Measuring Influenza Neuraminidase Inhibition Antibody Titers by Enzyme-linked Lectin Assay

Antibodies to neuraminidase (NA), the second most abundant surface protein on influenza virus, contribute toward protection against influenza. Traditional methods to measure NA inhibiting (NI) antibody titers are not practical for routine serology. This protocol describes the enzyme-linked lectin assay (ELLA), a practical alternative method to measure NI titers that is performed in 96 well plates coated with a large glycoprotein substrate, fetuin. NA cleaves terminal sialic acids from fetuin, exposing the penultimate sugar, galactose. Peanut agglutinin (PNA) is a lectin with specificity for galactose and therefore the extent of desialylation can be quantified using a PNA-horseradish peroxidase conjugate, followed by addition of a chromogenic peroxidase substrate. The optical density that is measured is proportional to NA activity. To measure NI antibody titers, serial dilutions of sera are incubated at 37 °C O/N on fetuin-coated plates with a fixed amount of NA. The reciprocal of the highest serum dilution that results in ≥50% inhibition of NA activity is designated as the NI antibody titer. The ELLA provides a practical format for routine evaluation of human antibody responses following influenza infection or vaccination.     

IVIg Treatment Reduces Catalytic Antibody Titers of Renal Transplanted Patients

Catalytic antibodies are immunoglobulins endowed with enzymatic activity. Catalytic IgG has been reported in several human autoimmune and inflammatory diseases. In particular, low levels of catalytic IgG have been proposed as a prognostic marker for chronic allograft rejection in patients undergoing kidney transplant. Kidney allograft is a treatment of choice for patients with end-stage renal failure. Intravenous immunoglobulins, a therapeutic pool of human IgG, is used in patients with donor-specific antibodies, alone or in conjunction with other immunosuppressive treatments, to desensitize the patients and prevent the development of acute graft rejection. Here, we followed for a period of 24 months the levels of catalytic IgG towards the synthetic peptide Pro-Phe-Arg-methylcoumarinimide in a large cohort of patients undergoing kidney transplantation. Twenty-four percent of the patients received IVIg at the time of transplantation. Our results demonstrate a marked reduction in levels of catalytic antibodies in all patients three months following kidney transplant. The decrease was significantly pronounced in patients receiving adjunct IVIg therapy. The results suggests that prevention of acute graft rejection using intravenous immunoglobulins induces a transient reduction in the levels of catalytic IgG, thus potentially jeopardizing the use of levels of catalytic antibodies as a prognosis marker for chronic allograft nephropathy.     

一种丙肝病毒滴度测定方法

目的:建立一种测定丙肝病毒(HCV)滴度的方法,为HCV的基础研究及临床药物评估提供有效的实验手段。方法:对HCV JFH1病毒株进行倍比稀释并感染Huh7细胞,甲醇固定后,采用抗HCV核心蛋白一抗进行孵育,加入二抗后采用DAB显色,通过计数病毒斑得到HCV的滴度。结果:采用甲基纤维素限制HCV于细胞间感染,利用免疫组化技术使被病毒感染的细胞形成肉眼可见的清晰斑块。结论:建立了一种简单、经济的HCV滴度测定方法。     

鼻咽癌患者血清中抗Epstein-Barr病毒特异性DNA酶抗体检测方法的探讨

本文报道了鼻咽癌患者血清中抗Epstein-Barr病毒特异性DNA酶抗体的检测方法,并对影响检测结果的几种因素进行了探讨。用抗酶率来表示抗体滴度可在酶用量为0.08—0.25U范围内进行检测,使实验条件容易控制。     

Addition of Valproic Acid to CHO Cell Fed-Batch Cultures Improves Monoclonal Antibody Titers

