EGFRvIII/PEP-3 cDNA与HBcAg重组基因原核表达载体的构建、表达与免疫分析

目的:构建EGFRvIII与HBcAg重组基因的原核表达质粒,进行原核表达、纯化,观察表达蛋白的免疫原性和抗原性。方法:通过双酶切从pGEM-TEasy/PEP-3载体获得编码EGFRvIII突变区的cDNA片段,插入去除了主要免疫优势区的重组质粒pGEMEX/HBcAg中,获得重组质粒pGEMEX/c-PEP-3-c,将重组基因亚克隆于原核表达载体pET28a( )中,通过IPTG诱导蛋白表达,利用Ni2 -NTA亲和层析法对融合蛋白进行纯化;用融合蛋白常规免疫BALB/c小鼠,用间接ELISA检测小鼠血清中抗体的滴度。结果:经酶切鉴定、序列测定证实融合基因已插入pET28a( )载体中,融合基因序列与设计序列完全一致。原核表达后表达量占沉淀全菌蛋白的56%,纯化产物占总蛋白的92%,浓度约8g/L。BALB/c小鼠经4次免疫后,其血清中中和抗体的滴度可达1∶16000,第6次免疫后血清中中和抗体的滴度达到1∶2.56×105,抗PEP-3抗体滴度达到1∶1.28×105,抗HBcAg抗体滴度小于1∶4×103。结论:融合基因PEP-3-HBcAg在大肠杆菌中可获得高效表达,表达的融合蛋白可以诱导小鼠产生高滴度和高特异性中和抗体,从而为高表达EGFRvIII恶性肿瘤基因工程疫苗的研究奠定基础。     

恙虫病东方体Sxh951株56kD蛋白的表达及其在ELISA中的应用

目的　构建含OrientiatsutsugamushiSxh95 1株 (Ot.Sxh95 1)相对分子质量 (Mr)为 5 6×10 3外膜蛋白基因 (sxh5 6 )的重组质粒pQE30 5 6 ,表达Mr5 6× 10 3蛋白并观察其在ELISA中的应用。方法　IPTG诱导sxh5 6重组质粒 ;观察SDS PAGE和免疫印迹结果 ;经Ni NTA亲和层析纯化后的重组蛋白分别与Ot.Gilliam、Ot.Karp、Ot.Kato感染鼠血清进行ELISA和免疫印迹检测。结果　SDS PAGE和免疫印迹检测显示有一Mr 约为 5 6× 10 3的特异蛋白带 ;ELISA和免疫印迹结果显示该重组蛋白只与Ot.Gilliam感染鼠血清呈阳性反应 ;重组蛋白用作ELISA包被抗原检测小鼠血清抗体IgG ,特异性为 10 0 % ,敏感性 96 .6 7% ;检测人血清抗体IgG的敏感性为 88.0 8% ,特异性 96 .36 %。结论　表达的重组Sxh5 6蛋白具有良好的免疫反应性 ;其作为ELISA包被抗原 ,具有良好的特异性和敏感性。     

人前蛋白转化酶枯草溶菌素9的cDNA克隆和表达及多克隆抗体的制备

目的 构建人前蛋白转化酶枯草溶菌素9(pcsk9)的原核表达载体并诱导其表达,制备其蛋白的多克隆抗体.方法 聚合酶链式反应扩增人工合成的pcsk9 cDNA序列;扩增产物经TA克隆、双酶切、连接,构建pET-28b-pcsk9表达载体;将重组质粒转化人大肠杆菌表达菌BL21(DE3)的感受态细胞中,诱导表达;用纯化检测过的目的蛋白免疫新西兰兔,最后采集全血收集血清,获得多克隆抗体,并对该抗体进行免疫印迹及效价测定.结果 PET-28b-pcsk9原核表达载体构建成功,用诱导表达的目的蛋白免疫动物,收集血清获得多克隆抗体.对多克隆抗体进行纯化,免疫印迹检测结果证明自制多克隆抗体特异性良好,效价测定为1∶13 200.结论 成功构建了pcsk9原核表达载体pET-28b-pcsk9,且获得了其蛋白的多克隆抗体.     

