小鼠血清中杀O139霍乱弧菌抗体检测方法的初步建立

目的研究探索O139霍乱弧菌杀弧菌抗体的检测方法。方法用微孔板培养和琼脂平板克隆计数相结合的杀弧菌抗体检测方法,对实验菌株及稀释度、补体浓度等关键参数进行筛选;对50份小鼠免疫血清进行杀弧菌抗体滴度检测,并与O139群霍乱弧菌LPS Ig G抗体滴度进行相关分析;对该方法的特异性、线性和精密性进行了验证。结果筛选出最佳菌株为20100603菌株,最佳稀释度倍数为2 000倍,补体最佳稀释倍数为16倍。O139群霍乱弧菌小鼠免疫血清检测到较高的杀弧菌抗体滴度而PBS小鼠免疫血清未检测到杀弧菌抗体滴度。小鼠免疫血清杀弧菌抗体滴度与O139群霍乱弧菌LPS Ig G抗体滴度之间存在正相关关系。验证结果显示,在抑制剂浓度达到1.0~2.0 A600时,抑制率100%;线性回归方程为y=-1.093x+5.058,其相关系数为-0.999,P0.05;方法批内CV值为15.72%,批间CV值为23.47%。结论初步建立了O139霍乱弧菌杀弧菌抗体的检测方法,该方法具有较高的特异性、线性和精密度。     

Haemolytic and bactericidal activity of serum from the ring dove (Streptopelia risoria) after treatment with exogenous prolactin

The purpose of this study was to elucidate the effect of exogenous prolactin on the haemolytic and bactericidal capacity of serum obtained from ring doves (Streptopelia risoria) previously injected with either bovine serum albumin or saline solution. Haemolytic activity was measured in CH-50 units (which represents the capacity of serum complement to lyse 50% of sheep red blood cells in the presence of specific antibody) and the bactericidal activity was estimated from the number of colony-forming units ofStaphylococcus aureus which survived after 24 h of incubation in the presence of serum. The results indicated that: (1) bovine serum albumin stimulated both haemolytic and bactericidal activity, the highest values occurring 24 h and 4 days after administration, respectively. (2) Prolactin induced an increase in the haemolytic activity of complement. (3) The administration of bovine serum albumin to animals previously treated with prolactin produced a greater stimulation than either bovine serum albumin or prolactin alone.Abbreviations BSA bovine serum albumin - CFT complement fixation test - CFU colony-forming units - CH-50 units, the reciprocal of the complement dilution with 50% lysis of the hemolysin-treated erythrocytes - IU international units - PBS phosphate-buffered saline solution - SS saline solution     

A simple and rapid method of determining the titer of herpes simplex virus antibodies in a study by immunoenzyme analysis of a single serum dilution

The result obtained in the study of the possibility of using the method for the determination of the titer of antibodies to herpes simplex virus by EIA techniques in a single dilution of the serum under test are presented. This method is based on the determination of the optical density of the serum titer (rcut) in different groups of sera with the use of the assay system, permitting the evaluation of the positive results obtained in the determination of their final dilution. The results obtained with the use of this method showed that error was 50% for high-titer sera, 60% for medium-titer sera and 30% for low-titer sera.     

Echinococcus granulosus: study of the in vitro complement activation by protoscoleces by measuring the electric potential difference across the tegumental membrane.

