Preparation and characterization of a new monoclonal antibody against CXCR4 using lentivirus vector

CXCR4 is a member of chemokine receptors and plays a vital role in numerous diseases and cancer processes, which makes the CXCR4/CXCL12 chemotactic axis a potential therapeutic target. In this study, we used lentiviral vectors as a novel technology to produce a monoclonal antibody against CXCR4. Lentivirus vector pLV-CXCR4-Puro was successfully constructed and a hybridoma cell line 1A4 was generated. The CXCR4 monoclonal antibody (MAb) 1A4 had high titer and affinity, and the isotype was identified as IgG1a. The recombinant lentivirus vector could effectively stimulate the production of 39 kDa CXCR4 antibody in vivo after immunization. Western blot analysis showed that the MAb could recognize the CXCR4 antigen expressed on transfected 293T cells as well as various human cancer cell lines. Immunofluorescence assays showed that MAb 1A4 mainly localized and strongly stained on the membrane of transfected 293T cells. Immunohistochemistry assays demonstrated that 1A4 could recognize strong expression of CXCR4 on the hepatocellular carcinoma (HCC). Thus, the method using lentiviral vectors may have application on effective and large-scale production of the CXCR4 monoclonal antibody, which will be a potential tool for the diagnosis and treatment of human cancers.     

Local and systemic antibody response to rotavirus WC3 vaccine in adult volunteers

An evaluation of the safety and immunogenicity of WC3 rotavirus vaccine was evaluated in adult volunteers. Pre- and post-vaccination titers of neutralizing antibody to WC3 and to the four human rotavirus serotypes as well as serum and stool rotavirus IgA levels were measured. Vaccination was safe and did not induce elevation of liver enzymes. None of the 12 volunteers receiving WC3 vaccine shed detectable amounts of virus although antibody rises were detected in 11 of 12 vaccines. Nine developed and increase in WC3 neutralizing antibody, one additional subject had a rise in Wa (human serotype 1) neutralizing antibody while another subject only developed a rise in stool rotavirus IgA. All of the vaccine recipients with a rise in WC3 neutralizing antibody also developed a rise in neutralizing antibody against at least one of the four most common human rotavirus serotypes. A stool IgA rotavirus antibody response was detected in 6 of 9 WC3 recipients with measurable stool antibody. None of the control subjects developed significant rises in any of the antibody titers measured. WC3 rotavirus vaccine appears to be safe and induces systemic and local immune responses in adults suggesting that further evaluation of WC3 should be considered in infants.     

Induction of protective and therapeutic anti-cancer immunity by using bispecific anti-idiotype antibody G22-I50 for nasopharyngeal carcinoma

Increasing evidence has suggested that bispecific and multivalent antibodies which have more antigen binding sites will improve their immunogenicity. The bispecific anti-idiotype antibody vaccine G22-I50 was obtained through genetic engineering to enhance the immunogenicity of anti-idiotype antibody vaccines G22 and I50. G22-I50 vaccination could induce anti-tumor immunity in the Balb/c mouse model. The protective and therapeutic efficacy of G22-I50 was also evaluated using the hu-PBL–SCID mouse model injected three times with G22-I50, G22, or I50 mixed with Freund's adjuvant. Results demonstrated that the protective anti-tumor effect of G22-I50 could be relevant with the production of Ab3 antibody and activation of CD8⁺ cytotoxic T-lymphocytes. In preventive and therapeutic experiments, G22-I50 could reduce tumor size and prolong the survival time of HNE2-bearing mice (p < 0.05). Human CD8⁺ T lymphocytes infiltrated the tumor sites, and high levels of human IFN-γ, TNF-α, and caspase-3 were also detected in the tumors from G22-I50-vaccinated and -treated mice. Therefore, the bispecific anti-idiotype antibody vaccine G22-I50 can induce strong humoral and cell-mediated immune responses. This vaccine can be potentially applied to prevent and treat nasopharyngeal carcinoma.     

