Statistical Evaluation of Diluents and Automatic Diluting and Pipetting Machines in Influenza Serology

The use of three diluents (i.e., 0.01 m phosphate-buffered saline, PBS; PBS with 0.2% gelatin, PBS/GEL; and PBS with 0.4% bovine plasma albumin) and three methods (i.e., the standard tube macro-procedure, TUBE; the manual microtechnique, MANUAL; and the semiautomatic microtechnique, AUTO) were statistically compared for their reproducibility and sensitivity in determining hemagglutinin (HA) and hemagglutination-inhibition (HI) antibody titers. In the HA test, analyses of between-cell variances of the different methods showed the AUTO microtiter procedure to be more reproducible than the standard TUBE method. The MANUAL microtiter procedure was the least reproducible. In the HI test, the TUBE method was the most reproducible. No significant difference in the reproducibility of the diluents was observed in either the HA or HI test. When a comparison of the sensitivity of test methods and diluents was made for determining HA titers, the AUTO microtiter procedure and PBS/GEL diluent appeared to be the method and diluent of choice. Evaluation of another instrument, the autopipetter, which standardizes the volume of diluent to be added in the microtechnique, suggests that the reproducibility of the AUTO microtiter procedure might be further increased.     

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.     

Titration of tetanus antitoxin by passive hemagglutination. I. Titration of guinea-pig antitoxins at various periods of immunization.

An improved technique for passive hemagglutination (HA) for titration of tetanus antitoxin was described. The use of highly purified tetanus toxoid and of improved diluent increased the specificity and reproducibility of the test. Several hundreds of specimens of guinea-pig serum taken at various stages of immunization were titrated by HA and toxin neutralization (NT) in mice. The ratio of HA to NT titers varied significantly depending on the immunization stage; higher at early stages and lower at later stages. The high HA/NT ratio was not due to the IgM antitoxin, which is very rare in guinea pigs. The variation in discrepancy between HA and NT titers decreased considerably by grouping the serum specimens with respect to the stage of immunization. Thus, it is possible to predict the in vivo titer of a tetanus antitoxin accurately enough for clinical study. The HA test may be useful as an alternative method for titrating tetanus antitoxin in the field trials. Moreover, it can be used for the study of characteristics of antitoxins.     

Studies on the growth-stimulatory activity of pigeon milk-comparison and synergistic effects with serum

Pigeon milk, a nutritive secretion from the crop of breeding pigeons, was tested (on v/v basis) for growth factor activity either separately or in combination with other growth supplements. Synthesis of DNA in confluent monolayers of quiescent Chinese hamster ovary cells was enhanced by the homogenates of pigeon milk in the presence of both fetal bovine serum and bovine serum albumin, although the response with fetal bovine serum was greater than that with bovine serum albumin. The in vitro growth stimulation by pigeon milk was also reflected in the increase in cell number. Specific activity of pigeon milk growth factor, measured against both Chinese hamster ovary cells and mouse embryo fibroblasts, was found to be higher than that of fetal calf serum, fetal bovine serum, and goat, horse, pig and human serum. The growth-stimulatory property of pigeon milk did not change in the first 5 days of its secretion.Abbreviations BSA bovine serum albumin - CHO Chinese hamster ovary cells - DMEM Dulbecco's modified minimum essential medium - DNA deoxyribonucleic acid - EDTA ethylenediaminetetraacetic acid - EGF epidermal growth factor - FBS fetal bovine serum - FCS fetal calf serum - GF growth factor - GS goat serum - NIH/3T3 mouse embryo fibroblasts - PBS phosphate-buffered saline - PDGF platelet-derived growth factor - PM pigeon milk     

Identification of amino acids of influenza virus HA responsible for resistance to a fusion inhibitor, Stachyflin

We have recently described a novel hemagglutinin (HA) conformational change inhibitor of human influenza virus, Stachyflin (Yoshimoto et al, Arch. Virol., 144, 1-14, 1999). Stachyflin-resistant variants of human influenza A/WSN/33 (H1N1) virus were isolated in vitro and the nucleotide sequences of their HA genes were determined. The relation of amino acid substitutions and Stachyflin resistance was analyzed with in vitro membrane fusion between HA-expressing cells and octadecylrhodamine (R18)-labelled chick erythrocytes (RBC). The amino acid substitutions, lysine to arginine at position 51 or lysine to glutamic acid at position 121 of the HA2 subunit of the HA protein was enough to confer a Stachyflin-resistant phenotype of HA protein. The molecular mechanism of anti-HA conformational change activity of Stachyflin is discussed.     