大鼠细胞因子信号转导抑制因子-1基因的原核克隆表达和免疫学分析

目的 克隆表达大鼠细胞因子信号转导抑制因子-1基因(SOCS-1).方法 将大鼠SOCS-1全长编码基因的PCR产物,克隆到原核表达质粒pET-28a(+)中,构建重组质粒pET-28a(+)-ratSOCS-1.转化大肠埃希菌BL-21/DE3,用异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达,应用镍离子金属螯合剂亲和层析柱从表达产物中纯化重组蛋白,通过十二烷基磺酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)对表达产物和重组蛋白进行鉴定.应用纯化的重组蛋白免疫新西兰大白兔,抗体滴度达到1:10000以上后进行末次免疫,2周后心脏取血、测定滴度,分离制备免疫血清.用红细胞裂解法制备大鼠外周血白细胞裂解全蛋白.将阴性血清(未用重组蛋白免疫的新西兰大白兔的血清)、免疫血清及兔抗组氨酸标签抗体转入各蛋白条带,用Western免疫印迹检测重组蛋白的免疫学活性.结果 PCR、双酶切及DNA测序分析均表明重组质粒pET-28a(+)-ratSOCS-1构建成功.SDS-PAGE结果可见转化了重组质粒的大肠埃希菌全菌和经超声裂解后的菌体沉淀的样品均在相对分子质量约26 000处有一明显的蛋白条带,经亲和层析获得的纯化重组蛋白有特异的单一条带与上述条带一致,而在超声裂解后的菌体上清中相同位置却未见有蛋白表达条带.Western免疫印迹表明重组蛋白、大鼠外周血白细胞裂解蛋白中相对分子质量约24000的蛋白条带、兔抗组氨酸标签抗体均可被免疫血清识别,分别出现清晰的相对分子质量26000、24 000、26000的反应条带,表明其具有免疫活性.结论 SOCS-1基因可以在大肠埃希菌BL-21/DE3中获得表达,其纯化重组蛋白具有良好的抗原性和免疫活性.     

人内质网分子伴侣BiP抗原表位筛选及其在RA血清学诊断中的作用研究

目的:筛选鉴定人内质网分子伴侣BiP抗原表位多肽,探讨其在类风湿性关节炎(Rheumatoid arthritis,RA)血清学诊断中的价值.方法:运用计算机软件分析BiP蛋白的结构特点及抗原决定簇分布,根据结果设计合成系列多肽.比较合成多肽与RA患者血清(BiP抗体阳性)IgG的反应强弱,鉴定抗原抗体反应最强的抗原表位多肽.进一步以获得的BiP抗原表位多肽作为包被抗原,检测79例RA患者、34例系统性红斑狼疮(SLE)患者、66例干燥综合征(SS)患者和173例正常人血清中抗BiP抗原表位多肽IgG抗体水平.结果:筛选到一条与RA患者血清反应最强的多肽(N403),抗N403多肽IgG抗体在RA患者中的阳性率(67%),明显高于SS患者(29%)、SLE患者(0%)、和正常人(0%)(P＜0.01).抗N403多肽IgG抗体在RA血清学诊断中的敏感性为67%,特异性为93%.而且该抗体的检测对于RF、CCP、HRF、RA33、AKA、APF等抗体阴性的RA患者的诊断具有重要意义.结论:筛选获得的BiP抗原表位多肽N403,其检测抗体在RA临床血清诊断中具有很好的应用价值.     

梅毒螺旋体TP0772蛋白的原核表达和免疫反应性鉴定

目的 利用原核基因工程技术克隆表达梅毒螺旋体TP0772基因,探讨其在梅毒血清学诊断中的应用.方法 PCR扩增获得TP0772基因,构建pET-28b-TP0772重组质粒,转化到大肠杆菌BL21中,以IPTG诱导蛋白质表达,经镍柱纯化后通过质谱技术鉴定.采用免疫印迹法测定其与梅毒患者血清的免疫反应性.建立基于重组TP0772抗原的ELISA间接法并对30份TPPA阳性血清和25份TPP阴性血清进行方法学评价.结果 PCR扩增获得约850 bp的基因片段,成功构建原核表达载体pET-28b-TP0772.目的蛋白分子量约为32kDa,以包涵体的表达形式存在,约占菌体总蛋白的30%.经质谱技术和免疫印迹分析证实重组蛋白为TP0772蛋白,并能够与梅毒患者血清发生特异性结合反应.ELISA测定TPPA阳性血清和阴性血清的符合率分别为93%（28/30）和96%（24/25）.结论 通过DNA重组技术成功获得了重组TP0772蛋白,其与梅毒阳性血清具有良好的免疫反应性,为优化梅毒的血清学诊断方法奠定基础.     