假病毒在中和抗体检测中的应用研究进展

目前成功的病毒性疫苗大多通过诱导广谱有效的中和抗体来保护机体免受感染,因此检测血清中和抗体水平对疫苗的免疫效果评价具有重要意义.中和抗体检测的传统方法包括微量细胞病变效应抑制法、蚀斑减少试验等,均需进行活病毒操作.近年来,随着逆转录病毒载体和重组病毒表达技术的发展,假病毒得到越来越多的研究,并被广泛应用在新型疫苗研发、中和抗原表位鉴定、细胞嗜性研究、抗病毒药物筛选、中和抗体检测、基因治疗等方面.此文主要对假病毒在中和抗体检测中的应用进行综述,并总结相关领域的研究进展.     

Hela抗原和Ehrlich抗原检测抗RA33、RA36抗体的比较研究

目的 探讨Ehrlich抗原能否取代Hela抗原检测抗RA33、36抗体，为临床提供简便、可靠的检测手段。方法 分别以Hela细胞核提取物和Ehrlich腹水瘤细胞核提取物为抗原，用免疫印迹法，对281例类风湿关节炎（RA）、89例其他风湿病以及50名正常人血清进行检测。结果 两种抗原均可以检出抗RA33、抗RA36抗体，但检测结果一致率只有65．5％。抗RA33和抗RA36抗体不仅见于RA，也见于系统性红斑狼疮等其他风湿病。281例RA中，Hela抗原和Ehrlich抗原检测结果对RA诊断的敏感性和特异性分别为34．8％和83．5％对44．1％和71．9％。两者联合，敏感性达52．3％，特异性70．5％。两种抗原检测的阳性结果一致者，其病情活动性和预后不良的指标多数高于结果均阴性者。结论 抗RA33抗体和抗RA36抗体都不是RA的标志性抗体。Hela抗原和Ehrlich抗原不能互相代替。同时使用两种抗原进行抗RA33／36抗体的检测能提高对RA诊断的敏感性，并有助于RA病情活动性的判断和预后估计。     

抗前列腺干细胞抗原单克隆抗体的制备及初步鉴定

目的　制备抗前列腺干细胞抗原 (PSCA)单克隆抗体 (McAb) ,并对其特性进行鉴定。方法　应用PC 3细胞免疫BALB/c小鼠 ,按常规方法融合并经间接ELISA方法筛选 ,制备稳定的抗PSCAMcAb ,并对McAb的亚型、组织特异性进行鉴定。结果　制备出抗PSCA单克隆抗体 ,免疫扩散鉴定为IgG1亚类 ,ELISA法测定其腹水效价为 5× 1 0 -5。组织学检查与前列腺癌组织呈阳性反应 ,与其他肿瘤组织均呈阴性反应 ,和人体正常组织细胞无一出现阳性反应。结论　初步制备出一株抗PSCA的单克隆抗体细胞株 ,能特异性识别前列腺癌组织 ,对前列腺癌的诊断和治疗提供有用的工具。     

Successful treatment of collagen-induced arthritis in non-human primates by chimeric anti-osteopontin antibody

The presence of thrombin-cleaved form of osteopontin well correlated with various inflammatory disease activities in not only rodents, but also humans. We previously demonstrated that the blocking of the interaction of a cryptic epitope within osteopontin, which is exposed by thrombin cleavage, with its integrins by specific antibody recognizing cryptic epitope of mouse osteopontin, could significantly inhibits the development of arthritis in mice. We generated a murine monoclonal antibody, 2K1, specifically recognizing a cryptic epitope of human osteopontin, SVVYGLR. We constructed a chimeric antibody, C2K1 in which variable region of 2K1 was fused with human IgG1 constant region. In the present study, we investigated whether the therapeutic administration of C2K1 could ameliorate the established collagen-induced arthritis in cynomolgus monkey. Thus, C2K1 was injected after the onset of arthritis. The inhibition of joint swelling by C2K1 became evident at 4 to 5 weeks after initiation of arthritis, when blood level of C2K1 was peaked. Joint swelling reappeared along with the sharp decline of C2K1 blood levels at 6 weeks. Importantly, destruction of bone and cartilage in joints was still significantly prevented at 10 weeks when blood level of C2K1 was quite low if any and anti-C2K1 antibody emerged. These results demonstrate that neutralizing antibody against the cryptic epitope of osteopontin can be a future therapeutic choice for patients with rheumatoid arthritis.     

