测定呼吸道合胞病毒抗体方法敏感性的比较

用ELISA、间接免疫荧光和中和试验三种方法测定兔免疫血清的呼吸道合胞病毒抗体。结果中和试验测定的免疫血清几何平均抗体滴度为1:32;间接免疫荧光试验为1:508,比中和试验高16倍;ELISA测得的抗体滴度1:4,000～1:5,000,为中和试验的125～156倍。以ELISA敏感性最高,间接免疫荧光试验次之,中和试验景差。     

Detection of neutralizing antibodies against α-toxin of different Clostridium septicum strains in cell culture

Clostridium septicum, a ubiquitious organism, is the pathogen which causes the classical malignant edema after injuries. Because of its strong cytotoxic alpha-toxin, infections are often lethal. To prevent losses in animals, vaccination with alpha-toxoid vaccines is carried out. Quality control of the vaccines is done by a neutralization test in mice. A cytotoxin test and as an alternative method to detect neutralizing antibodies, a cytotoxin inhibition test was standardized. In the studies, alpha-toxin of the C. septicum reference strain (NC 547) from the National Collection of Type Cultures was compared with alpha-toxin of a field strain from an outbreak in Germany. Sera from five heterologous polyvalent and three monovalent vaccines from eight rabbit groups were available. Each vaccination had been carried out according to the procedure of the German Pharmacopoeia. In three out of the five sera of the groups vaccinated with the heterologous polyvalent vaccine, cytotoxin neutralizing antibodies were detected. High antibody titers were observed in sera of rabbits vaccinated with a vaccine of strain NC 547, lower titers in the sera of rabbits vaccinated with a vaccine of the field strain. No cytotoxin neutralizing antibodies could be found in the sera of rabbits vaccinated with the monovalent C. chauvoei vaccine. The toxins of all strains showed the same ranking of the vaccines. Vaccines which caused high antibody titers in the animals were detected by all toxins as such, as well as vaccines which had medium or low antibody inducing capacity. The results were independent of the C. septicum strain used for the production of alpha-toxin.     

SEROLOGICAL ATTRACTION OF NONTOXIC EGG COMPONENT TO STAPHYLOCOCCAL ANTI-ENTEROTOXIN

Raw whole liquid and dried eggs which were purported to contain staphylococcal enterotoxin were analyzed by a number of enzyme-linked immunosorbent assay (ELISA)-based methods. The initial evaluation was to establish whether the purported positive ELISA reactions were a result of toxin-anti-enterotoxin serological activity. A secondary consideration was to determine whether the putative protein occurred in the yolk and/or white portions of the eggs. A manual polyvalent detection system, manual monovalent ELISA and an automated polyvalent enzyme-linked fluorescent immunoassay were used to investigate this component in eggs. The ELISA results were instantaneous and showed strong positive reactions (false positives) with the manual and automated polyvalent systems, suggesting nonspecific binding of the egg-reactive component to one or more staphylococcal enterotoxin antibodies. Further analysis with the monovalent ELISA showed false-positive reactions when egg extracts were tested separately for enterotoxins A–E. The putative protein from fertilized eggs had caused false-positive reactions and the eggs did not contain preformed staphylococcal enterotoxin.

PRACTICAL APPLICATIONS

The analysis of raw whole liquid and dried eggs for staphylococcal enterotoxin must be performed with circumspection when applying serological systems which use whole antibody, as the resulting positive reaction could be a false-positive reaction due to a nontoxic component in eggs which reacts with the staphylococcal anti-enterotoxins.     