Complement activation by protoscoleces of Echinococcus granulosus was studied by analyzing the damage to their tegumental membrane produced by incubation in both normal and hydatid human sera. The state of the apical tegumental membrane was evaluated by measuring the electric potential difference with microelectrodes. Protoscoleces incubated in Ringer-Hepes or in heat-decomplemented normal human serum in the presence or absence of specific antibodies did not show significant variations in the electric potential difference throughout the experiment (P > 0.4 in all cases) and their mean values were -46 +/- 3, -43 +/- 4, and -56 +/- 5 mV, respectively. In contrast the potential difference of protoscoleces incubated in 1:2 diluted normal human serum showed a significant variation (P < 0.001), reaching -10 +/- 6 mV after 30 min, and the median depolarization time was estimated to be 21 +/- 3 min. The capacity of normal human serum to depolarize the tegumental membrane of protoscoleces was abolished by treatment at 50 degrees C during 20 min or by 10-fold dilution. In addition, protoscoleces incubated in 1:10 diluted hydatid human serum plus 1:10 diluted normal human serum or Factor B-inactivated normal human serum showed a significantly faster depolarization (0.01 < P < 0.02 and P < 0.001, respectively): the potential difference reached -13 +/- 5 mV after 15 min and the median depolarization times were 9 +/- 5 and 5 +/- 3 min, respectively. Our results suggest that following the time course of the potential difference is a useful tool for studying complement activation in the host-parasite interface and they show that the tegumental membrane of protoscoleces can activate the alternative pathway of human complement.     

Micromethod for Assaying Serum Levels of Erythromycin

A micromethod for assaying serum levels of erythromycin is described. The assay had the following characteristics: detection of 0.03 to 0.035 mug/ml, a long-range curve which minimizes sample dilution, 0.04 ml for a single measurement (0.3 ml required for full coverage), and utility for a variety of body fluids. The method employs radial diffusion from small paper discs which were saturated by capillary action rather than by dipping or pipetting. Although the method was designed to handle serum in which the sample volume is limited, statistical analysis demonstrated that the method has satisfactory precision for routine use. A study of 10 consecutive assays indicates a precision of +/-12% at the 95% confidence limits. The method of least squares was used to calculate the line of best fit, and the statistics were developed on this basis. The assay method is applicable to a variety of antibiotics.     

Serodiagnosis of M. pneumoniae infections by enzyme-linked immunosorbent assay (ELISA)

The enzyme-linked immunosorbent assay (ELISA) has been used for detection of antibodies against Mycoplasma pneumoniae. The results of ELISA and its sensitivity compared with other serological methods, such as complement fixation (CF), metabolic inhibition (MI), mycoplasmacidal test (MC), and radioimmunoprecipitation (RIP) are reported. ELISA and MC showed greater sensitivity than CF and MI, while RIP showed serum titer two- to 16-fold higher. ELISA was specific as determined using other human mycoplasma. A simplified method based on the determination of ELISA antibody end-point titer by a single serum dilution has been proposed. ELISA presented several advantages: sensitivity, rapidity, and low cost and, if adequately standardized, could become a reliable method for the serodiagnosis of M. pneumoniae infection.     

Recording and interpretation of the results obtained by using the ELISA Sevatest commercial immunoenzyme kit for detecting Toxoplasma gondii antibodies

The results of the repeated tests of 295 serum samples from patients, previously examined by means of enzyme immunoassay screening and traditional serological tests (complement fixation, indirect immunofluorescence, and indirect hemagglutination) and 115 serum samples from healthy donors, studied by enzyme immunoassay techniques with the use of the commercial kits Sevatest ELISA, have been analyzed. The methodological approach permitting the determination of the final titer of the serum under study, taken in a dilution of 1:800, by its optical density has been used. The mean geometric titer for the control group of donors, determined in the enzyme immunoassay, has been 1:800. This fact suggests that titers exceeding 1:1,600 should be considered diagnostically significant.     