A novel peptide isolated from phage display peptides library recognized by an antibody against connective tissue growth factor (CTGF)

The aim of this study was to isolate a peptide binding to an antibody against CTGF C-terminal domain from the peptide library and to evaluate its immunological and biological activities. A phage display 12-mer peptide library was screened using anti-CTGF/C antibody as the target. Ten of the positive clones were sequenced after three rounds bio-panning. The DNA encoding peptide ZD521 was cloned and expressed as the fusion protein(TrxA-ZD521). The specificity of ZD521 to anti-CTGF/C antibody was determined by competitive inhibition assay. Mice were immunized with purified fusion protein(TrxA-ZD521) and the anti-peptide or anti-CTGF response of antiserum was also tested by enzyme-linked immunoabsorbent assay (ELISA) and Western blot. The inhibition effect of anti-serum on proliferation of kidney mesangial cells was evaluated by MTT. A peptide ZD521(GEPQTKLFSFPL) that could specifically recognize anti-CTGF/C antibody was isolated. No sequence homology was found between ZD521 and CTGF/C. The purified TrxA-ZD521 could specifically bind to anti-CTGF/C antibody and block the binding of anti-CTGF/C antibody to CTGF/C and native CTGF(mesangial cell lysate). Moreover, the antiserum from mice immunized with TrxA-ZD521 could also bind to CTGF/C recombinant protein and native CTGF, as well as significantly inhibit the proliferation of kidney mesangial cells induced by CTGF/C. Therefore, ZD521 might be a conformational epitope of CTGF which is potentially useful to be developed as a vaccine for prevention and treatment of fibrosis disorders.     

Chlamydia trachomatis in symptomatic and asymptomatic men: detection in urine by enzyme immunoassay

BACKGROUND: Infection with Chlamydia trachomatis is known to be a common cause of urethritis and cervicitis. The standard methods of detection require the collection of intra-urethral and/or cervical swabs, which may be submitted for culture, antigen detection or nucleic acid amplification. The collection of swabs is suitable only within the context of a health care facility. Recent reports have indicated that antigen detection can be used with urine specimens, and because these can be self-collected, this may be particularly useful for the detection of asymptomatic carriage. OBJECTIVE: To determine the sensitivity and specificity of urine antigen assays in the detection of chlamydial infection in men. SETTING: Two groups of men were investigated; men with urethritis attending clinics or private practitioners, and healthy adult men enrolled into either urban or rural HIV prevention projects. METHODS: Urine samples from men in both groups were collected and assayed for the presence of chlamydial antigen using a commercial enzyme immunoassay (EIA) kit. For symptomatic men an intra-urethral swab was also collected and assayed for antigen detection using a commercial EIA. For asymptomatic men, a ligase chain reaction was carried out on the same urine sample. RESULTS: The prevalence of chlamydial antigen in symptomatic men was 15% (39/257), and in asymptomatic men was 4% (15/349). The sensitivity and specificity of urine EIA for symptomatic men was 87% and 83% respectively. For asymptomatic men, the sensitivity of urine EIA was 86%, and the specificity was 100%. CONCLUSION: Urine EIA is a relatively inexpensive method for the detection of chlamydial infections in men. The true specificity in symptomatic men may be higher, as the "gold standard" that we used may give false negative results. Antigen EIA for examination of urine specimens from community surveys of asymptomatic men may be particularly useful because of the low cost of assays, and because urine samples can be self-collected without discomfort to study subjects. The prevalence of C. trachomatis that we describe here is consistent with other studies of chlamydial epidemiology in Zimbabwe.     

Topical delivery of plasmid DNA using biphasic lipid vesicles (Biphasix)

The development of non-invasive methods for the delivery of vaccines through the skin will greatly improve the safety and the administration of human and veterinary vaccines. In this study we examined the efficiency of topical delivery of plasmids by assessing the localization of gene expression using luciferase as a reporter gene and induction of immune responses using a plasmid encoding for the bovine herpesvirus type-1 glycoprotein D (pgD). Topical administration of plasmids in a lipid-based delivery system (biphasic lipid vesicles--Biphasix) resulted in gene expression in the lymph node, whereas with intradermal injection, antigen expression was found in the skin. Following administration of plasmid with the gene gun, antigen expression was observed in both the skin as well as in the draining lymph nodes. Transcutaneous immunization with pgD formulated in biphasic lipid vesicles elicited gD-specific antibody responses and a Th2-type cellular response. In contrast, immunization by the intradermal route resulted in the stimulation of a Th1-type response. These findings have implications for both vaccine design and tailoring of specific immune responses.     