新疆出血热病毒核蛋白多克隆抗体的制备及其免疫学评价

目的 原核细胞中表达、纯化新疆出血热病毒BA88166毒株核蛋白(NP)并制备及鉴定抗NP蛋白的多克隆抗体.方法 采用逆转录-聚合酶链式反应(RT-PCR)扩增出BA88166毒株S基因的cDNA片段,将其构建到原核表达载体pET-32a上,形成重组原核表达质粒pET-88166S.构建好的质粒在大肠杆菌BL21(DE3)中进行诱导表达,经镍柱亲和层析法纯化NP-His融合蛋白,SDS-PAGE分析蛋白相对分子质量(Mr).用该纯化蛋白免疫新西兰白兔制备抗血清,ELISA和Western blot法检测血清效价和特异性.结果 双酶切鉴定和DNA测序证实构建的pET-88166S重组表达载体正确,目的基因序列与GenBank中公布的序列一致,在E.coli BL21中表达的NP-His融合蛋白经SDS-PAGE分析,其Mr约为66000.ELISA检测抗体效价高于1:25600,蛋白免疫印迹实验结果表明抗体能特异性识别新疆出血热病毒YL04057毒株的NP蛋白及其截短蛋白.结论 成功获得新疆出血热病毒NP-His融合蛋白,得到了特异性兔抗NP蛋白多克隆抗体.     

重组人BMP-2的基因克隆及原核表达

目的 构建重组人骨形态发生蛋白2(recombinant human bone morphogenetic protein-2,rhBMP-2)的原核表达质粒并在大肠埃希菌中诱导表达.方法 采用RT-PCR法,从骨髓细胞总RNA中扩增获得人BMP-2成熟肽cDNA,将其克隆入表达载体pBV220,构建BMP-2的重组原核表达质粒.重组质粒经酶切和测序鉴定后,在大肠埃希菌中诱导表达目的蛋白,表达产物采用Western印迹和ELISA进行鉴定.结果 测序表明,重组基因序列与人BMP-2成熟肽基因完全一致.Western印迹和ELISA检测显示,表达产物的相对分子量(Mr)与预期结果相符,与相应抗体有结合活性.结论 获得了人BMP-2成熟肽的编码基因,并构建了含有该基因的重组质粒pBV220/BMP-2,在大肠埃希菌中可高效表达人BMP-2.     

小鼠精子Sp17与IL-5融合蛋白的构建、表达及其免疫原性鉴定

目的 构建和表达小鼠精子蛋白Sp17与白介素5(IL-5)融合蛋白,并对其进行纯化和免疫原性鉴定.方法 采用PCR技术从质粒pGEM-1-IL-5中扩增IL-5基因片段,将其克隆至pET/Sp17原核表达载体中与Sp17基因融合,构建重组质粒pET/Sp17-IL-5,经酶切鉴定后转化大肠杆菌B121(DE3),IPTG诱导表达,采用Ni2+-NTA Agarose 纯化,SDS-PAGE检测、N端测序及Western blot;重组蛋白Sp17.IL-5免疫小鼠后ELISA检测小鼠血清特异性抗体.结果 成功构建小鼠精子蛋白Sp17与IL-5融合蛋白的高效表达质粒pET/Sp17-IL-5,重组工程菌pET-28a(+)-sp17-IL-5/BL21经IPTG诱导目的蛋白表达率约28%,PAGE初步测定目的蛋白相对分子量(Mr)约39 000,纯化后蛋白纯度达91%,免疫后小鼠血清中检测到特异性抗体.结论 Sp17-IL-5蛋白经基因克隆获得了较高的表达量,并初步显示了较好的免疫活性,为新型Sp17避孕疫苗的研制奠定基础.     