Validated capillary gas chromatographic-mass spectrometric assay to determine 2-methylcitric acid I and II levels in human serum by using a pulsed splitless injection procedure

BACKGROUND: Despite some clinical applications of 2-methylcitric acid (2-MCA) determination in urine and amniotic fluid, a diagnostic use of 2-MCA levels in serum is not common practice. This could be related to the complexity of the assay, or possibly to unawareness of other feasible clinical applications. METHODS: The levels of the diastereomers 2-MCA I and II in human serum were determined by GC-MS based on a method using a pulsed splitless injection technique. A stable isotope dilution principle was modified considering the diastereomer ratio and impurities of the internal standard. Precision parameters as well as recovery rates of the assay were determined. Reference intervals for 2-MCA(total), 2-MCA I and II levels were obtained in 52 healthy volunteers (31 female, 21 male, mean age 41.7+/-14.4 years). RESULTS: 2-MCA was readily detected in each sample of serum, as well as in urine, cerebrospinal fluid and amniotic fluid. The limit of detection was 10 nmol/l for 2-MCA(total). The internal standard showed a diastereomer ratio of 2-MCA II-d3 to 2-MCA I-d3 of 0.83+/-0.05, its chemical purity had to be corrected to 90.5+/-0.5%. In concentrations of 446, 750 and 1256 nmol/l 2-MCA(total), recovery rates of 98.5, 93.7 and 88% with a mean intra-assay RSD of 1.5% were determined. The day-to-day precision was 10% RSD (SD 40 nmol/l) for 2-MCA(total) obtained with a pooled serum sample at a concentration of 401 nmol/l 2-MCA(total) over a period of 5 months (n=17). The normal range for 2-MCA(total) in human serum was calculated as 81-266 nmol/l confirming previous findings. CONCLUSIONS: The GC-MS assay using a pulsed splitless injection procedure ensures a good response to differing concentrations of 2-MCA in various specimens. Considering exact determination of the diastereomer ratio as well as the purity of the internal standard, the assay offers good precision and recovery for 2-MCA I and II levels in serum.     

Plasmid-mediated serum resistance and alterations in the composition of lipopolysaccharides in Salmonella dublin

Survival rates of Salmonella dublin in rabbit serum after culture for 1 h at 37 degrees C were compared between a wild-type strain (5240) carrying a 50 MDa plasmid, a plasmid-cured strain (C524), and a cured strain containing the 50 MDa plasmid tagged with Tn1 (5241). Strain C524 was more susceptible to the bactericidal activity of normal serum than its parent strain 5240 (percentage survival less than 1% and 52.5 +/- 9.2%, respectively). On the other hand, the percentage survival of strain 5241 was significantly increased (90.4 +/- 4.0%), indicating that the reintroduction of the plasmid into the cured strain restored the serum resistance. Moreover, this change in the serum resistance properties correlated with changes in the neutral sugar composition of the lipopolysaccharides (LPS) of these strains, suggesting that the 50 MDa plasmid is necessary for O-side chain expression in the LPS of S. dublin.     

Agglutinating and Bactericidal Properties of Fractions of Rabbit Anti-Vibrio cholerae Serum

The major portion of the agglutinating and bactericidal activity of the sera of rabbits immunized with live Vibrio cholerae or with cholera vaccine was found in the gammaM fractions during the early stages of immunization. After 5 weeks or more, gammaG fractions accounted for more than half of the agglutinating activity. When late antibody was measured as the amount of protein precipitated by somatic antigens, nearly 3 times as much gammaG as gammaM was required for agglutination, and about 30 times as much gammaG as gammaM was required to kill 50% of a standard inoculum in the presence of complement. The ratio of vibriocidal to agglutinin titer of gammaG fractions at different stages of immunization was more variable than that of gammaM fractions. More complement was required for a vibriocidal effect by gammaG than by gammaM. Increasing the amount of complement decreased the amount of both gammaG and gammaM required to kill, but smaller amounts of gammaM required disproportionately larger amounts of complement. Less time was required by gammaM than by gammaG to kill 50% of the inoculum. Removal of the group-reactive antibody from anti-Ogawa serum and serum fractions by absorption with Inaba reduced the vibriocidal titer by more than one-half.     

Cortisol production rate measurement by stable isotope dilution using gas chromatography-negative ion chemical ionization mass spectrometry.