An Improved Diluent for Rubella Hemagglutination and Hemagglutination-Inhibition Tests

Rubella hemagglutinating (HA) antigen was prepared in BHK-21 tissue as 5% cell suspensions and from unconcentrated and 20× concentrated infected supernatant fluids. In some instances, unconcentrated fluids were treated with Tween 80 and ether; cell suspensions were treated with ether alone. Preparations were tested for HA activity in dextrose-gelatin-Veronal (DGV) buffer solutions; 0.85% NaCl; Sorenson's phosphate-buffered saline, pH 7.2; and a diluent of 0.9% NaCl, 0.1% CaCl₂ (anhydrous), and 0.1% MgSO₄·7H₂O. HA titers were consistently two- to fourfold higher in the saline with added Ca⁺⁺ and Mg⁺⁺ than in DGV. Hemagglutination-inhibition titers of paired human sera were the same in either diluent. It is suggested that the interaction between rubella HA antigen and the red cells of young (less than 1-day-old) chicks may be at least partially ion dependent and that titers are enhanced by increased quantities of divalent cations.     

pH-dependent binding of the fluorophore bis-ANS to influenza virus reflects the conformational change of hemagglutinin

Binding of the fluorophore 1,1-bis(4-anili-no) naphthalene-5,5-disulfonic acid (bis-ANS) to influenza virus A/PR 8/34 is strongly enhanced at low pH. Binding is accompanied by a significant increase in fluorescence intensity. The binding and the fluorescence increase are associated with the low-pH induced conformational change of the viral spike protein, hemagglutinin, exposing hydrophobic binding sites. The data indicate that in addition to the hydrophobic N-terminus of HA2 other hydrophobic sequences of the HA ectodomain become accessible to bis-ANS at low pH. It is shown that the time course of the fluorescence increase of bis-ANS at low pH is determined by the conformational change of HA. The application of this assay for continuously monitoring the kinetics of the structural alteration in HA is discussed and its relevance for elucidating the temporal relationship between the conformational change of HA and virus-membrane fusion is outlined.Abbreviations HA hemagglutinin - BHA bromelain-solubilized ectodomain of HA - N-HA2 N-terminus of the HA2 subunit - PBS phosphate buffered saline - bis-ANS (1,1-bis(4-anilino)naphthalene-5,5-disulfonic acid) - R18 octadecylrhodamine B chloride - FDQ fluorescence dequenching - RBC red blood cell Correspondence to: A. Herrmann     

Adaptation of a duck influenza A virus in quail

Quail are thought to serve as intermediate hosts of influenza A viruses between aquatic birds and terrestrial birds, such as chickens, due to their high susceptibility to aquatic-bird viruses, which then adapt to replicate efficiently in their new hosts. However, does replication of aquatic-bird influenza viruses in quail similarly result in their efficient replication in humans? Using sialic acid-galactose linkage-specific lectins, we found both avian (sialic acid-α2-3-galactose [Siaα2-3Gal] linkages on sialyloligosaccharides)- and human (Siaα2-6Gal)-type receptors on the tracheal cells of quail, consistent with previous reports. We also passaged a duck H3N2 virus in quail 19 times. Sequence analysis revealed that eight mutations accumulated in hemagglutinin (HA) during these passages. Interestingly, many of the altered HA amino acids found in the adapted virus are present in human seasonal viruses, but not in duck viruses. We also found that stepwise stalk deletion of neuraminidase occurred during passages, resulting in reduced neuraminidase function. Despite some hemagglutinin mutations near the receptor binding pocket, appreciable changes in receptor specificity were not detected. However, reverse-genetics-generated viruses that possessed the hemagglutinin and neuraminidase of the quail-passaged virus replicated significantly better than the virus possessing the parent HA and neuraminidase in normal human bronchial epithelial cells, whereas no significant difference in replication between the two viruses was observed in duck cells. Further, the quail-passaged but not the original duck virus replicated in human bronchial epithelial cells. These data indicate that quail can serve as intermediate hosts for aquatic-bird influenza viruses to be transmitted to humans.     