Detection of pneumococcal antibodies in children with acute pneumonia and pleurisy by an immunoenzyme method

The samples of sera and pleural fluid from sick children have been analyzed by means of EIA techniques. To detect the time course of antibody production, the antigenic preparations of pneumococci (monovalent capsular polysaccharides, polyvalent polysaccharide vaccine and complex pneumococcal antigen) have been used. Antibody response observed in the forms of pneumococcal infection, studied in this investigation, has proved to be highly variable. It is expedient to determine antibodies to polysaccharide antigens not earlier than on days 10-12 from the beginning of the disease. But, besides the positive dynamics of antibodies, their unchanged level is sometimes observed in the patients at the beginning of the disease. As a rule, there is a coincidence between the dynamics of antibody formation in response to polysaccharide antigens and to complex pneumococcal antigen.     

猪和牛轮状病毒抗原和抗体的检测

应用ELISA直接双抗体夹心法检查轮状病毒抗原,24份仔猪和29份犊牛的腹泻粪样,分别有12和16份阳性。用病毒RNA电泳分析检查阳性粪样,各出现两种病毒RNA电泳型,用中和试验检查17份成年牛和16份成年猪血清,分别有16和15份病毒抗体阳性。将其与ELISA间接法和结合法进行了比较。     

Effects of Homologous O-Antibody on Host Responses to Lipopolysaccharide from Yersinia enterocolitica: Neutralization of Its Pyrogenicity

The pyrogenic activity of lipopolysaccharide (LPS) obtained from Yersinia enterocolitica was neutralized by the homologous O-antiserum at the optimal antigen/antibody ratio. The level of neutralization in either antigen excess or extreme antibody excess was significantly lower than that at the optimal ratio. These facts suggested that the structure of the so-called “lattice” of LPS/antibody complex might influence the neutralization of pyrogenic activity. Moreover, when LPS was gradually added to the antiserum to reach the optimal antigen/antibody ratio, the pyrogenic activity of LPS was only slightly neutralized by the antibody. The similar event to Danysz phenomenon that has been known in the diphtheria toxin-antitoxin system was also observed in the LPS-anti-LPS system.     

Determination of the internal association constant as a measure of the affinity of erythocyte-fixed antibodies that interact with mono- and multivalent antigens]

The fraction of antibody-bound determinants of a polyvalent antigen is determined by the fraction of free antigen molecules and the valence of the antigen. The internal association constant for antibodies fixed onto erythrocytes can be found by the suspension method, the index obtained in this process being similar when both monovalent and polyvalent antigens are used. The sensitivity of the antibody-erythrocyte diagnostic preparation in the passive hemagglutinin test depends on the affinity of the active centers of antibodies.     

Antibody responses elicited by a polyvalent vaccine containing synthetic diphtheric, streptococcal and hepatitis peptides coupled to the same carrier

Three synthetic peptides copying fragments of the diphtheria toxin, the M protein of the streptococcus type 24 and the hepatitis B virus surface antigen (HBs) have been conjugated together to the tetanus toxoid. This polyvalent vaccine has been administered to mice. High antibody titers were obtained against the three antigens. No cross-reactivity could be observed between them as demonstrated by the ability of each peptide to inhibit only the antibodies against the natural M protein and the synthetic M protein peptide indicated that the avidity of the antibodies raised against a monovalent streptococcal vaccine were identical to those raised following injection of the polyvalent vaccine. Antibodies raised against the polyvalent streptococcal vaccine were also protective as shown by opsonophagocytic assays.     

Efficient neutralization of foot-and-mouth disease virus by monovalent antibody binding.

Neutralization of an aphthovirus by monovalent binding of an antibody is reported. Foot-and-mouth disease virus (FMDV) clone C-S8c1 was neutralized by monoclonal antibody (MAb) SD6, which was directed to a continuous epitope within a major antigenic site of the G-H loop of capsid protein VP1. On a molar basis, the Fab fragment was at most fivefold less active in neutralization than the intact antibody, and both blocked virus attachment to cells. Neither the antibody nor the Fab fragment caused aggregation of virions, as evidenced by sucrose gradient sedimentation studies of the antibody-virus complex formed at antibody to virion ratios of 1:50 to 1:10,000. The results of neutralization of infectivity and of ultracentrifugation are fully consistent with structural data based on X-ray crystallographic and cryoelectron microscopy studies, which showed monovalent interaction of the antibody with a critical receptor binding motif Arg-Gly-Asp. The conclusions of these neutralization studies are that (i) bivalent binding of antibody is not a requisite for strong neutralization of aphthoviruses and (ii) aggregation of viral particles, which has been proposed to be the dominant neutralization mechanism of antibodies that bind monovalently to virions, is not necessary for the neutralization of FMDV C-S8c1 by MAb SD6.     