Presented here is a stable isotope dilution technique for determining cortisol production rate (CPR). The method involves extraction and derivatization of cortisol isoforms from serum (0.5 ml), separation of derivatives by gas chromatography, and detection by using negative ion chemical ionization mass spectrometry. This method provides 50-100-fold greater sensitivity than positive ion mass spectrometry and allows for estimations of cortisol production rate with the use of small amounts of pooled serum, even in the presence of high concentrations of lipophilic contaminants. The area under the curve for the total selected ion chromatogram of fluoroacyl derivatives of cortisol (d0, m/z 782) and deuterated cortisol (d3, m/z 785) were used to determine the isotopic dilution ratio in three types of samples: 1) standards: d0/d3 ratios ranging from 1 to 8%; 2) controls: d3-cortisol added to serum with known cortisol concentration; 3) subjects: 24-h pooled serum samples (q 30 min over 24 h) from healthy children (male 10-13 years; female 7-11 years) receiving continuous infusions of d3-cortisol at 2-4% of their estimated CPR. Recovery after the solid phase extraction and derivatization process was >90%, as determined by thin-layer chromatography. Expected versus measured ratios for d3/d0 in standards and serum controls were highly correlated (r2(standard) = 0.99; r2(control) = 0.99) over a wide range of d3-cortisol enrichment (1.0-10.0%). Mean 24-h CPRs were 4.8 +/- 0.6 mg/m2/24 h (mean +/- SEM, n = 7) in male children and 4.4 +/- 0.5 mg/m2/24 h in female children (n = 4). These CPR values are lower than those derived by radio tracer methods, but are in agreement with previous isotopic dilution studies. This technique is an important tool for assessing CPRs in a wide range of disease states affecting cortisol production.     

The Stability of Complement-Mediated Bactericidal Activity in Human Serum against Salmonella

The complement cascade includes heat-labile proteins and care is required when handling serum in order to preserve its functional integrity. We have previously used a whole human serum bactericidal assay to show that antibody and an intact complement system are required in blood for killing of invasive isolates of Salmonella. The aim of the present study was to evaluate the conditions under which human serum can be stored and manipulated while maintaining complement integrity. Serum bactericidal activity against Salmonella was maintained for a minimum of 35 days when stored at 4°C, eight days at 22°C and 54 hours at 37°C. Up to three freeze-thaw cycles had no effect on the persistence of bactericidal activity and hemolytic complement assays confirmed no effect on complement function. Delay in the separation of serum for up to four days from clotted blood stored at 22°C did not affect bactericidal activity. Dilution of serum resulted in an increased rate of loss of bactericidal activity and so serum should be stored undiluted. These findings indicate that the current guidelines concerning manipulation and storage of human serum to preserve complement integrity and function leave a large margin for safety with regards to bactericidal activity against Salmonella. The study provides a scheme for determining the requirements for serum handling in relation to functional activity of complement in other systems.     

A rapid microtiter plate serum bactericidal assay method for determining serum complement-mediated killing of Mannheimia haemolytica

In this study, we describe a rapid microtiter serum bactericidal assay (RMSBA) that can be used to measure the functionality of immune sera. It quantifies bactericidal activity of immune sera in the presence of complement against a homologous bacterium, M. haemolytica in this case. There is high correlation between data from RMSBA and standard complement-mediated bacterial killing assay (r=0.756; p<0.0001). The RMSBA activity of sera can be generated in less than 5 h instead of overnight incubation. RMSBA costs substantially less in terms of time, labor, and resources and is highly reproducible.     