甲型H1N1流感病毒血凝素HA1在糖基工程毕赤酵母中的表达

目的:建立用糖基工程酵母制备流感血凝素的方法 ,研究其免疫原性,为酵母表达流感疫苗提供基础。方法:通过PCR的方法扩增编码H1N1流感病毒血凝素HA1(1～330 aa)的基因片段,将HA1基因克隆到表达载体pPIC9质粒上,电转化到糖基工程酵母中,甲醇诱导表达并用镍亲和层析柱纯化重组蛋白,N-糖苷酶F(PNGF)酶切分析N-糖链,Western印迹验证纯化蛋白,免疫小鼠并测定HA1诱导抗体的滴度。结果:获得HA1基因的酵母重组表达菌株,SDS-PAGE分析可见野生型GS115表达的重组HA1相对分子质量约为100×103,而糖基工程酵母GJK01表达的HA1约为60×103,PNGF酶切后相对分子质量均降至45×103左右;经Western印迹检测,这些条带均为目的蛋白条带,野生型和糖基工程酵母表达的HA1分子大小不同是由于不同的N-糖基化修饰引起的。重组HA1免疫小鼠可产生抗HA1抗体,随着抗原剂量的增加,其产生的抗体滴度相应增加;3次免疫后,4μg HA1诱导小鼠产生的抗体滴度最高。结论:利用糖基工程酵母表达制备了低糖化的流感病毒血凝素HA1,该重组蛋白可以诱导小鼠产生HA1抗体,且产生的抗体滴度具有HA1剂量依赖性。     

Oligosaccharide composition of an influenza virus hemagglutinin with host-determined binding properties

We have previously reported that the binding properties of the hemagglutinin (HA) of the WSN-F strain of influenza A are affected by the cells in which the virus is grown (Crecelius, D. M., Deom, C. M., and Schulze, I.T. (1984) Virology 139, 164-177); at 37 degrees C chick embryo fibroblast-grown F virus has a greater affinity for host cells than does the same virus grown in Madin-Darby bovine kidney (MDBK) cells. In an attempt to explain this host-determined property, we have characterized the carbohydrate put onto the viral HA by these two cells. Experiments using tunicamycin indicate that the HA made by MDBK cells contains about 4000 daltons of carbohydrate in excess of that on the HA from chick embryo fibroblast. Serial lectin affinity chromatography of the asparagine-linked oligosaccharides on the HA subunits, HA1 and HA2, detected a number of host-dependent differences in the complex oligosaccharides. Both HA1 and HA2 from MDBK cells contained more highly branched (i.e. tri- and tetraantennary) complex oligosaccharides than did the subunits from chick embryo fibroblasts. In addition, the HA subunits from the two sources differed in the amount of galactose-containing "bisected" complex oligosaccharides and in the presence of certain fucosylated triantennary oligosaccharides. Profiles of the asparagine-linked oligosaccharides from the host cells did not show these differences, indicating that the HA subunit profiles were not necessarily representative of the structures found on the cellular glycoproteins. The data support the conclusion that bulky oligosaccharides on the MDBK-HA subunits of WSN-F reduce the affinity of the virus for cellular receptors.     

Avian smooth muscle: Morphometric analysis and comparison of different types

Summary Smooth muscle from the pigeon gizzard and intestine, and quail gizzard were investigated using ultrastructural morphometric analysis and compared on the basis of volume percent mitochondria, dense bodies, sarcoplasmic reticulum, number of mitochondria/m², and fiber diameter. One-way analysis of variance tests showed significant differences in (1) volume percent mitochondria between pigeon intestine and quail gizzard and between pigeon and quail gizzards; (2) mitochondrial number between all three muscles; and (3) fiber diameter between pigeon gizzard and intestine and pigeon intestine and quail gizzard.     