New equine antitoxins to botulinum neurotoxins serotypes A and B

Hyperimmune monovalent antitoxins to botulinum neurotoxin serotypes A and B have been produced by immunizing horses with newly developed formalin toxoids. After primary immunization, horses developed acceptable prophylactic antibody titers (1-5 IU/mL). Three horses received additional toxoid booster injections to induce hyperimmune antibody titers with antitoxin-A and antitoxin-B titers reaching peaks of approximately 2000 IU/mL and 150-625 IU/mL, respectively. Titers were quantified throughout the process by antigen-capture ELISA and by in-vivo neutralization. ELISA titers and neutralization titers correlated (R2 ～0.62-0.92), however, unique correlations between in-vitro and in-vivo titers were observed for each horse. Monovalent antitoxin pools were made by combining plasma that had been collected twice via plasmaphoresis several months after primary immunization. Neutralizing units were established for each pool relative to the current US and WHO reference standards. Titers were determined at the L(+)/10 and L(+)/40 toxin dose for Toxin types A and B, respectively, and U.S. and international units were assigned to each monovalent antitoxin. Avidity of the new Anti-A pool was equivalent to the WHO Anti-A reference at the L(+), L(+)/10 and L(+)/30 dose. Each monovalent plasma pool failed to cross-neutralize other botulinum neurotoxin serotypes indicating a high degree of specificity of each antitoxin for the toxin serotype used during immunization.     

Characteristics of the low molecular antigens of pertussis bacteria]

The authors elaborated a method of obtaining pertussis soluble antigenic complex by dialysis through the cellophane membrane against the physiological saline at a temperature of 4 degrees C. An antigen which was active in the passive hemagglutination and neutralization of antibodies tests was revealed in the dialyzate. The amount of this antigen in the dialyzate increased gradually up to the 7th day and then became stabilized. The serological activity of the antigen after evaportation increased 4-16 times. The results of the antibody neutralization test pointed to the presence in the dialysate of substances common to those contained in the 1a and 1Da fractions isolated from the pertussis bacteria with the aid of ammounium sulfate.     

Detection of postvaccination mumps virus antibody by neutralization test, enzyme-linked immunosorbent assay and sensitive hemagglutination inhibition test

A group of 251 children aged 2-3 years given live attenuated mumps virus vaccine PAVIVAC of Czechoslovak production were tested for antiparotitis antibody levels in pre- and postvaccination sera by neutralization test (NT), enzyme-linked immunosorbent assay (ELISA) and sensitive hemagglutination inhibition test, enhanced by heterologous antibody to human immunoglobulin G (E-HIT). The prevaccination findings were as follows: positive ELISA IgG titres, neutralization antibodies and hemagglutination inhibition antibodies were present in, respectively, 35%, 25.9% and 27.9% of the sera. Postvaccination seroconversions were evaluated in 159 susceptible vaccinees whose prevaccination sera had been negative by all three tests. The lowest seroconversion was detected by NT (74.2%), seroconversions by ELISA and E-HIT were appreciably higher (82.4% and 86.8%, respectively). The seven children showing a seroconversion by E-HIT but not by ELISA had a 4 fold increase of anti-mumps ELISA IgG antibodies as well, but the rise of antibody titres was at a level falling in the range below the positivity criterion for ELISA. The statistically evaluated detection rate for antibodies was significantly higher (significance test "t") by ELISA as compared with neutralization test. However, antibody levels (geometric mean titres) were 8-10 times lower in postvaccination sera than in convalescent sera of 30 children with mumps in all three tests.     

