Immunoadsorbent purification of carcinoembryonic antigen using a monoclonal antibody: a direct comparison with a conventional method

Carcinoembryonic antigen (CEA) has been extracted and purified by two different methods from a single aqueous homogenate of liver metastases from a primary adenocarcinoma of the colon, thus enabling direct comparison to be made between a conventional technique using gel filtration and a monoclonal antibody immunoadsorbent method. Yield, immunoreactivity and purity have been determined in both cases by direct weighing, enzyme-linked immunoadsorbent assay, radioimmunoassay and SDS-polyacrylamide gel electrophoresis. Reactivity of the antigen with monoclonal and polyclonal anti-CEA antibodies recognising different epitopes was analysed by Western blotting. There appears to be no significant difference in immunoreactivity or purity by these criteria, but the immunoadsorbent method gave a higher yield of CEA for far less expenditure of time and effort. A variant of CEA with a lower molecular weight was also identified in both preparations.     

The minimum concentration of fibrinogen needed for platelet aggregation using ADP.

OBJECTIVE: Determine the minimum concentration of plasma fibrinogen needed to stimulate the aggregation of platelets, collected from normal subjects, using ADP. DESIGN: Platelet rich plasmas (300 x 10(9) platelets/L) were made and adjusted to final fibrinogen concentrations of 75, 19, 5, and 0 mg/dL using fibrinogen free serum. Each fibrinogen concentration in all twelve subjects was aggregated with ADP SETTING: Research laboratory in the Department of Clinical Laboratory Science at Saint Louis University. PARTICIPANTS: Twelve healthy volunteers of both genders, between the ages of 18 and 60 years who were not pregnant and weighed at least 110 pounds were included in the study. Subjects were excluded from the study if they had ingested aspirin within one week prior to blood collection. In addition, subjects with a history of bleeding disorders such as afibrinogenemia, hypofibrinogenemia, von Willebrand disease, and Bernand-Soulier disease were rejected from the study. MAIN OUTCOME MEASURES: Platelet aggregation tracings were analyzed for amplitude and compared across plasma fibrinogen concentrations. In addition, the type of curve (monophasic vs. biphasic), smoothness and aggregation stability were also noted. RESULTS: The results show that aggregation occurred with every dilution of fibrinogen tested and that the amplitude of the aggregation curves appears not to be dependent on plasma fibrinogen. CONCLUSIONS: The results indicate that platelets from healthy individuals previously exposed to normal fibrinogen levels will aggregate equally well in decreasing plasma fibrinogen concentrations and even in the absence of plasma fibrinogen using ADP as the aggregator.     

Buffered lidocaine as a local anesthetic: an investigation of shelf life.

STUDY OBJECTIVE: To determine whether buffered lidocaine must be prepared just before use. DESIGN: Randomized, double-blind, prospective trial. SETTING: University hospital. PARTICIPANTS: Twenty-four adult volunteers. INTERVENTIONS: Three buffered lidocaine solutions prepared seven days, one day, and just before use were compared with a control solution. Subjects received 0.5 mL intradermal injections of each solution. Pain of infiltration and extent and duration of anesthesia were measured. MEASUREMENTS AND MAIN RESULTS: Pain of infiltration was less with all buffered solutions than control (P less than .0001). Mean maximum diameter of anesthesia ranged from 29 to 33 mm for the buffered solutions compared with 31 mm for control. Mean duration of anesthesia was 33 minutes for control and 30 minutes for all of the buffered solutions. There was no statistically significant difference in extent or duration of anesthesia for any of the solutions (P greater than .5, beta = .15 for delta = 10%). CONCLUSION: Buffered lidocaine stays effective for up to one week after preparation. It is therefore convenient to use in emergency settings.     

Complications of the cardiopulmonary stress test in patients with depressed left ventricular systolic function.

BACKGROUND: The exercise test has a recognized lower risk of complications when used in the general population and in coronary artery diseased patients, but from a theoretical point of view should have a higher rate of complications when performed in patients with chronic heart failure (CHF). AIMS: To characterize and assess the type and incidence of complications during cardiopulmonary stress test (CPX) in patients with depressed left ventricular systolic function in comparison with a group of patients and individuals with normal function. METHODS: Retrospective analysis of the 334 consecutive CPX performed for risk stratification in 198 patients with a left ventricular ejection fraction below 40% (Group A) and 180 consecutive CPX performed in 78 subjects with normal function (Group B). The two groups were compared with respect to demographic data, CPX parameters and specific complications. Results: Major complications during the tests occurred only in 14 tests of Group A (4.2%, p = 0.012). Non-sustained ventricular tachycardia, <6 beats, occurred in 7 group A and 2 group B tests. The absence of coronary artery disease was the only independent predictor for complications. CONCLUSIONS: Major CPX complications occurred only in patients with impaired left ventricular systolic function. Heart failure patients showed a low probability (around 4%) for complications during CPX, significantly higher and more severe than the risk in the group of patients with normal ventricular function, allowing us to recommend that CPX in patients with heart failure should be performed in a hospital setting under the supervision of a physician with specific training.     

乙型肝炎表面抗原“免疫逃避”突变体在哺乳动物细胞中的表达

利用乙型肝炎病毒ＤＮＡ开放框架上的ＢａｍＨＩ和ＨｐａＩ位点，酶切消化质粒载体ＰＥｃｏｂ６（含双拷贝ＨＢＶＤＮＡ），得到约９００ｂｐ的ＨＢＶ－Ｓ基因片断。将其插入到噬菌粒载体ＰＢｌｕｅｓｃｒｉｐｔｓｋ＋的ＳｍａＩ位点上。然后通过体外寡核苷酸介导的人工定点突变获得一系列（共１２种）Ｓ基因“免疫逃避”突变型。再通过ＥＢ病毒真核表达载体ｐＭＥＰ４上的ＢａｍＨＩ和Ｋｐｎｌ位点将噬菌粒ｐＢｌｕｅｓｃｒｉｐｓｋ＋上的Ｓ基因突变型片断定向克隆到ＰＭＥＰ４上，从而构建了含乙肝Ｓ基因突变型的重组质粒ｐＭＥＰ４ＨＢＳＭ。用其转染人肝癌传代细胞系ＨｅｐＧ２，经潮霉素选择，三周后获得抗性细胞克隆。经用抗ＨＢｓ单克隆抗体（含针对ＨＢｓＡｇ“ａ”抗原决定簇）检测除含变异体１４５（即１４５位上甘氨酸为精氨酸替代）外其余抗变异体ＨＢｓＡｇ均为阳性。经Ｗｅｓｔｅｒｎｂｌｏｔ证实变异体１４５，在分子量约为２３ＫＤ处有一特异ＨＢｓＡｇ蛋白带。     

The processing of antigens delivered as DNA vaccines

Summary: The ability of DNA vaccines to provide effective immunological protection against infection and tumors depends on their ability to generate good CD4⁺ and CD8⁺ T‐cell responses. Priming of these responses is a property of dendritic cells (DCs), and so the efficacy of DNA‐encoded vaccines is likely to depend on the way in which the antigens they encode are processed by DCs. This processing could either be via the synthesis of the vaccine‐encoded antigen by the DCs themselves or via its uptake by DCs following its synthesis in bystander cells that are unable to prime T cells. These different sources of antigen are likely to engage different antigen‐processing pathways, which are the subject of this review. Understanding how to access different processing pathways in DCs may ultimately aid the rational development of plasmid‐based vaccines to pathogens and to cancer.