弓形虫ROP2基因的克隆及在大肠杆菌中的表达

目的 :构建弓形虫棒状体蛋白 (ROP2 )基因重组质粒并在大肠杆菌中表达 ,用于筛选弓形虫新的诊断抗原和疫苗分子。方法 :根据ROP2基因已知序列 ,设计合成一对引物 ,用PCR方法从弓形虫RH株基因组DNA中扩增出ROP2基因片段 ,再克隆到pGEX 4T 1质粒 ,重组质粒经酶切及PCR鉴定 ,而后进行测序 ;重组质粒在大肠杆菌BL2 1 (DE3)中进行ITPG诱导表达 ,表达产物经SDS PAGE及WesternBlot分析。结果 :ROP2基因PCR产物大小与预期相符 ,约 1 0 4 3bp ,重组质粒经酶切及PCR鉴定表明获得正确重组子 ,测序结果与已知序列基本吻合 ;SDS PAGE及免疫印迹显示ROP2融合蛋白表观分子量约为 5 5kDa ,表达产物约占菌体总蛋白1 7% ,具有一定的免疫反应性。结论 :克隆并表达了弓形虫ROP2基因 ,为下一步弓形虫诊断及疫苗研究奠定了基础     

Autoantigen BiP and autoimmune diseases

Immunoglobulin Binding Protein (BiP) is a member of heat shock protein 70 famaily, and is also known as an autoantigen in rheumatoid arthritis (RA) patients. Serum anti-BiP antibody is detected up to 60% of RA patients, and recent reports demonstrated that serum anti-BiP antibody is also detected in systemic lupus erythematosus patients. Notably, anti-citrullinated BiP antibody is revealed as another member of anti-citrullinated protein/peptide antibodies (ACPAs). Since ACPAs are supposed to be closely associated with RA pathogenesis, immune responses to citrullinated BiP could play an important role in RA. Indeed, immunization of citrullinated BiP exacerbated inflammatory arthritis in mice. Moreover, T cell responses to BiP were reported in human RA and mice models. In mice models, native BiP administration induced IL-4 and IL-10 producing CD4(+) T cells and regulated inflammatory arthritis. In this way, immune responses to BiP are various, and dysregulation of the balances between pro-inflammatory and regulatory responses to BiP could lead to the autoimmune responses and diseases.     

Antibodies to extractable nuclear antigens in rheumatoid arthritis: relationship to vasculitis and circulating immune complexes

Antibodies to extractable nuclear antigens (ENA) were analysed in the sera of fifty-two patients with severe rheumatoid arthritis (RA) who were divided into two categories: twenty-five with arthritis only and twenty-seven with extra-articular disease. Using haemagglutination, counterimmunoelectropheresis (CIE) and double diffusion, antibodies to ENA were detected in three (12%) of the patients with arthritis only. In the group with extra-articular disease, antibodies were found in sixteen (59%) of the patients. In ten patients the antibodies reacted with an RNase and DNAse resistant, but trypsin sensitive protein component of ENA. These patients all had extraarticular disease with digital vasculitis being a particularly common feature. Their sera also contained circulating immune complexes detected by elevated cryoprecipitate protein levels associated with relatively low complement levels. It is suggested that antibodies to soluble proteins of nuclear origin may be markers of circulating immune complexes in extra-articular RA.     

Detection of autoantibodies in a quantitative immunoassay using recombinant ribonucleoprotein antigens.

A human cDNA expression library was screened with anti-ribonucleoprotein (RNP) antibodies from patients with connective tissue diseases. Three cDNA clones were isolated encoding 70 kD, A and B" ribonucleoprotein autoantigens which were expressed as beta-galactosidase fusion proteins. Antigens were purified and used to develop sensitive ELISAs suitable for the routine screening of large series of sera from patients with connective tissue diseases. More than 400 sera were tested both by ELISA and by immunoblotting. The ELISA was found to be at least as sensitive as immunoblotting and very specific. Anti-70 kD antibodies were found in 94% of patients with mixed connective tissue disease (MCTD), in 4% of patients with other connective tissue diseases but not in normal controls. Furthermore, the use of recombinant 70 kD antigen enabled us to discriminate between anti-70 kD antibodies present in anti-Sm and in anti-(U1) RNP sera. Recombinant A antigen contained at least two autoantibody-reactive sites; one unique for the A protein and another cross-reactive with anti-B" antibodies. Antibodies reactive with the unique site were found in 83% of MCTD patients, in 4% of patients with other connective tissue diseases and not in normal controls. Antibodies against the cross-reactive B" epitope present on A and B" recombinant antigens, were found in high titres in a small percentage of patients with systemic lupus erythematosus (SLE, 5%) and rheumatoid arthritis (RA, 2%).     