人源抗狂犬病毒中和性全抗体在昆虫细胞中的表达

将来源于噬菌体抗体库的人源狂犬病毒糖蛋白特异性单抗G10Fab的基因 ,克隆入杆状病毒人源IgG抗体表达载体 ,通过转染将重组质粒导入昆虫细胞 ,以全抗体的形式表达了一株人源抗狂犬病毒基因工程抗体G10。用亲和层析的方法纯化了表达产物 ,经与一株鼠源糖蛋白特异性单抗竞争证实 ,该单克隆抗体特异性识别狂犬病毒糖蛋白 ,亲和力约为 10 -9M。体外中和实验证明 ,该单抗对狂犬病毒aG株具有体外中和活性     

Modification of the ELISA for the estimation of tetanus antitoxin in human sera

The use of indirect ELISA for the quantitation of tetanus toxin neutralizing antibodies in human sera is limited by marked overestimations in low titered sera. The reasons for the discrepancy between the results obtained by ELISA and by in vivo assay and modifications of the ELISA to overcome the problem were investigated. Catching ELISA and indirect ELISA using trays coated with the contaminant proteins in toxoid preparations indicated that antibodies to contaminants were only partly responsible for the discrepancy and the introduction of these modifications did not solve the problem. In ELISA competition experiments with toxin neutralizing monoclonal antibodies, the human immunoglobulins irrelevant in toxin neutralization, but detectable in indirect ELISA, were found to be difficult to inhibit in their binding to the solid antigen phase. These might represent antitoxins bound bivalently to the solid phase but with affinities in monovalent binding insufficient for toxin neutralization or other coupled antibodies due to conformational changes of the antigen. A competition ELISA with toxin in solution was therefore developed to assess selectively the antitoxin capable of binding the antigen in solution and by this approach the in vivo activities of even low titered sera were accurately predicted. This antigen competition ELISA may be easily introduced into routine tetanus serology and the principle may also be of value for the in vitro detection of functional antibodies to other antigens.     

Immunodiffusion test in avian encephalomyelitis. II. Detection of precipitating antibody in infected chickens in comparison with neutralizing antibody.

The sensitivities of double-immunodiffusion (DID) and neutralization tests to detect avian encephalomyelitis (AE) antibody in chickens were studied. Two antigens were employed in the tests. Concentrated antigen gave a higher titer of antiserum than crude antigen, which reacted only to serum having a neutralization log-index (NI) of 3.4approximately4.0 or more. Antibody responses were examined in four growing chick groups inoculated with AE virus by the intracerebral, subcutaneous and oral routes by the DID test with concentrated antigen and by the neutralization test for 1 or over 2 years after inoculation. When concentrated antigen was used, most sera having an NI of over 1.0 were positive for precipitating antibody. Therefore, the sensitivity of the DID test was nearly equal to that of the neutralization test. The DID test was considered to be applicable to the diagnosis of AE and an antibody survey in the field.     

ELISA for the routine determination of antitoxic immunity to tetanus

Serum samples from 727 persons with different vaccination histories were assessed for tetanus antitoxin content in an enzyme linked immunosorbent assay (ELISA) and tested for tetanus toxin neutralization activity in mice in order to compare the results obtained by the two methods. Neutralizing antibody activities in sera from individuals previously completely vaccinated correlated well with results obtained by ELISA and the accuracy increased with increasing antitoxin concentration in serum. This correlation was observed in sera from persons vaccinated recently as well as in sera from persons vaccinated many years ago. In sera from persons with an incomplete vaccination history ELISA was found to be an unreliable tool for the prediction of in vivo results. Many of these sera had antitoxin levels by ELISA far above the in vivo values, probably due to the presence of non specific or low avidity antitoxin which is detected in ELISA. The lowest ELISA value reliably predictive of protective antibody activity in serum irrespective of vaccination history was found to be 0.16 IU/ml. It was concluded that ELISA is useful for larger population studies as an initial test, but sera with an antitoxin content below 0.16 IU/ml should also be assessed in a neutralization system.     

