Serological studies with the ficin-antificin system.

Rabbit antisera prepared against ficin reacted with it in a series of serologic tests. Upon immunodiffusion analysis, ficin was found to consist of at least nine antigenic components. Ficin will adsorb spontaneously to erythrocytes which can be used in passive hemagglutination tests. The enzymatic activity of ficin was not abolished by antificin sera.     

Comparative evaluation of the commercial Sevatest ELISA immunoenzyme set and other serological tests for detecting Toxoplasma gondii antibodies

This work analyzes the results of 4 serologic tests used for the diagnosis of toxoplasmosis: the complement fixation (CFT), indirect immunofluorescence (IIF), passive hemagglutination (PHAT) tests, and the enzyme-linked immunosorbent assay (ELISA). The last-mentioned one was made with the use of the commercial kits Sevatest ELISA IgG/Toxo Micro I. The results of ELISA were in good correlation with those yielded by the traditional tests: 70% coincidence with CFT, 80% with IIF, 84% with PHAT; besides, ELISA has shown a higher sensitivity in the screening of sera.     

Experience with a bacteriologic and serologic study of a tularemia epizootic in rodents in a natural focus of the meadow and field type

The autumn-winter (1977-1978) tularemia epizootic in small murine rodents was revealed and studied at the natural focus of the meadow and field type in the south of the Moscow Region. The efficacy of the serologic method (the antibody neutralization test) of studying the organs of the caught rodents and the bodies of dead rodents was found to be greater than that of the traditional bacteriologic methods (26.6% and 9.6%, respectively). The serologic study of 908 specimens of avian excrements collected during the period from autumn to spring (1977-1978) revealed that tularemia antigen could be constantly detected, starting from October. The serologic method was effective when used both for the early and retrospective detection of the infective agent and allowed to characterize the epizootic process in greater detail.     

Serologic Relationship of Coxsackie A16 Viruses from the Epidemic of Hand-Foot-and-Mouth Disease in Japan, 1970, to the Prototype Strain

In 1970, a great outbreak of hand-foot-and-mouth disease (HFMD) due to Coxsackie A16 virus (Cox A16) occurred throughout Japan. When serologic relationship between the viruses isolated during the epidemic and the prototype strain of the serotype was examined by the tube neutralization test, the crude new isolates were found to be poorly neutralized by both an antiserum against the prototype strain and those against the isolates, although they were neutralized significantly in the plaque reduction test. However, about 2% of virus in crude suspensions of the isolates remained unneutralized even in the plaque reduction test and this fraction could be eliminated by filtering the virus materials through a 100 mμ Millipore filter. Therefore, the difficulty in neutralization of the isolates by the tube method could be accounted for by the presence of aggregated viruses. Even when filtered viruses were used, the reciprocal neutralization kinetic studies revealed a significant serological difference between the isolates and the prototype strain. Such serological properties of the isolates were not influenced by the cell types used for virus isolation or passages. All the results suggest that the Cox A16 isolates from the epidemic of HFMD in Japan, 1970, are serologically different from the prototype strain.     

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.     

A Modification of the Mycoplasma Serum-Drop Growth Inhibition Test

A modification of the serologic mycoplasma growth inhibition test is described. It is based on application of a drop of antiserum directly onto the centre of an inoculated agar plate provided with a slightly concave agar surface. The special apparatus devised for the preparation of these plates is described. This modified serum-drop method is more sensitive than other growth inhibition tests used today and is equally specific.     

H-Y antigen negative patients with testicular tissue and 46,XY karyotype

Summary H-Y antigen could not be detected on lymphocytes from two male pseudohermaphrodites with 46,XY karyotypes and testicular tissue. One of the patients had additional assays performed on fibroblasts grown from the skin, and the gonadal ridge—these were also negative. The H-Y antiserum was raised in rats, with Raji cells the target of cytotoxicity tests. In these patients, the substance that promoted testicular differentiation does not have serologic H-Y antigen detectable by the assay used. It appears that H-Y antigen that is commonly measured in neutralization reactions may not be the only form of testicular organizing factor present.     