Improving the productivity of a biopharmaceutical Chinese hamster ovary (CHO) fed-batch cell culture can enable cost savings and more efficient manufacturing capacity utilization. One method for increasing CHO cell productivity is the addition of histone deacetylase (HDAC) inhibitors to the cell culture process. In this study, we examined the effect of valproic acid (VPA, 2-propylpentanoic acid), a branched-chain carboxylic acid HDAC inhibitor, on the productivity of three of our CHO cell lines that stably express monoclonal antibodies. Fed-batch shake flask VPA titrations on the three different CHO cell lines yielded cell line-specific results. Cell line A responded highly positively, cell line B responded mildly positively, and cell line C did not respond. We then performed factorial experiments to identify the optimal VPA concentration and day of addition for cell line A. After identifying the optimal conditions for cell line A, we performed verification experiments in fed-batch bioreactors for cell lines A and B. These experiments confirmed that a high dose of VPA late in the culture can increase harvest titer >20 % without greatly changing antibody aggregation, charge heterogeneity, and N-linked glycosylation profiles. Our results suggest that VPA is an attractive and viable small molecule enhancer of protein production for biopharmaceutical CHO cell culture processes.     

Hemagglutination Inhibition with Arboviruses: Relationship Between Titers and Source of Erythrocytes

Antigens for Grand Arbaud, Hazara, and California arboviruses were able to agglutinate goose and either dog, hamster and guinea pig, or hamster red blood cells (RBC) to the same titer at the same pH; in hemagglutination-inhibition (HI) tests, titers for homologous and related sera were the same with these different types of RBC or occasionally one dilution higher with the mammalian cells. Antigens for St. Louis encephalitis and Eastern equine encephalitis viruses required use of lower antigen dilutions with human, guinea pig, and hamster RBC than with goose RBC. The results of comparative HI testing with these latter antigens and types of RBC indicate that HI titer is not directly related to the antigen dilution used with different types of RBC.     

Method for Determining Antineuraminidase Activity

A test was developed to screen drugs for antineuraminidase (influenza sialidase) activity in vitro. Neuraminidase prepared from Vibrio cholerae was added to a substrate containing ganglioside, prepared from calf brain. Sialic acid is a split product in the reaction. The presence of sialic acid was detected colorimetrically by use of Warren's Thiobarbituric Acid Assay after drugs had been added to inhibit the action of neuraminidase on the calf brain substrate.     

One-Tube Test for Determining Oxidative or Fermentative Metabolism of Bacteria

A one-tube test for distinguishing oxidation from fermentation in the metabolism of carbohydrates was examined on 1,200 gram-negative bacilli. There was a 100% correlation between the one-tube method and the standard two-tube procedure.     

Clinical Value of Teichoic Acid Antibody Titers in the Diagnosis and Management of the Staphylococcemias

Differentiation of endocarditic from nonendocarditic Staphylococcus aureus (SA) septicemia is prognostically and therapeutically important. A study of 68 cases of either SA or streptococcal sepsis, including 50 cases of SA sepsis of both cardiac and noncardiac origin, was done to determine the presence and titer of serum teichoic acid antibodies (TAA''s) by double immunodiffusion. Thirty-seven uninfected controls were also examined. There was no statistical difference in either incidence or peak TAA titers in endocardial versus deepseated, extracardiac SA sepsis. However, in both of these groups, incidence and peak titers were significantly higher than in intravascular catheter-related SA sepsis, streptococcal endocarditis and controls (P<0.05). Peak TAA titers in SA sepsis develop on admission or shortly thereafter (6 to 11 days) and permit early decisions on degree of tissue infection, likelihood of metastatic seeding and necessity for higher-dose, longer-term antibiotic therapy.Cases of catheter-related SA sepsis with no clinical evidence of metastatic SA seeding and with negative or low-titered (1:1) TAA''s were classified as superficial sepsis. Treatment consisted of short-term, low-dose antistaphylococcal regimens and catheter removal. In posttherapy follow-up after 6 to 12 weeks, all of the patients were cured and no signs of endocarditis or deepseated SA infection developed.     