Autoantibodies to DNA topoisomerase II in juvenile rheumatoid arthritis.

Sera from 58 children with juvenile rheumatoid arthritis were examined for the presence of antibodies to DNA topoisomerase II. Eight sera were reactive in immunoblotting with purified human topoisomerase II and a protein encoded by a cloned cDNA expressed in Escherichia coli which represents the carboxy-terminal domain of the human enzyme. In addition, the sera detect topoisomerase II in mitotic chromosomes and chromosome scaffolds. Five of the sera bind to the native enzyme in solution and deplete such solutions of the active enzyme. All eight sera also contain antibodies to nuclear antigens other than topoisomerase II.     

Antibodies from patients with rheumatoid arthritis and juvenile chronic arthritis analyzed with core histone synthetic peptides

Antihistone antibodies were detected in the sera of a randomly selected group of patients with rheumatoid arthritis (RA) and juvenile chronic arthritis (JCA) by an enzyme-linked immunosorbent assay (ELISA) with the five individual histones and by immunoblotting. In ELISA, the overall frequency of antihistone antibodies in RA and JCA was 51 and 44%, respectively. Antibodies present in these serum samples were further studied by ELISA by means of 17 core histone synthetic peptides. The fragment 1-21 of H3 was recognized by 60% of RA sera and by 62% of JCA sera. In addition, at least four terminal or internal peptides of H3 and H4 were recognized by more than a third of JCA sera, while only two of these peptides reacted with 20% of RA sera. Many of the sera that did not show any reactivity with the whole histone reacted with various histone peptides. This finding demonstrates the usefulness of synthetic peptides for identifying autoantibodies.     

The Major Rheumatoid Factor Cross-Reactive Idiotype in Rheumatic Disease

The major rheumatoid factor cross-reactive idiotype (RCRI), a tertiary structure formed by both light and heavy chains, is found on 60% of all monoclonal IgM kappa RFs. To determine if the RCRI is expressed in patients with rheumatic disease, we used polyclonal rabbit anti-idiotypic antibodies to detect RCRI in sera and in pokeweed mitogen cultures of blood mononuclear cells (PBM) from patients with rheumatoid arthritis (RA) and juvenile rheumatoid arthritis (JRA). We detected increased expression of RCRI + plasma cells in PWM cultures, and in sera from these patients. We have determined that some 7S IgM molecules from RF+ RA patients are RCRI +, and can bind IgG in a sensitive RF ELISA. We have also observed that the CD5+ B cell subset, which is responsible for autoantibody production, generates RCRI+ antibodies. We review these data and discuss the relationship of the idiotypic network of interacting antibodies with rheumatic disease.     

Autoantibodies directed against the amino-terminal domain I of human calpastatin (ACAST-DI Ab) in connective tissue diseases. High levels of ACAST-DI Ab are associated with vasculitis in lupus

To identify new autoantibody populations in patients with rheumatic diseases, a cDNA expression library was immunoscreened with a rheumatoid arthritis (RA) patient's serum which contains autoantibodies binding to uncharacterized polypeptides by Western-blotting. One clone encoding the amino-terminal region (Nt) [domain L and half of domain I] of human calpastatin was selected. Different fragments of the selected cDNA were prepared and the corresponding recombinant polypeptides were produced by in vitro translation and analysed by Western blotting. Most RA sera bound to recombinant amino-terminal region and domain I but not to domain L. This prompted us to use a recombinant polypeptide corresponding to the domain I of calpastatin as the antigen in a solid-phase ELISA to test sera from patients with various systemic rheumatic diseases and healthy controls.Anti-calpastatin domain I antibodies (ACAST-DI Ab), were detected by ELISA in RA, systemic lupus erythematosus (SLE), Sj?gren's syndrome and control sera at respective frequencies of 10, 9, 0 and 1%. These Ab did not have prognostic value in early RA; high levels were significantly associated with vasculitis in SLE. Antibodies reacting with the calpastatin amino-terminal region are produced during systemic rheumatic diseases and are predominantly directed against domain I. High levels of these Ab may constitute a marker of vasculitis in SLE.     