Immune aggregation and not antigen-induced conformational change accounts for enhanced reactivity between immunoglobulin G and protein A

Enhanced reactivity between affinity purified rabbit immunoglobulin G antibodies against the polyvalent antigen human serum albumin and protein A of $\text{Staphylococcus}\text{aureus}$ correlates with immune aggregation but not with antigen-induced allosteric changes in the antibody molecule. Absence of enhancement when immune complexes were prepared with the monovalent hapten methotrexate and anti-methotrexate antibodies is consistent with the inability of these complexes to undergo similar aggregation.     

Validation and use of an indirect enzyme-linked immunosorbent assay for detection of antibodies to West Nile virus in American Alligators (Alligator mississippiensis) in Florida

In October 2002, West Nile virus (WNV) was identified in farmed American alligators (Alligator mississippiensis) in Florida showing clinical signs and having microscopic lesions indicative of central nervous system disease. To perform seroepidemiologic studies, an indirect enzyme-linked immunosorbent assay (ELISA) was developed to determine exposure of captive and wild alligators to WNV. To validate the test, a group of WNV-seropositive and -seronegative alligators were identified at the affected farm using hemagglutination inhibition (HAI) and the plaque reduction neutralization test (PRNT). The indirect ELISA utilized a rabbit anti-alligator immunoglobulins polyclonal antibody as the secondary antibody, and inactivated WNV-infected Vero cells were used as the coating antigen. For all samples (n=58), the results of the ELISA were consistent with the HAI and PRNT findings. Plasma was collected from 669 free-ranging alligators from 21 sites across Florida in April and October 2003. Four samples collected in April and six in October were positive for WNV antibodies using HAI, PRNT, and the indirect ELISA. This indicated that wild alligators in Florida have been exposed to WNV. These findings can be used as a baseline for future surveys.     

人巨细胞病毒抗体检测方法的应用研究

目的确定用于筛选人巨细胞病毒(HCMV)IgG阳性血浆的ELISA试剂和用于HCMV免疫球蛋白成品检定的中和试验方法。方法比较目前国内外现有的人巨细胞病毒IgG抗体检测方法(3家ELISA试剂和3种病毒中和试验)的相关性。结果德国赛润和意大利德塞的ELISA试剂与成都蓉生微量中和试验及台湾宝血的蚀斑减少中和试验的相关性很好(r299%)。结论从成本和使用便利性考虑,建议使用意大利德赛的ELISA试剂盒用于原料血浆的筛选,使用成都蓉生的微量中和试验作为成品效价检定的方法。     

Efficiency and sensitivity of indirect immunoperoxidase methods

The peroxidase-antiperoxidase (PAP) complex method has repeatedly been claimed to be more sensitive and antibody efficient than the indirect peroxidase labeled antibody method. However, most studies comparing these methods used tissue sections as the test material. However, test systems with known amounts of antigen will allow more reliable comparison of these methods and quantitative evaluation of method sensitivity. We therefore compared the antibody efficiency and sensitivity of these methods for the detection of human chorionic gonadotropin in an enzyme linked immunosorbent assay (ELISA), an antigen spot test (AST) and tissue sections of choriocarcinoma. In the PAP technique rabbit PAP and goat anti-rabbit antibody were applied. The same antibody was peroxidase-labeled with the periodate technique and used in the labeled antibody method. In the ELISA the PAP method resulted in slightly higher antibody efficiency than the labeled antibody method. At low primary antibody dilutions the intensity of the reaction decreased with the PAP method but remained high with the labeled antibody method, in the ELISA as well as on tissue sections. In the AST the labeled antibody method and the PAP method appeared to be equally sensitive.