Variables of the Rubella Hemagglutination-Inhibition Test System and Their Effect on Antigen and Antibody Titers

A systematic study was made of certain variables of the rubella hemagglutination-inhibition (HI) test system and their effect on antigen and antibody titers. Erythrocytes from pigeons and 1-day-old chicks gave similar antigen and antibody titers, but goose erythrocytes gave lower titers. Indicator erythrocytes could be stored in Alsever's solution at 4 C for as long as 2 weeks without losing sensitivity in hemagglutination (HA) and HI tests. Antigen titers varied by eightfold or more in different diluent systems; titers were generally higher at pH 6.2 than at pH 7.2. A diluent without Ca²⁺ gave antigen titers as high as those obtained in diluents with added Ca²⁺ ions. Antibody titers also varied in different diluent systems. HEPES diluents at pH 6.2 gave higher antibody titers than those obtained in other diluents, but occasional “false-positive” inhibition reactions were seen. Kaolin suspended in borate saline at pH 9.0 effectively removed inhibitor from sera without absorbing specific antibody, but at pH 7.3 it removed various amounts of specific antibody. Antibody titers of sera treated with kaolin at pH 9.0 were similar to those of sera treated with heparin-MnCl₂; treatment with dextran sulfate-CaCl₂ gave lower antibody titers. Antigens varied widely in sensitivity for detecting HI antibody and in the ability to detect diagnostically significant increases in antibody. Sensitivity in detecting antibody was not related to the HA titer of the antigens. Tween-ether-treated antigens gave lower antibody titers but were more reliable than corresponding untreated antigens for serological diagnosis of infection.     

Simplified Method for Determining Acetylator Phenotype

A chemical method has been developed for estimating the acetylator phenotype without the use of special equipment. One urine specimen taken after ingestion of sulphadimidine, sulphapyridine, or sulphasalazine is required. The acetylator phenotype was assessed correctly in 150 urine specimens from 100 healthy subjects. Between 15 and 25 specimens can be determined hourly. Urine specimens for the test can be stored for two weeks at 37°C without any loss of drugs or their metabolites.     

淀粉酶活性测定方法的改进

以香蕉果实为试材,对常规的淀粉酶活性的测定方法进行了改进,取得了更好的效果.     

Comparative Analysis of Hemagglutination Inhibition Titers Generated Using Temporally Matched Serum and Plasma Samples

Influenza-specific hemaggluitination inhibition (HAI) antibody titer, an indicator of immunity to influenza, is often used to measure exposure to influenza in surveillance and immunogenicity studies. Traditionally, serum has been the specimen of choice for HAI assays, but a desire to reduce the amount of blood collected during studies and the availability of plasma in archived sample collections warrant the evaluation of plasma for HAI titer. Therefore, the relationship between serum and plasma HAI titer values is of great interest. Here, we compare HAI titers determined on temporally matched serum and plasma (citrated and heparinized) using influenza A and B viruses. Bland-Altman plots, McNemar''s test, and geometric coefficient of variation were used respectively for evaluating agreement, correlation and variability in the serum-plasma titer results. We observed a high degree of agreement (80.5%–98.8%) and correlation (r = 0.796–0.964) in the serum and matched plasma titer values although plasma titers were generally lower than corresponding serum titers. Calculated seropositive (HAI ≥40) rates were higher using serum titers than with plasma titers, but seroconversion rates were unaffected by sample type. Stronger agreement and decreased variability in titers were seen between serum and citrated plasma than between serum and heparinized plasma. Overall, these data suggest that serum or plasma can be used in serodiagnostic HAI assays, but seropositive rates may be underestimated using plasma HAI titers. The type of anticoagulant present in plasma may affect HAI titer values and warrants further investigation.     

Molecular Method for Determining Sex of Walruses

Abstract: We evaluated the ability of a set of published trans-species molecular sexing primers and a set of walrus-specific primers, which we developed, to accurately identify sex of 235 Pacific walruses (Odobenus rosmarus divergens). The trans-species primers were developed for mammals and targeted the X- and Y-gametologs of the zinc finger protein genes (ZFX, ZFY). We extended this method by using these primers to obtain sequence from Pacific and Atlantic walrus (O. r. rosmarus) ZFX and ZFY genes to develop new walrus-specific primers, which yield polymerase chain reaction products of distinct lengths (327 and 288 base pairs from the X- and Y-chromosome, respectively), allowing them to be used for sex determination. Both methods yielded a determination of sex in all but 1–2% of samples with an accuracy of 99.6–100%. Our walrus-specific primers offer the advantage of small fragment size and facile application to automated electrophoresis and visualization.     