Production of the islet cell antigen ICA69 (p69) with baculovirus expression system: analysis with a solid-phase time-resolved fluorescence method of sera from patients with IDDM and rheumatoid arthritis.

Islet cell antigen 69 (ICA69), previously implicated as an autoantigen in autoimmune insulin-dependent diabetes mellitus (IDDM), was produced using baculovirus-mediated expression in Spodopterafrugiperda (Sf9) insect cells. In these cells the protein was effectively expressed and ICA69 carrying C-terminal histidine-hexapeptide could be efficiently purified using immobilized metal chelate affinity chromatography. Screening of patient and control sera using this protein as an antigen in time-resolved fluoroimmunoassay (TR-FIA) identified 4/50 of patients with IDDM and 6/73 of patients with rheumatoid arthritis (RA) to be positive for ICA69 antibodies. The number of positives did not differ significantly between patients and control subjects but the level of binding was higher in sera from RA patients compared to that of control sera (P = 0.003). The results show that some subjects have specific autoreactive antibodies against the ICA69 protein produced with recombinant technology.     

Antibodies against macaque monoclonal immunoglobulin G in rheumatoid arthritis

The presence of rheumatoid factors (RF) in the serum of rheumatoid arthritis (RA) patients is commonly evidenced by agglutination tests: the Waaler-Rose assay, based on the use of human red blood cells (RBCs) coated with rabbit anti-RBC antibodies, and the latex test, which uses latex particles coated with denatured human immunoglobulin G (IgG). The aim of the present study was to characterize the RF able to agglutinate human RBCs coated with macaque antihuman RBC IgG antibodies secreted from macaque-mouse heterohybridomas (two from rhesus monkey and one from crab-eating macaque). Human RBCs coated with macaque monoclonal antibodies (MacMoAbs) were used for agglutination tests and these were carried out in parallel with standard tests (Waaler-Rose and latex agglutination tests) on sera from 82 RA patients, 86 patients with other forms of inflammatory chronic arthritis and 47 healthy human subjects. MacMoAb-coated RBCs identified RF in the sera of 66% patients with RA. By contrast, the frequency of positive sera in other inflammatory diseases was 5% and all 47 healthy controls were negative. Antimacaque IgG antibodies were found to be more specific for RF than standard tests, in the sera of patients with RA.     

Antibodies to a new viral citrullinated peptide, VCP2: fine specificity and correlation with anti-cyclic citrullinated peptide (CCP) and anti-VCP1 antibodies

Anti-citrullinated protein/peptide antibodies (ACPA) are a hallmark of rheumatoid arthritis (RA) and can be measured using different citrullinated substrates. In this paper we describe a new viral citrullinated peptide - VCP2 - derived from the Epstein-Barr virus-encoded protein EBNA-2 and analyse its potential as substrate for ACPA detection. Analysing sera from 100 RA patients and 306 controls, anti-VCP2 immunoglobulin (Ig)G were found in 66% of RA sera, IgM in 46% and IgA in 39%, compared with less than 3% of control sera. Anti-VCP2 IgG was associated with erosive arthritis, the presence of rheumatoid factor and anti-VCP1 and anti-cyclic citrullinated peptide (CCP) antibodies. Anti-VCP2 antibodies were detected in 1% and anti-VCP1 antibodies in 4% of CCP-negative RA sera; conversely, 3% of the VCP-negative sera were CCP-positive. Taken together, these data suggest that VCP2 could offer a valuable tool for ACPA detection. Inhibition assays showed that two non-overlapping epitopes - a citrulline-glycine stretch shared between VCP1 and VCP2 and the N-terminal portion of the VCP2 sequence - were targeted by anti-VCP2 antibodies. Moreover, in some RA sera that tested positive in CCP and VCP2 assays, preincubation with VCP2 inhibited binding to CCP, whereas in other sera the binding was unaffected. Thus, the reactivity with more than one ACPA substrate might be due in some RA patients to antibody populations with different specificities, and in others to cross-reactive antibody populations. Finally, affinity-purified anti-VCP2 antibodies immunoprecipitated deiminated Epstein-Barr virus nuclear antigen (EBNA-2) from an EBNA-2-transfected cell line, suggesting that viral sequences may be involved in the generation of the ACPA response.