Exploiting the plant secretory pathway to improve the anticancer activity of a plant-derived HPV16 E7 vaccine

The human papillomavirus 16 (HPV16) E7 oncoprotein can be considered a "tumor-specific antigen", and therefore it represents a promising target for a therapeutic vaccine against HPV-associated tumors. Efficient production of E7 protein with a plant-based transient expression system has been already described and it was demonstrated that E7-containing crude plant extracts confer partial protection against tumor challenge in a mouse model system. Before adopting the plant-based system as a cost-effective method for the production of an E7-based anti-cancer vaccine, some aspects, such as the oncoprotein yield, need further investigation. In the present study, we report the transient expression, mediated by a potato virus X (PVX)-derived vector, of the E7 protein targeted to the secretory system of Nicotiana benthamiana plants by using a plant-derived signal sequence. Targeting the antigen to the secretory pathway enhanced the E7 protein expression levels about five-fold. Mice immunized by s.c. administration with crude foliar extracts containing E7 showed strong stimulation of cell-mediated immune response after five boosters, as detected by ELISPOT. After challenging with the E7-expressing C3 tumor cells, tumor growth was completely inhibited in 80% of the vaccinated animals and a drastic reduction of tumor burden was observed in the remaining tumor-affected mice. These data demonstrate that, by enhancing E7 yield, it is possible to improve the anti-cancer activity of the plant-based experimental vaccine and open the way for a large-scale production of the E7 protein which could be purified or used as in planta formulation, also suitable for oral therapeutic vaccination.     

A combination of the TLR4 agonist CIA05 and alum promotes the immune responses to Bacillus anthracis protective antigen in mice

Anthrax is an infectious disease caused by Bacillus anthracis. The currently licensed human anthrax vaccines contain protective antigen (PA) as a major protective component and alum as an adjuvant. In this study, we investigated whether CIA05, a TLR4 agonist, is able to promote the immune response to an anthrax vaccine adjuvanted with alum. BALB/c mice were immunized intraperitoneally three times at 2-week intervals with a recombinant B. anthracis PA alone or in combination with CIA05 in the absence or presence of alum, and immune responses were determined 2 or 3 weeks after the third immunization. The results showed that the combination of CIA05 and alum significantly increased both serum anti-PA IgG antibody and toxin-neutralizing antibody titers, and the adjuvant effects were greater when lower antigen doses were used for immunization. Both CIA05 and alum stimulated PA-specific splenocyte secretion of interleukin (IL)-4, IL-5, and IL-6. A combination of the two yielded synergistic effects on IL-4 secretion, but CIA05 tended to repress IL-5 and IL-6 secretions induced by alum. Co-administration of CIA05 and alum also increased GL7 expression in B220(+)CD24(+) splenic cells, indicating the ability to activate B cells. These data suggest that CIA05, combined with alum, could be used to achieve higher immune responses to PA, leading to the development of an effective anthrax vaccine.     

RA33/36纯化抗原检测抗体在类风湿关节炎的应用

目的探讨RA33/36抗体对类风湿关节炎(RA)的临床价值。方法以纯化的RA33/36为抗原,用免疫印迹法检测RA498例、其他风湿性疾病患者514例、正常对照50名,比较RA33/36抗体在各组人群中的阳性率,并评价其与RA患者临床和实验室指标间的关系、早期诊断及联合诊断价值。结果RA33/36抗体在RA中的灵敏度为32.93%,特异性为88.52%,在RA早期病例中阳性率为32.03%,在72例类风湿因子(RF)阴性的RA患者中有21例阳性(29.17%)。RA33/36抗体与抗环瓜氨酸肽(CCP)抗体联合检测,特异度为98.46%。RA33/36抗体阳性组和阴性组的关节肿胀数、疼痛关节数、功能分级、X线分期及其他临床指标差异无统计学意义,RF、抗核周因子(APF)、抗角蛋白抗体(AKA)、抗Sa抗体及抗CCP抗体阳性率两组差异均无统计学意义。结论RA33/36抗体对RA有较好的灵敏度和特异度,且有早期诊断价值,并对RF有补充诊断价值,与抗CCP抗体联合诊断可提高诊断的特异性,可广泛应用于RA的临床诊断。     