In vitro activity of propolis against Streptococcus pyogenes

Propolis, a multifunctional substance used by bees to maintain the safety of their hives, is popular for its therapeutic potential against some micro-organisms. Ethanolic extracts of two propolis specimens, collected from different areas within a region in the north-west of Italy, were examined to evaluate their antimicrobial activity against 46 Streptococcus pyogenes strains. By both agar dilution and agar diffusion methods, the minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) were 相似文献   

Direct quantitative analysis of lysophosphatidic acid molecular species by stable isotope dilution electrospray ionization liquid chromatography-mass spectrometry

In order to better understand the role of lysophosphatidic acid (LPA) in physiology and pathophysiology, it is necessary to accurately determine the molecular species and amounts of LPA in biological samples. We have developed a stable-isotope dilution, liquid chromatography-mass spectrometry assay for the direct quantitative analysis of 1-acyl-LPA. This method utilizes a deuterium-labeled internal standard, LPA (18:0-d(35)), and a single liquid-liquid extraction with acidic butanol that allows >95% recovery of LPA, followed by online normal-phase liquid chromatography-mass spectrometry. This protocol allows for the accurate, sensitive, and reproducible analysis of the individual 1-acyl-LPA species present in biological samples. The utility of the assay is demonstrated through the analysis of LPA species in plasma and serum from human volunteers. Total LPA in EDTA plasma was 0.61 +/- 0.14 microM in males and 0.74 +/- 0.17 microM in females, which increased to 0.91 +/- 0.23 and 0.99 +/- 0.38 microM after incubation for 24 h at 25 degrees C. Total LPA in serum was 0.85 +/- 0.22 microM in males and 1.57 +/- 0.56 microM in females, which increased to 4.78 +/- 0.89 and 5.57 +/- 0.73 microM after incubation for 24 h at 25 degrees C.     

Effects of protein source and co-culture on bovine embryo development in synthetic oviductal fluid medium

Experiment 1 compared the development of 2- to 4-cell bovine embryos cultured in synthetic oviductal fluid with 20% fetal calf serum or 3.2% BSA and in the presence of oviductal cells, cumulus cells, or medium alone. More embryos developed in medium with serum, regardless of culture method (P = 0.063). Oviductal cell co-culture resulted in more embryos developing to at least the morula stage (P /= 0.400). Addition of serum to oviductal cell co-culture medium increased the number of excellent or good quality embryos (P = 0.019). Experiment 2 further compared the development of 2-cell or 3- to 4-cell embryos co-cultured with oviductal cell suspensions in serum-supplemented synthetic oviductal fluid or M-199 medium. More 3- to 4-cell than 2-cell embryos developed to at least the morula stage (P < 0.001). More embryos developed to at least the morula stage in synthetic oviductal fluid (P = 0.083). Neither initial embryo cell stage nor medium type influenced the percentage of developing embryos that achieved the blastocyst stage or final morphological quality of embryos (P >/= 0.535).     

Development and calibration of a standard for the protein content of granulocyte colony-stimulating factor products

This collaborative study characterizes a homogeneous standard for the protein content determination of granulocyte colony-stimulating factor (G-CSF) products with traceability of the measurement. The Kjeldahl method was used to determine the average protein content of G-CSF bulk as 2.505 mg/ml (95% C.I: 2.467-2.543 mg/ml, GCV 4.0%). Using G-CSF bulk as a traceability benchmark, the protein content of the final freeze-dried standard using reverse phase HPLC (RP-HPLC) was 215.4 μg protein per ampoule (95% C.I: 212.407-218.486 μg/ampoule, GCV 3.4%). A comparative study showed that there was no difference between using Filgrastim CRS (European Pharmacopeia G-CSF reference standard) and freeze-dried homogeneous standard when quantifying G-CSF protein content by RP-HPLC (P > 0.05). However, there were significant differences in the G-CSF protein content obtained using a serum albumin standard by Lowry assay and a G-CSF standard with RP-HPLC. Therefore, use of RP-HPLC with a freeze-dried homogeneous standard would eliminate the systematic errors introduced when using a serum albumin standard because of the differences in protein composition between the standard and the sample. It would also be helpful to use this method to compare the quality of G-CSF biosimilar products in situations where the protein content has been calibrated using various standards.     

