Advanced characterization and refinement of poly N-butyl cyanoacrylate microbubbles for ultrasound imaging

We aimed to develop and characterize poly n-butylcyanoacrylate (PBCA) microbubbles (MBs) with a narrow size distribution. MBs were synthesized by established emulsion polymerization techniques, size-isolated by centrifugation and functionalized for molecular imaging by coating their surface with streptavidin. The physical and acoustic properties of the parent solution, different-size isolated populations and functionalized MBs were measured and compared. As expected from negative zeta potentials at pH 7, cryo scanning electron microscopy showed no aggregates. In phantoms MBs were destructible at high mechanical indices and showed a frequency-dependent attenuation and backscattering. The MBs were stable in solution for more than 14 weeks and could be lyophilized without major damage. However, for injection, small needle diameters and high injection rates are shown to be critical because both lead to MB destruction. In summary, when being handled correctly, size-isolated PBCA MBs are promising candidates for preclinical functional and molecular ultrasound imaging.     

测定小鼠细胞因子的双抗体夹心ELISA法的建立及应用研究

目的　建立敏感的测定小鼠IL - 5、IL - 4和IFN -γ水平的生物素 -链霉亲和素双抗体夹心ELISA法 ,并探讨该法在IL - 5转基因小鼠感染模型中的应用价值。方法　含大鼠抗小鼠IL - 4、IL - 5和IFN -γ单克隆抗体的杂交瘤细胞培养上清 ,经PM 10膜超滤、5 0 %饱和硫酸铵沉淀及ProteinG -Sepharose亲和层析纯化。应用活化生物素制剂对纯化的TR FK4、BVD6和AN18单克隆体抗体进行生物素化。用纯化的 2 μg/mlTRFK5、8μg/ml 11B11和 4 μg/mlR4 - 6A2分别作为测定小鼠IL - 5、IL - 4和IFN -γ的包被抗体 ,然后逐步加入样品、生物素化TRFK4 (1μg/ml)、BVD6 (2 μg/ml)和AN18(1μg/ml)、SA -HRPO和OPD ,硫酸终止反应后读取A490 值。结果　所建立的生物素 -链霉亲和素双抗体夹心ELISA法特异性和敏感性高 ,用于检测感染巴西日圆线虫感染的IL - 5转基因小鼠和非转基因小鼠的血清标本取得了较好的效果。结论　该ELISA方法对于测定小鼠细胞因子具有应用价值     

Synthesis of bifunctional saxitoxin analogues by biotinylation

Saxitoxin (STX) contaminates seafood and freshwater catchments worldwide. Conjugation of STX with biotin would enable new biochemical methods to quantitate STX and its analogues as well as diversify its utility as a research tool. We conjugated biotin at the region of the toxin normally occupied by a carbamoyl and this conjugate could concurrently bind both avidin/streptavidin and saxiphilin. Increasing the length of the linker between biotin and the STX portion of the semisynthetic analogue increased potency of saxiphilin binding of the STX moiety.     

应用链霉亲和素在ELISA中检测HBe系统

应用链霉亲和素和生物素系统发展成ELISA的改良法,用于检测HBeAg和HBe,取得较为满意的效果。标记SA-HRP吋,两者的重量比以O.5～0.8/1为佳,浓度以7μg/ml时最为理想;SA-HRP的反应时间不同,亦会影响检测结果,其中以30min为适宜;本法、ABC法及普通法进行检测抗-HBe的灵敏度比较,本法相对灵敏度高于后两者。用Abbott试剂作为标准,检测HBeAg100人份、抗-HBe87人份,其阳性符合率、阴性符合率及总符合率均高于ABC法和普通ELISA,BSA法,提高了方法的特异性及灵敏度,在应用上有着广阔的前景。     

生物素-链霉抗生物素蛋白硷性磷酸酶体系标记检测DNA探针

本室采用近年发展的生物素—链霉抗生物素蛋白体系检测特异互补的核酸序列,先经缺口翻译制备生物素DNA探针,分子杂交后,再与链霉抗生物素蛋白—生物素—硷性磷酸酶复合物反应和显色,检测限度约50pg靶DNA,并将其结果与~(35)S标记相同的探针进行了比较。     

Modified tobacco mosaic virus particles as scaffolds for display of protein antigens for vaccine applications