瓜氨酸合成蛋白抗体、抗RA33抗体检测在类风湿关节炎诊断中的应用

目的 探讨瓜氨酸合成蛋白抗体（CPA）和抗RA33抗体检测在类风湿关节炎（RA）诊断中的意义。方法 用ELISA法分别检测94例RA和97例非RA风湿病患者血清中的CPA、抗RA33抗体和类风湿因子（RF）水平。结果 CPA、抗RA33抗体和RF对RA诊断的敏感性分别为73．4％、31．9％和72．3％;特异性分别为96．9％、75．3％和77．3％CPA与抗RA33抗体或RF联合可提高诊断的特异性;CPA和RF滴度呈正相关。结论 CPA对RA的诊断有良好的特异性和敏感性,较抗RA33抗体和RF为优,可作为RA诊断的血清学指标,与RF联合检测效果最佳。     

Serum IgG subclass antibody responses in children vaccinated with influenza virus antigens by live attenuated or inactivated vaccines

To ascertain whether live attenuated or inactivated vaccines can be considered equivalent, we examined the primary antibody response of children following vaccination with influenza virus antigens in three different formulations. Nine children received cold recombinant vaccine (CRV) containing A/Korea/82 (H3N2) and A/Dunedin/83 (H1N1) variants. Eight of these children responded to HA of the H3N2 subtype and the major portion of the elicited antibody was in the IgG1 subclass. Antibody of low titer in the IgG2 and IgG3 subclasses was detected in two and six serum specimens, respectively. Six of the nine children administered with CRV responded to the H1 antigen and only IgG1 antibody was detected. Serum specimens from eight children less than one year of age (5 less than 6 months of age) who had developed an antibody response to trivalent inactivated vaccine (TIV) vaccination were examined. High levels of IgG1 antibody to purified H3 were detected in all eight children. Low titers of antibody in IgG2 and IgG3 subclasses were detected in two and five children, respectively. Antibody responses to purified H1 showed a similar subclass distribution. In order to examine secondary response, eight children primed by immunization with TIV vaccine were subsequently given a single booster dose of purified hemagglutinin (HA) conjugated to diphtheria toxoid (HA-D). In 6/8 specimens antibody rises were detected to purified H3 and H1 antigens. Prior to the HA-D immunization, low levels of HA specific IgG1 antibody were detected in all serum specimens and vaccine induced responses were primarily of the IgG1 subclass.(ABSTRACT TRUNCATED AT 250 WORDS)     

Substances with affinity to a monoclonal aflatoxin B1 antibody in Danish urine samples

Using a competitive enzyme immunoassay, one or more substances recognized by a monoclonal antibody against aflatoxin B1 were detected in human urine samples collected in Denmark. The concentration of urinary aflatoxin-like substances was equivalent to 0.0-6.5 ng aflatoxin B1/mg creatinine. A truly competitive interaction in the immunoassay was found between aflatoxin-like substances and aflatoxin B1. Aflatoxin-like substances could be isolated in small quantities from urine by affinity chromatography. The quantity of urinary aflatoxin-like compounds in the samples collected showed a skewed normal distribution (80 individuals). In order to explain the seemingly high level of aflatoxin-like material in urine samples from people living in a cold temperate climate, the source of aflatoxin-like compounds was investigated. In a dietary restriction study, potential dietary factors leading to excretion of aflatoxin-like compounds were investigated. Our data indicate that the excretion of these compounds by healthy Danes depends mainly on the food ingested 24-48 hr before urine samples were collected. In particular, the excretion of aflatoxin-like substances was increased when diets include beer, dairy products or meat. A map of the epitope recognized by the antibody was constructed from the results of competition studies with several AFB1 analogues. The epitope map was used to draw chemical structures representing the minimal requirements for antibody recognition. An on-line search was conducted among the 98.2 x 10(6) structures in the Chemical Abstracts and Registry Databases (STN, Columbus, OH) and provided strong evidence that only aflatoxins or aflatoxin derivatives are recognized by the antibody. The possible chemical structures of the aflatoxin-like substances are discussed.     