Dengue Virus Plaque Development in Simian Cell Systems: I. Factors Influencing Virus Adsorption and Variables in the Agar Overlay Medium

Chemical and physical variables influencing the plaquing of all dengue serotypes in two simian cell systems were studied. Calf serum in the nutrient overlay may be replaced by mouse ascitic fluid or bovine plasma albumin when employing the rhesus monkey kidney LLC-MK(2) cell system for plaquing all dengue serotypes. Doubling the serum concentration in the overlay had little effect in modifying dengue types 1, 2, 3, and 4 plaque titers. Newborn agamma, 4-week-old and 8-week-old calf serum gave comparable titers with all dengue virus serotypes. Dengue virus titers, plaque size, and development time were unaffected by sodium bicarbonate concentrations ranging from 1.1 to 4.4 mg/ml of overlay. A twofold increase (0.00332 g%) in the amount of either autoclaved or filtered-sterilized neutral red reduced the dengue 2 virus titer as much as 2.2 logs. An increased Mg(++) and decreased Ca(++) concentration in the overlay medium increased the efficiency of the plaquing system.     

Selective inactivation of the infectivity of freeze-dried Sendai virus by heat

The infectivity of freeze-dried Sendai virus was destroyed after heating at 100 ° C for 20 min while the hemagglutinin (HA) titer and the hemolytic (HL) activity were not affected. The HA titer was unaltered after heating at up to 140 ° C for 30 min. The HL activity was increased after freeze-drying, further increased after heating of freeze-dried virus at 115 ° C for 20 min, but was destroyed after heating for 30 min at 140 ° C.The selective heat inactivation of freeze-dried Sendai virus could be of use in the production of myxovirus vaccines and inactivated virus for cell-fusion studies.     

Airway sizes and proportions in children quantified by a video-bronchoscopic technique

Background

Detecting serum antibody against inhaled antigens is an important diagnostic adjunct for hypersensitivity pneumonitis (HP). We sought to validate a quantitative fluorimetric assay testing serum from bird fanciers.

Methods

Antibody activity was assessed in bird fanciers and control subjects using various avian antigens and serological methods, and the titer was compared with symptoms of HP.

Results

IgG antibody against pigeon serum antigens, quantified by fluorimetry, provided a good discriminator of disease. Levels below 10 mg/L were insignificant, and increasing titers were associated with disease. The assay was unaffected by total IgG, autoantibodies and antibody to dietary hen's egg antigens. Antigens from pigeon serum seem sufficient to recognize immune sensitivity to most common pet avian species. Decreasing antibody titers confirmed antigen avoidance.

Conclusion

Increasing antibody titer reflected the likelihood of HP, and decreasing titers confirmed antigen avoidance. Quantifying antibody was rapid and the increased sensitivity will improve the rate of false-negative reporting and obviate the need for invasive diagnostic procedures. Automated fluorimetry provides a method for the international standardization of HP serology thereby improving quality control and improving its suitability as a diagnostic adjunct.     

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.     

Factors Affecting the Passive Hemagglutination Titration: Dilution Loops, Titration Trays, Vibration, Diluents

Serial dilution with Takatsy loops resulted in exaggerated passive hemagglutination titers with most of the anti-bovine serum albumin sera tested. It appears that certain types of agglutinins adhere to the loop surface and are released only gradually. This adherence, or carry-over effect, was prevented by presoaking loops in gelatin or gelatin-rabbit serum-albumin solutions. Hemolysins did not adhere to loops. In general, hemagglutination reactions performed on plastic trays gave higher titers than those performed in glass test tubes. The quality of the hemagglutination pattern was dependent to a great extent on the type of plastic tray used. As much as a 100-fold difference in titers was obtained depending on the composition of the antiserum diluent. The increase in vibration, in terms of linear displacement (approximately twofold), resulted in an eightfold decrease in titers.     

Comparison of the sensitivities of WaterLOGSY and saturation transfer difference NMR experiments

The WaterLOGSY (WL) and saturation transfer difference (STD) NMR experiments have proven to be extremely useful techniques to characterize interactions between small molecules and large biomolecules. In this work we compare the relative sensitivities of WL and STD NMR using 3 experimental systems: ketoprofen (KET)–bovine serum albumin (BSA), tert-butyl hydroquinone (TBHQ)–hemagglutinin (HA), and chloramphenicol (CAM)–ribosome (70S). In all cases we find that WL is more sensitive than STD for a given experimental time with the ratios ranging from 3.2 for KET–BSA to 16 for TBHQ–HA and CAM–70S. We attribute the increased sensitivity of WL to be due to simultaneous saturation of multiple sources of cross correlation, including direct NOEs of ¹H of water and exchangeable groups and indirect NOEs of ¹H–C groups. We suggest that the outstanding sensitivity of WL make it ideally suited for drug screening efforts targeting very large biomolecules at relatively low concentrations.     