Micro-neutralization test for mumps virus using the 96-well tissue culture plate and PAP (peroxidase-antiperoxidase) staining technique

A rapid micro-test method for mumps virus neutralization was developed. In this method, a 96-well tissue culture plate was used for preparation of cell monolayers and the PAP staining technique was used for visualization of mumps virus infected cells. Clusters of infected cells were observed as a focus and the numbers of foci could be counted by the naked eye 2 days after the infection. A linear relationship between virus dilutions and focus numbers was observed. When neutralizing antibodies in sera from cases of natural mumps infection were assayed, a good correlation was observed between those obtained by the focus reduction method applying the micromethod and those obtained by the ordinary plaque method. Our results indicate that this micromethod is useful in mumps virus neutralization tests and it has many advantages over other methods previously reported.     

Radioimmunoassay of human serum antibody specific for adenovirus type 5-purified fiber.

A radioimmunoassay (RIA), utilizing a second antibody to separate immune complexes, was developed to provide a sensitive and specific measure of serum antibody to adenovirus type 5 (Ad 5) fiber. Purity of fiber antigen was ascertained by sodium dodecyl sulfate urea-polyacrylamide gel electrophoresis and isoelectric focusing in ampholyte pH gradients. After labeling with 125I to high specific activity, the iodinated fiber did not exhibit loss of antigenic reactivity and remained stable for 3 weeks when stored at minus 20 degrees C with supplemental protein. Rabbit anti-Ad 5 serum with a neutralization titer of 1:320 precipitated 50% of the labeled fiber at a serum dilution of 1:50,000 when tested by the RIA. In competition assays as little as 0.5 ng of unlabeled fiber per millimeter was sufficient to inhibit the 125I fiber-antibody reaction. Serum specimens from 20 volunteers, obtained before and after vaccination with purified Ad 5 fiber or hexon subunit vaccine, were tested by RIA, hemagglutination-inhibition (HI), and neutralization tests. A comparison of mean antibody titers of post-inoculation sera showed that the RIA was 300 and 1000 times more sensitive than the HI and neutralization tests, respectively. Moreover, 19 of the men who were negative by the standard serologic tests before vaccination were shown to have anti-fiber antibody, with a mean RIA titer of 1:1028. Specificity of the RIA was demonstrated by the lack of an increase in antibody to Ad 5 fiber among those individuals vaccinated with the hexon subunit. Thus, the development of a highly sensitive and reproducible RIA allows for the detection of antibody specific for the Ad 5 fiber in serum which contains antibodies to the different virion antigenic determinants associated with Ad 5.     

Serologic and mucosal immune response to rotavirus infection in the rabbit model.

We examined the humoral immune response to rotavirus infection in specific pathogen-free rabbits inoculated and challenged orally with rabbit Ala rotavirus (7.5 x 10(5) to 1 x 10(7) PFU). The humoral immune response in both serologic and mucosal samples was monitored by using total antibody enzyme-linked immunosorbent assays (ELISAs), isotype-specific ELISAs, and plaque reduction neutralization assays. Following a primary infection, all rabbits shed virus and serologic and mucosal antibody responses were initially detected by 1 week postinoculation. Intestinal immunoglobulin M was detected by 3 days postinoculation, and secretory immunoglobulin A was detected by 6 days postinoculation. Following challenge, rabbits were protected (no detectable virus shedding) from infection. An anamnestic immune response was observed only with mucosal neutralizing antibodies, and all serologic and mucosal immune responses persisted at high levels until at least 175 days postchallenge (204 days postinoculation). Detection of neutralization responses was influenced by the virus strain used in the neutralization assay; all inoculated rabbits developed detectable serum and intestinal neutralizing antibodies against the infecting (Ala) virus strain. Neutralization activity in both serum and mucosal samples was generally, but not exclusively, homotypic (VP7 serotype 3) after both primary and challenge inoculations with Ala virus. Heterotypic serum neutralization activity was observed with serotype 8 (9 of 12 rabbits) and 9 (12 of 12 rabbits) viruses and may be based on reactivity with the outer capsid protein VP4 or on a shared epitope in the C region of VP7. Comparisons of heterologous (serotype 3) and heterotypic neutralizing responses in mucosal and serologic samples revealed that 43% (21 of 49) of the responses were discordant. In 19 of 49 (39%) of these cases, a heterotypic serologic response was seen in the absence of a heterotypic mucosal response, but in 2 of 49 (4%) instances, a heterotypic mucosal response was seen in the absence of a concomitant serologic response. These results provide insight into factors which may affect detection of heterotypic responses.     