A New Method for the Detection of Neutralizing Antibodies against Mumps Virus

Neutralization test is the most reliable method of evaluating immunity against viral diseases but there is no standard procedure for mumps virus, with tests differing in the infectivity of the challenge virus, 50% plaque reduction or complete inhibition of cytopathic effects (CPE), and usage of complement. A reliable, easy, and simple neutralization test for mumps virus was developed in this study. A recombinant mumps virus expressing GFP was generated as a challenge virus. Complement was added to the neutralizing mixture at 1∶200 when stocked serum samples were used. Neutralizing antibody titers were expressed as the reciprocal of the highest dilution that did not exceed two-fold of FU values (GFP expression) of the cell control wells. A total of 1,452 serum samples were assayed by inhibition of GFP expression in comparison with those examined by conventional 100% inhibition of CPE. 1,367 (94.1%) showed similar neutralizing antibody titers when examined by both methods. The GFP expression inhibition assay, using a recombinant mumps virus expressing GFP, is a simple and time- saving method.     

Schistosoma mansoni: killing of transformed schistosomula by the alternative pathway of human complement

The interaction of mechanically transformed schistosomula of Schistosoma mansoni with the alternative pathway of human complement was studied in vitro. To detect early changes in transformation, the schistosomula were prepared at a low temperature and used immediately. As shown previously, freshly transformed schistosomula were highly susceptible to killing by normal human serum and by C4-depleted normal human serum. This serum activity was concentration dependent and was markedly reduced on a twofold serum dilution. Upon incubation at 37 C in defined synthetic medium, schistosomula rapidly became refractory to killing by the alternative pathway of complement. After 1 hr of incubation at 37 C, the percentage of schistosomula which were resistant to killing increased from 16 to 85. This conversion was accompanied by a fivefold decrease in deposition of C3b on schistosomula which had been exposed to 37 C for 1 hr and then further incubated with C4-depleted normal human serum. The following events occurred concomitantly during incubation of freshly transformed schistosomula at 37 C with a half-life of 30-60 min: (1) Decrease in activation and consumption of the alternative pathway of complement by schistosomula; (2) appearance of a strong complement consuming activity in the supernatant of incubating schistosomula; and (3) shedding of protein- and carbohydrate-containing substances from the surface of schistosomula into the supernatant. Isolated external membranes of freshly transformed schistosomula consumed the alternative pathway of complement to a greater extent than membranes of schistosomula preincubated in medium at 37 C. The results demonstrate that transformed schistosomula acquire resistance to complement killing via the alternative pathway by shedding complement-activating substances.(ABSTRACT TRUNCATED AT 250 WORDS)     

Measuring the 50% Haemolytic Complement (CH50) Activity of Serum

The complement system is a group of proteins that when activated lead to target cell lysis and facilitates phagocytosis through opsonisation. Individual complement components can be quantified however this does not provide any information as to the activity of the pathway. The CH₅₀ is a screening assay for the activation of the classical complement pathway (Fig 1) and it is sensitive to the reduction, absence and/or inactivity of any component of the pathway. The CH₅₀ tests the functional capability of serum complement components of the classical pathway to lyse sheep red blood cells (SRBC) pre-coated with rabbit anti-sheep red blood cell antibody (haemolysin). When antibody-coated SRBC are incubated with test serum, the classical pathway of complement is activated and haemolysis results. If a complement component is absent, the CH₅₀ level will be zero; if one or more components of the classical pathway are decreased, the CH₅₀ will be decreased. A fixed volume of optimally sensitised SRBC is added to each serum dilution. After incubation, the mixture is centrifuged and the degree of haemolysis is quantified by measuring the absorbance of the haemoglobin released into the supernatant at 540nm. The amount of complement activity is determined by examining the capacity of various dilutions of test serum to lyse antibody coated SRBC. This video outlines the experimental steps involved in analysing the level of complement activity of the classical complement pathway.Download video file.^{(81M, mp4)}