Régulation des excédents dans le développement du bourgeon de membre de l'embryon d'oiseau. Analyse expérimentale de combinaisons xénoplastiques caille/poulet

Summary In order to support the demonstration of the regulative capacity of the chick limb bud, already stressed by one of us (Kieny, 1964, 1967), heterospecific combinations were made between chick and quail tissues, the cells of the latter bearing a distinctive nuclear marker. A Japanese quail whole limb bud (stage-18 to 21 of H. H., wing or leg) was grafted distally onto the prospective zeugopod of a chick (stage-22) wing bud sectioned at the prospective wrist level. Thus, from a heterospecific surplus recombinant containing five prospective limb segments (stylopod and zeugopod from the chick host; stylopod, zeugopod and autopod from the quail graft), it was possible to obtain a normally shaped appendage that comprised either upper arm, lower arm and hand in the case of a wing bud graft, or heteromorphic upper arm, lower leg and foot in the case of a hind-limb bud graft. In these cases, regulation for excess appeared to take place mainly within the host tissues. The three proximal segments of the recombinant, namely the chick stylopod and zeugopod of the host's stump and the quail stylopod of the graft, became reorganized and gave rise to a single stylopodial segment, which usually contained a double stylopodial bone element, one of chick, the other of quail origin.The absence of development of the squeezed prospective zeugopod can be interpreted as follows: owing to an interaction with the stylopodial graft tissues, the zeugopodial cells of the juxtaposed stump boundary have shifted proximally their originally more distal positional values, so that they changed their prospective pattern of differentiation to that of stylopod. These reset zeugopodial cells combine with the stylopodial cells of host and graft and form a huge composite stylopod, in which, due to an asynchronous determination in the two species, chick and quail tissues do not cooperate fully for the development of a single bone.

Ce travail a été effectué avec l'aide de la D.G.R.S.T. (Action complémentaire coordonnée: Biologie de la reproduction et du développement, convention n^o 73-7-1661)     

Antibody to testicular cells in the serum of normal mice

The mixed hemadsorption hybrid antibody (MHA.HA) test was applied successfully to the detection of antigens on the surface of testicular cells separated into sub-populations by velocity sedimentation at unit gravity in the staput apparatus. Normal serum from mice of all strains tested, both male and female, was found to contain a natural autoantibody that reacts with testicular cells of all mice tested, but not with sperm or other cells. This autoantibody is detectable at an age of 4–6 weeks in females, and reaches a plateau at about 10 weeks of age. The corresponding antigen is denoted TCDA, because it is evidently a Testicular Cell Differentiation Antigen. Microscopy of the cells forming rosettes in the MHA.HA test confirmed that the TCDA⁺ cells belong to the gametogenetic series. Because females as well as males produce the autoantibody we presume that TCDA is also present on female gametic cells although it was not feasible to test this adequately. The anti-TCDA autoantibody is not related to the natural autoantibody against sperm, which according to MHA.HA test occurs in the serum of males but not of virgin females.Abbreviations used in this paper are as follows MHA.HA mixed hemadsorption hybrid antibody - TC testicular cells - TCDA testicular cells differentiation antigen - BALB BALB/c - BSA bovine serum albumin - Sp sperm - SpA sperm antigen - Ig immunoglobulin - PBS phosphate-buffered saline - NMS normal mouse serum - FBS fetal bovine serum - SRBC sheep red blood cells     

Domain-specific monoclonal antibodies to ovotransferrin indicate conservation of determinants involved in avian transferrin receptor recognition

1. Three of five monoclonal antibodies produced to chicken ovotransferrin bound quail ovotransferrin but none of the antibodies bound human, bovine or equine serum transferrin. 2. Equilibrium binding experiments indicate that both quail and chicken ovotransferrin bind to transferrin receptors on chick reticulocytes although the quail protein binds to 40% fewer sites with an affinity which is three times lower than chicken ovotransferrin. 3. The antibodies that recognize quail ovotransferrin block binding of both radiolabelled chicken and quail ovotransferrin to chick reticulocytes. 4. Quail NH2-terminal half-molecule domain appears to be unable to form a functional hybrid holo-ovotransferrin with chicken C-terminal half-molecule domain.