Serology of Rubella—Comparison of Fluorescent Antibody,Complement Fixation and Neutralization Tests for Diagnosis of Current Infections and Determination of Sero-immunity

Neutralization, complement fixation (CF) and indirect fluorescent antibody (FA) assays for rubella virus were compared for sensitivity in the serologic diagnosis of infection, for demonstrating antibody in the sera of infants with suspected rubella syndrome, and in the detection of antibody elicited by past infection (determination of immunity status). The combination of CF and FA tests was shown to be the most useful for serologic diagnosis of infection, largely eliminating the need for the slower and more cumbersome interference neutralization test.Neutralizing antibodies were found to appear rapidly in the course of infection, antibodies demonstrable by immunofluorescent staining appeared slightly later, and CF antibodies were rarely demonstrable in sera collected earlier than 14 days after onset of illness. Antibodies detected by all three techniques showed good correlation in infants with clinical evidence of rubella syndrome and corresponding maternal sera. The indirect FA technique compared favorably with the neutralization test for the detection of antibody elicited by past infection (determination of immunity status) and offered distinct advantages in ease of technical performance and more rapid results. In both current and past infections, FA titers tended to be higher than neutralizing antibody titers.     

Antibodies to bluetongue and epizootic hemorrhagic disease viruses from white-tailed deer blood samples dried on paper strips.

The feasibility of using dried blood samples for serologic testing of white-tailed deer (Odocoileus virginianus) for antibodies to bluetongue virus (BTV) and epizootic hemorrhagic disease virus (EHDV) was tested with matched samples of serum and eluted dried whole blood. Results from matched serum virus neutralization (SN) tests indicated that a 1-ml elution from a 1- x 2-cm section of filter paper strip containing dried blood approximated a 1:10 serum dilution. Neutralizing antibody titers detected from 34 matched titrations of serum and dried blood samples were equivalent in 25 (74%) titrations and were within a single dilution in the remaining nine (26%) titrations. Eluted blood samples from SN-positive deer, however, did not produce detectable precipitin lines on agar gel immunodiffusion tests for antibodies to either BTV or EHDV. In a trial using serum and dried blood samples from 108 hunter-killed deer from five locations in Georgia (USA), antibody prevalence and serotype distribution results were similar. Use of dried blood samples for serologic testing for antibodies to BTV and EHDV provides a reliable alternative to serum but should be considered only when serum collection is not feasible.     

Potency control of diphtheria component in adsorbed vaccines by in vitro neutralization tests.

Samples from 20 lots of dT vaccine and from 20 lots of DTP vaccine were used to standardize and validate the Vero cell and the toxin binding inhibition (ToBI) tests for the potency control of diphtheria component. For the Vero cell method, violet crystal solution was used to stain the cells and estimate the endpoint of diluted diphtheria antitoxin. Diphtheria anatoxin was used for performing the ToBI test instead of toxin. The results obtained by both in vitro tests were similar to those obtained by in vivo toxin neutralization test in guinea pigs. The various analysis and the chi(2) test applied to evaluate the reproducibility and homogeneity, respectively, among in vitro tests and in vivo toxin neutralization test did not detect statistical significant difference for both analysed vaccines. An excellent correlation among in vitro tests and in vivo neutralization test was observed by Spearman's correlation coefficient.     

The importance of neutralization in the evaluation of triclosan-containing products

A variety of bactericidal tests are available for evaluating the antimicrobial activity of products. An often overlooked variable in these types of studies is adequate neutralization. Triclosan is a widely used antimicrobial agent and has been shown to be difficult to neutralize. Incomplete neutralization may overestimate the efficacy of triclosan-containing products. Received 3 July 1997/ Accepted in revised form 22 August 1998     

Microneutralization Test for Determination of Rhinovirus and Coxsackievirus A Antibody in Human Diploid Cells

A method for determination of serum-neutralizing antibody titers to rhinovirus type 16 and coxsackievirus type A-4 in human diploid cells (WI-38) grown in Microtiter plates is described. A good correlation was observed when comparing neutralization tests in WI-38 and other cells. The WI-38 Microtiter method is relatively simple and economical. It is suitable for those laboratories which are required to conduct large-scale serological evaluation of antibodies for rhinoviruses and coxsackie A viruses. This system also can be used for cytomegalovirus and varicella-zoster virus, which grow relatively slowly.     

