Agar-Gel Precipitin-Inhibition Technique for Histoplasmosis Antibody Determinations

The agar-gel precipitin-inhibition technique has been modified to detect antibodies of Histoplasma capsulatum in sera from human clinical cases and experimental animals infected with this organism. By use of this modified technique, the histoplasmosis can be detected more consistently and reliably than with the direct agar-gel diffusion test, and titers are comparable to those attained by the complement-fixation serological technique.     

Agar-Gel Precipitin-Inhibition Technique for Plague Antibody Determinations

An application of the agar-gel precipitin-inhibition technique we described previously can be used to detect plague antibodies in human and animal sera after a series of plague vaccine inoculations or after exposure to Pasteurella pestis. Determination of the minimal reacting concentrations of the plague antigen and antibody reagents, methods for combining reagents, and length of incubation periods are discussed.     

Agar-Gel Precipitin-Inhibition Technique for C-Reactive Protein Determinations

An application of the agargel precipitin-inhibition technique of Ray and Kadull detects the C-reactive protein present in the acute-phase human sera. The sensitivity of this procedure is compared with the capillary tube-precipitin method of Selman and Halpern.     

Agar-Gel Precipitin-Inhibition Technique for C-Reactive Protein Determinations: II. Degree of Sensitivity and Reproducibility

The agar-gel precipitin-inhibition test (AGPI) for C-reactive protein determinations proved more sensitive than the capillary tube-precipitin procedure. Furthermore, titers of positive CRP sera were obtained by this AGPI technique. This technique was compared with other indices of infectivity, namely, the white blood cell count and the erythrocyte sedimentation rate. The ease and reproducibility of this recommended test procedure were demonstrated by technicians unfamiliar with this procedure.     

Agar-Gel Precipitin-Inhibition Technique for C-Reactive Protein Determinations: III. Quantitation of C-Reactive Protein in Serum Specimens

A quantitative C-reactive protein serological procedure has been developed. By use of this method, which is performed in agar-gel plates, from 2 to 654 μg of C-reactive protein per ml of titrated human serum can be detected. The method is based on the inhibition of a specific C-reactive protein antigen-antibody precipitate formed in agar-gel by the minimal reactive dilutions of each reagent in 48 hr. It is simple, sensitive, and readily reproducible.     

Agar-Gel Precipitin-Inhibition Test for Coccidioidomycosis: I. Preliminary Evaluation of the Complement-Fixation and Agar-Gel Precipitin Tests in the Serodiagnosis of Human Coccidioidomycosis

The agar-gel precipitin-inhibition serological test for coccidioidomycosis was a more sensitive indicator of Coccidioides immitis antibodies than the tube precipitin, the agar-gel immunodiffusion, in the complement-fixation tests in assaying monkey sera, whether these sera were from prechallenge-vaccinated or postchallenged animals. When applying this technique to the assay of human sera, an analogous finding generally persisted. However, some human sera were positive by the complement-fixation test and negative by the agar-gel precipitin-inhibition test. These sera were diffused in agar-gel against various coccidioidin complement-fixation, tube precipitin, and agar-gel precipitin-inhibition test antigens with essentially negative results.     

Arbovirus Identification by an Agar-Gel Diffusion Technique

A double diffusion-in-agar test was used to investigate precipitation reactions of 75 arboviruses. Specific reactions were regularly observed with members of arbovirus groups B, California, Simbu, Turlock, Hart Park, vesicular stomatitis, and several other arboviruses as well as with a member of the Tacaribe group and a herpesvirus. The results demonstrated the feasibility of applying this technique to the identification of arboviruses.     

Agar-Gel Precipitin-Inhibition Test for Coccidioidomycosis: II. Serological Test Antigen Studies in Agar-Gel

In this study of saprophytic and parasitic growth-phase extracts of Coccidioides immitis, an antigen from the spherule culture supernatant fluid detected a specific antibody in heretofore serologically negative suspect coccidioidomycosis human sera when diffused in agar-gel. This antigen-antibody reaction occurred also in some of the serologically positive human coccidioidomycosis sera. This study indicates that this antigen-antibody reaction should be utilized as a possible routine serological test for complete serodiagnosis.     

A Novel Technique for Viable Cell Determinations

We have developed a simple method to determine cell viability using two fluorescent dyes, Hoechst 33258 and acridine orange. When these dyes are used in combination, dead cells fluoresce brilliant blue and live cells fluoresce green. This method works over a range of dye concentrations (Hoechst 33258, 0.25-2 μg/ml; acridine orange, 1-5.0 μg/ml) and the fluorescence spectra of the two dyes are such that only one set of filters is required to visualize the effects of both dyes simultaneously. It is insensitive to a wide range of exogenous serum concentrations and is read with greater uniformity by different observers.     

