obtain evidence of thisincreased primary response to haptens after carrier proteinpreimmunization [17–23]. It is also known that the phe-nomenon of carrier-induced epitopic suppression is depen-dent on the dose of the carrier protein. In the present work,we attempted to use a lower dose of antigens to optimizethe immunization protocol, and thus, epitopic suppressionwas prevented. Furthermore, it was observed that anti-BPPs were generated in a dose-dependent manner (Figs. 1and 4). The results indicate that a suitable antibody titercan be obtained even using low antigen doses.Immunization with free BPP or BPP conjugated to car-rier (Figs. 2 and 4, respectively) produced similar antibodytiters. It is relevant to consider that di V  erent amounts of peptides were used for the various immunization schedules.For antibodies obtained with free BPP, the dose adminis-tered corresponded to the total amount of peptides,whereas in the immunization with BPP–KLH, the totalprotein concentration was considered for the whole conju-gate. We might conclude that the amount of peptides forthe immunization with free peptides is higher than in thesecond case; which means that high antibody titers wereobtained using lower antigen doses for the immunizationwith BPP–KLH.The immunoglobulin class of anti-hapten antibodies ischaracteristic of the mode and time of immunization with ae Comparison of IgG 1  and IgG 2a  levels of anti-BPP5a from KLH-carrier-primed mice and unprimed mice after immunization with BPP5a–KLH a Days after immunization with BPP5a–DVS–KLH.Days a Antibody titer (log 2 )Carrier primedUnprimedIgG 1 IgG 2a IgG 1 IgG 2a 1314.8 § 1.012.6 § 1.110.3 § 2.94.1 § 1.37115.3 § 1.314.0 § 1.810.6 § 1.13.8 § 1.0
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