Identification of group A coxsackieviruses by immune adherence hemagglutination

We investigated to find whether the immune adherence hemagglutination (IAHA) test could be used for identification of group A coxsackieviruses (Cox. A). By using homogenate of suckling mouse torsos infected with each of nine prototype viruses (Cox. A 2, 3, 4, 5, 6, 8, 9, 10 and 16) and 46 isolates as antigens and hyperimmune mouse ascitic fluids to the prototype viruses, we compared IAHA with complement fixation (CF) for serotyping of these viruses. The results of identification tests by IAHA were the same as those by combined use of CF and neutralization tests on all the 46 strains. By CF alone, however, six of 46 strains were not identified because of lower antigen titers; IAHA antigen titers were generally higher by 16-fold or more than CF tests. Furthermore, IAHA had a higher type-specificity than CF; a weak cross-reaction was found by IAHA only between Cox. A 3 and Cox. A 8. Nonspecific reactions encountered in IAHA were reduced more readily by kaolin than fluorocarbon treatment of the torso homogenates. From these results, we conclude that IAHA is an alternative method to CF and neutralization for serotyping of Cox. A viruses.     

Isolation of the most immunoreactive antigenes of echinococcus granulosus from sheep hydatid fluid.

This paper describes a simplified procedure for obtaining purified Echinococcus granulosus antigens from sheep hydatid fluid by using affinity chromatography on concanavalin A-Sepharose. The presence of two "major" antigens (4 and 5) was confirmed. Antigen 5 was isolated by preparative polyacrylamide gel electrophoresis. Antigen 4, eluted by diffusion from the gel, was seen to be "contaminated" by antigen 5 and was isolated by using anti-5 Sepharose-linked serum. These two major antigens were then tested separately against the sera of hydatidosis patients by using very simple immunolgic tests. The best results were obtained in passive hemagglutination with antigen 4. Antigen 4 is the most immunoreactive parasitic antigen; antibodies against it were found in the sera of all hydatidosis patients showing positive reaction. Apart from the direct use of this antigen in serologic tests, it appears possible to standarize the most frequently used and commerically available antigenic materials by titrating this component.     

Characteristics of the immunologic response of animals to Clostridium perfringens anatoxin]

Experiments were conducted on guinea pigs, rabbits and mice (mongrel and inbred); immunogenic properties of Cl. perfringens toxoids of different purity were studied. Toxin neutralization and passive hemagglutination tests were used to determine the antitoxic immunity level. It appeared that in the immunization of guinea pigs and rabbits the degree of immunogenicity of the preparations increased with the elevation of their specific activity. Under the same conditions both the mongrel and the inbred mice displayed the maximum immune response in the immunization with the least purified preparations, and the minimum after the injection of a highly purified antigen.     

Experimental plague in rock squirrels, Spermophilus variegatus (Erxleben)

Experimental infections with Yersinia pestis were followed in groups of rock squirrels. Development of coagulopathy and pneumonia were observed in 2-4% and 11-12% of the test animals, respectively. Susceptibility to experimental infection was heterogeneous with some animals surviving inoculation with large numbers of organisms and others succumbing after inoculation with small numbers. Production and longevity of serum antibody titers, as measured by passive hemagglutination tests, were variable as well, and apparently unrelated to dose. The data presented attest to the need for care in interpreting serologic tests results for individual animals.     

Vaccinia Neutralization Test Using a Radioisotope Assay for Virus-induced Enzyme Activity

Vaccinia virus induction of a metabolic activity in host cell cultures forms the basis of a new assay for neutralizing antibodies. A direct relationship between the amount of vaccinia virus infecting cell cultures and the induced incorporation of tritium-labeled thymidine into the acid-insoluble fraction of the cells provided an indicator system. A liquid scintillation spectrometer was used to determine radioactivity associated with cell materials, and it provided a method for partial automation of an immunological procedure. Reproducibility of the method was satisfactory, and agreement with conventional vaccinia serum neutralization tests was demonstrated.