CHO细胞表达的抗炭疽保护性抗原人源化抗体的纯化及质量控制分析

目的:建立CHO细胞表达的抗炭疽保护性抗原人源化单抗纯化工艺和质量控制方法。方法:收获50 L生物反应器中无血清悬浮培养的CHO工程细胞培养液,通过高速离心去除细胞及碎片后超滤浓缩上清液,经亲和层析、SPFF阳离子交换层析后,将所得目的蛋白质经G25凝胶柱更换缓冲液以完成纯化;对纯化的产品进行单抗鉴别(Western印迹)、相对分子质量(SDS-PAGE和MOLDI-TOF)、纯度(SEC-HPLC)、生物学活性(毒素中和试验)、产品相关杂质(SEC-HPLC检测聚集体、降解产物)、工艺相关杂质(ELISA分析残余宿主蛋白、残余蛋白A)、安全性(凝胶法检测内毒素、薄膜过滤法考察无菌)分析。结果:样品回收率达61.7%;单抗鉴别实验阳性;MOLDI-TOF测定完整分子的相对分子质量为147 995,与预期相符;单体比例为99.25%,二聚体比例为0.75%;EC50值为0.1516μg/m L。残余宿主蛋白、残余蛋白A、内毒素、无菌检查结果符合药典要求。结论:初步建立了纯化工艺和质量控制方法,为抗炭疽人源化单抗药物的研制奠定了基础。     

Anthrax in animals

Anthrax is the archetype zoonosis; no other infectious disease affects such a wide range of species, including humans, although most susceptible are herbivorous mammals. Although the disease appears to have been recognized for centuries, it has yet to be established scientifically how animals contract it. While primarily a disease of warmer regions, it has long been spread to cooler zones through the trade of infected animals or contaminated animal products. Today it is still endemic in many countries of Africa and Asia and non-endemic countries must remain alert to the possibility of imports from such endemic areas resulting in outbreaks in their own livestock. The epidemiology of anthrax is becoming understood better with new systems coming on stream for distinguishing different genotypes and this is covered in detail. Clinical signs and pathology in animals are described.     

胞内抗体技术及医学应用

胞内抗体技术是近年来随抗体工程技术发展起来的一项新型基因治疗方法．它将单链抗体表达于非淋巴细胞内,并使之定向分布于细胞核、细胞浆或内质网等部位,特异性阻断、干扰分布于该部位的大分子物质生物活性或加工分泌过程．实验证明该技术能抑制生长因子受体的表达,灭活原癌基因蛋白,抑制病毒复制．在生物科学及医学研究中具有很大应用潜力．     

Technique for Viral Neutralization Antibody Surveys in Primary Microcultures

A simplified, microplate tissue culture procedure is described which facilitates neutralization antibody surveys on large numbers of sera. A comparison of the micromethod with that employing standard tube cultures indicates it to be as sensitive and far more economical.     

A Heterodimer of a VHH (Variable Domains of Camelid Heavy Chain-only) Antibody That Inhibits Anthrax Toxin Cell Binding Linked to a VHH Antibody That Blocks Oligomer Formation Is Highly Protective in an Anthrax Spore Challenge Model

Anthrax disease is caused by a toxin consisting of protective antigen (PA), lethal factor, and edema factor. Antibodies against PA have been shown to be protective against the disease. Variable domains of camelid heavy chain-only antibodies (VHHs) with affinity for PA were obtained from immunized alpacas and screened for anthrax neutralizing activity in macrophage toxicity assays. Two classes of neutralizing VHHs were identified recognizing distinct, non-overlapping epitopes. One class recognizes domain 4 of PA at a well characterized neutralizing site through which PA binds to its cellular receptor. A second neutralizing VHH (JKH-C7) recognizes a novel epitope. This antibody inhibits conversion of the PA oligomer from “pre-pore” to its SDS and heat-resistant “pore” conformation while not preventing cleavage of full-length 83-kDa PA (PA83) by cell surface proteases to its oligomer-competent 63-kDa form (PA63). The antibody prevents endocytosis of the cell surface-generated PA63 subunit but not preformed PA63 oligomers formed in solution. JKH-C7 and the receptor-blocking VHH class (JIK-B8) were expressed as a heterodimeric VHH-based neutralizing agent (VNA2-PA). This VNA displayed improved neutralizing potency in cell assays and protected mice from anthrax toxin challenge with much better efficacy than the separate component VHHs. The VNA protected virtually all mice when separately administered at a 1:1 ratio to toxin and protected mice against Bacillus anthracis spore infection. Thus, our studies show the potential of VNAs as anthrax therapeutics. Due to their simple and stable nature, VNAs should be amenable to genetic delivery or administration via respiratory routes.     

Crystal Structure of the Engineered Neutralizing Antibody M18 Complexed to Domain 4 of the Anthrax Protective Antigen

The virulence of Bacillus anthracis is critically dependent on the cytotoxic components of the anthrax toxin, lethal factor (LF) and edema factor (EF). LF and EF gain entry into host cells through interactions with the protective antigen (PA), which binds to host cellular receptors such as CMG2. Antibodies that neutralize PA have been shown to confer protection in animal models and are undergoing intense clinical development. A murine monoclonal antibody, 14B7, has been reported to interact with domain 4 of PA (PAD4) and block its binding to CMG2. More recently, the 14B7 antibody was used as the platform for the selection of very high affinity, single-chain antibodies that have tremendous potential as a combination anthrax prophylactic and treatment. Here, we report the high-resolution X-ray structures of three high-affinity, single-chain antibodies in the 14B7 family; 14B7 and two high-affinity variants 1H and M18. In addition, we present the first neutralizing antibody-PA structure, M18 in complex with PAD4 at 3.8 Å resolution. These structures provide insights into the mechanism of neutralization, and the effect of various mutations on antibody affinity, and enable a comparison between the binding of the M18 antibody and CMG2 with PAD4.