单纯疱疹病毒-2型抗原gG-2在大肠埃希菌中的表达及纯化

目的用基因工程方法表达单纯疱疹病毒-2型(HSV-2型)血清型特异性抗原,代替传统的病毒检测抗原。方法采取PCR和蛋白质原核细胞表达方法。结果成功地克隆出了HSV-2型特异性基因,采用该抗原包被试剂盒,能检测出生殖器疱疹感染者血清中的IgM抗体。结论HSV-2型特异性抗原基因的成功表达,为HSV-2型感染者的特异性诊断和HSV-2型疫苗的研究打下基础。     

A humanized anti-osteopontin antibody protects from Concanavalin A induced-liver injury in mice

Osteopontin has been implicated in various inflammatory diseases including rheumatoid arthritis, multiple sclerosis, Crohn's disease, and fulminant hepatitis. Increased expression of osteopontin has been detected in pathological foci of these diseases. RA and fulminant hepatitis have been successfully treated by administration of neutralizing anti-osteopontin antibody in mice. However, rodent antibodies are highly immunogenic in humans and therefore limited in their clinical application. Here, a murine monoclonal antibody 23C3 against human osteopontin, was humanized by complementarity-determining region grafting method based on computer-assisted molecular modeling. The humanized version of 23C3, denoted as Hu23C3, was shown to possess affinity comparable to that of its parental antibody. Hu23C3 could also inhibit monocyte migration in response to osteopontin in vitro. Furthermore, in vivo data showed that Hu23C3 significantly protects mice from Concanavalin A (Con A) induced-liver injury in association with the reduction of transaminase activities and improvement of liver injury. Mechanistic studies demonstrated that Hu23C3 inhibited T and NKT cell infiltration, and activation of nuclear factor κB (NF-κB) in the liver, resulting in reduction of TNF-α and IFN-γ production. Thus, our data strongly support that the humanized anti-osteopontin antibody, Hu23C3, may have a potential for the treatment of T cell mediated-hepatitis in human.     

Drug-monoclonal antibody conjugates for cancer therapy: potentials and limitations

The development of monoclonal antibodies against antigens associated preferentially with human tumors has reawakened interest in the use of antibodies as site-specific targeting agents for cancer therapy. There are now many reports of the construction of conjugates of drugs and toxins with antibodies which have in vitro toxic properties directed by the antibody moiety. Only recently, however, have limitations to the in vivo therapeutic application of these conjugates become appreciated. These include limited levels of antibodies accumulating in tumor deposits in patients, sometimes marked differences in the biodistribution of antibody-drug conjugates from those seen with free antibody, limited penetration of antibody and drug-antibody conjugates into tumor tissue together with heterogeneity of antigen expression within tumor, and limitation to the cytotoxicity of conjugates caused by the low drug to antibody molar ratios possible without denaturation. It is now becoming appreciated that effective development of this approach will require more than chemical conjugation of existing or even novel drugs or toxins to antibodies so that they are cytotoxic in vitro. Additional strategies will probably have to be employed, including the use of intermediate carriers to increase drug to antibody ratios in conjugates, the production of conjugates without extreme overall charge or conformation changes, techniques to increase antibody localization into tumors by, for example, regional perfusion, blood flow enhancement, or increase in antigenic expression, or the use of conjugates containing mixtures of antibodies or fragments directed against different tumor-associated antigens.     

Review article: clinic-based testing for Helicobacter pylori infection by enzyme immunoassay of faeces,urine and saliva

Enzyme immunoassays have been used to detect Helicobacter pylori infection in human body materials such as faeces, urine and saliva. The stool antigen assay (HpSA), which uses polyclonal anti-H. pylori antibody as a capture reagent, has been widely used in the pre-treatment diagnosis of the infection in adults and children. Although the assay has the potential for monitoring eradication therapy, there are controversies over its use, especially at an early stage after treatment. The efficacy of the stool antigen assay can be modified by using monoclonal antibodies towards well characterized H. pylori faecal antigens. Two types of enzyme immunoassays (enzyme-linked immunosorbent assay [ELISA] and immunochromatography) have been used to detect antibodies to H. pylori in urine. Immunochromatography of urine is a rapid assay well suited for epidemiological studies. The salivary ELISA, used in a number of studies, has shown inconsistent results with less than optimum sensitivity and specificity. Urinary and salivary immunoassays may not distinguish between past and present infections, thus limiting their potential to monitor eradication therapy.