Display of peptides or proteins in an ordered, repetitive array, such as on the surface of a virus-like particle, is known to induce an enhanced immune response relative to vaccination with the "free" protein antigen. The coat protein of Tobacco mosaic virus (TMV) can accommodate short peptide insertions into the primary sequence, but the display of larger protein moieties as genetic fusions to the capsid protein has not been possible. We employed a randomized library approach to introduce a reactive lysine at the externally located amino terminus of the coat protein, which facilitated biotinylation of the capsid. To characterize display of heterologous proteins on the virion surface, we bound a model antigen (green fluorescent protein (GFP)-streptavidin (SA), expressed and purified from plants) to the biotinylated TMV particles, creating a GFP-SA decorated virus particle. A GFP-SA tetramer loading of 26% was obtained, corresponding to approximately 2200 GFP moieties displayed per intact virion. We evaluated the immunogenicity of GFP decorated virions in both mice and guinea pigs and found augmented humoral IgG titers in both species, relative to unbound GFP-SA tetramer. Next, we fused an N-terminal fragment of the Canine oral papillomavirus L2 protein to streptavidin. With TMV display, the L2 protein fragment was significantly more immunogenic than uncoupled antigen when tested in mice. By demonstrating the presentation of whole proteins, this study expands the utility of TMV as a vaccine scaffold beyond that which is possible by genetic manipulation.     

抗人纤维蛋白单链抗体-链亲和素融合蛋白的稳定性及对动物纤维蛋白的识别

目的：探索抗人纤维蛋白单链抗体链亲合素融合蛋白的应用前景。方法：构建抗人纤维蛋白单链抗体ｓｃＦｖ８Ｅ５基因与链亲和素基因的重组表达载体ｐＳＴＥ２８Ｅ５ ,表达的融合蛋白可特异性识别人纤维蛋白,同时有生物素的结合位点。转化ＪＭ１０９ 后用ＩＰＴＧ 诱导表达,ＥＬＩＳＡ 观察融合蛋白的稳定性。结果：抗人纤维蛋白单链抗体链亲合素融合蛋白稳定性好,可耐受至少２ ｗ ,４ ℃保存、数次反复冻融或一定时间的３７ ℃孵育。它能与大鼠和豚鼠的纤维蛋白结合,而与猪纤维蛋白反应不明显。结论：抗人纤维蛋白单链抗体链亲和素融合蛋白适用于大鼠或豚鼠血栓病模型,是临床前研究的有用材料。     

Addition of redox function to streptavidin by chemical conversion of the tyrosine residues to 3-aminotyrosine residues

Tyrosine residues of streptavidin were chemically converted to 3-aminotyrosine residues by nitration followed by reduction. Although the biotin binding activity of the modified streptavidin reduced by about a half, no conformational change was observed in CD spectroscopy. Furthermore, electron transfer between the modified streptavidin with 3-aminotyrosine residues and the biotin-modified gold electrode was detected by a voltammetry technique. This chemical conversion method will be a general and promising method for providing an electron transfer function to various proteins.     

A magnetism/laser-auxiliary cascaded drug delivery to pulmonary carcinoma

Although high-efficiency targeted delivery is investigated for years, the efficiency of tumor targeting seems still a hard core to smash. To overcome this problem, we design a three-step delivery strategy based on streptavidin–biotin interaction with the help of c(RGDfK), magnetic fields and lasers. The ultrasmall superparamagnetic iron oxide nanoparticles (USIONPs) modified with c(RGDfK) and biotin are delivered at step 1, followed by streptavidin and the doxorubicin (Dox) loaded nanosystems conjugated with biotin at steps 2 and 3, respectively. The delivery systems were proved to be efficient on A549 cells. The co-localization of signal for each step revealed the targeting mechanism. The external magnetic field could further amplify the endocytosis of USPIONs based on c(RGDfK), and magnify the uptake distinctions among different test groups. Based on photoacoustic imaging, laser-heating treatment could enhance the permeability of tumor venous blood vessels and change the insufficient blood flow in cancer. Then, it was noticed in vivo that only three-step delivery with laser-heating and magnetic fields realized the highest tumor distribution of nanosystem. Finally, the magnetism/laser-auxiliary cascaded delivery exhibited the best antitumor efficacy. Generally, this study demonstrated the necessity of combining physical, biological and chemical means of targeting.     

Hybridoma production by simplified avidin-mediated electrofusion

A simplified method for producing cell hybrids by avidin-mediated electrofusion has been developed. First, biotin was attached both to immunogen and to the surface of murine myeloma cells using N-hydroxysuccinimidobiotin. Biotinylated immunogen was incubated with splenocytes derived from immunized mice and allowed to bind to surface immunoglobulins of B cells. Using streptavidin, biotinylated myeloma cells then were bridged to those B cells bearing biotinylated immunogen. Selective fusion of bridged cells was accomplished by their limited exposure to high-voltage potentials. A high frequency of hybridomas was obtained all of which secreted high titers of antibodies to a selected model immunogen, keyhole limpet hemocyanin.