检测小鹅瘟感染抗体的Dot-ELISA方法研究*

以GPV VP1-VP3非重叠序列重组原核表达多肽为检测抗原,建立了鉴别GPV感染抗体的Dot-ELISA方法,试验确定检测抗原包被浓度分别为700ng; 兔抗鹅IgG-HRP-标记抗体的最适稀释度为1∶200;被检血清最佳稀释度为1∶400。检测GPV血清抗体的阳性率为100%;检测鸡抗GPV VP3禽痘重组病毒血清的阳性率为0 %。     

Rapid Method for Determining Serum Bactericidal Activity

To screen large numbers of sera, a method was devised which utilizes the Steers-Foltz replicator which is usually used to determine minimal inhibitory concentration for antibiotics. Each of the wells (9 by 15 mm) of the replicator is filled with 0.06 ml of serum, 0.02 ml of a 10(5) suspension of organisms, and 0.02 ml of diluent (tris(hydroxymethyl)aminomethane-hydrochloride buffer, pH 8.4). The mixtures are incubated for 3 h, and samples are taken at 0, 1, 2, and 3 h by stamping duplicate nutrient agar plates (approximately 0.04 ml from each well). Plates are incubated overnight, and bactericidal activity is estimated by visual inspection of bacterial growth at each site for each sampling time. Results obtained with 28 serum-organism pairs paralleled standard pipetting-pour plate methods. The replicator method for determining bactericidal activity allows for the testing of a large number of samples and requires negligible amounts of serum.     

高效液相测定同型半胱氨酸方法的建立

为测量包括同型半胱氨酸在内的 1 8种氨基酸 ,用高效液相紫外检测法 ,在氨基酸混合标样中 ,加进Hcy标准品 ,仍用 4 5min程序分析 ,对AccqTag方法进行了修改 ,衍生温度改为常温 ,衍生后用醋酸酸化 ,并保存于 0～ 1℃ ,同型半胱氨酸出峰的时间为第 33min左右 ,结果得到了分离良好的 1 8种氨基酸图谱。     

一种构建改形单域抗体的方法

为验证一种构建改形单域抗体的实用新方法,与以往方法不同的是,该方法不需要对抗体进行空间结构模拟,以确定人源抗体的FRs接受序列及在人源FRs接受序列中哪些氨基酸残基需要突变,并且该方法将抗体的改形与亲和力成熟于同一过程完成,利用该方法构建了改形抗CD28重链单域抗体,根据一种鼠源抗CD28重链单域抗体的氨基酸序列,于GenBank中查得两条与之最同源的人源抗体序列,利用其中一条的FRs作为改形抗体的主框架进行改形构建,将鼠源抗体的CDR区插入到人源FR区后,对人源FR区的一些氨基酸残基进行替换突变,替换的氨基酸残基数及替换原则主要是根据对查到的人源抗体序列,鼠源抗体序列,以及这些序列与Kabat分类中的种属序列进行的比较,为了增加改形抗体基因的多样性,对要被替换的氨基酸残基在基因合成中采用简并的方式,使要被替换的氨基酸残基和替换的氨基酸残基都有机会出现,二者出现的几率各为50%,同时,在将大小不同的合成核苷酸片段采用重叠PCR扩增以获得完整改形抗体基因时,采用高Mg^2 浓度下和使用TaqDNA聚合酶,以进一步随机引入突变,利用重叠PCR产物构建了一个噬菌体抗体库,经过3轮淘选后,获得了几个具有较高免疫学活性的改形抗体,对其中的两个抗体进行了进一步研究,将两个抗体的基因在大肠杆菌BL21（DE3）中表达,复性后的表达蛋白仍具有较高的免疫学活性,结果表明该方法是有效可行的。