Toxin Neutralization Test Research Articles

Analysis of antigenicity of Clostridium botulinum type C1 and D toxins by polyclonal and monoclonal antibodies.

Clostridium botulinum type C1 toxin was purified from C-Stockholm (C-ST), and D toxin was purified from D-1873 and D-South African. Polyclonal antibodies against these toxins were prepared in rabbits. Twenty-eight monoclonal antibodies to these toxins were also prepared with BALB/c myeloma cells. The antibodies were analyzed by both enzyme-linked immunosorbent assay (ELISA) and a toxin neutralization test. ELISA was performed with the three purified toxins and heavy-chain (Hc) and light-chain (Lc) components derived from C-ST and D-1873 toxins. A neutralization test was carried out with 11 toxin preparations (7 from type C and 4 from type D cultures). ELISA results indicated that there exists at least one common antigenic determinant on each of the Hc and Lc components of the three purified toxins. The results of the neutralization test also indicated that type C1 and D toxin preparations contain several common antigenic sites in their molecules. Some are common to toxins from several specific cultures, whereas others are common to toxins from a large number of cultures. It was speculated that toxins from two type C strains are composed of Hc and Lc components which are somewhat similar to those of D-1873 and C-ST toxins, respectively.

Prevalence of Clostridium botulinum type C in substrates of phosphate-mine settling ponds and implications for epizootics of avian botulism.

Prevalence and conditions for occurrence of Clostridium botulinum type C were examined on phosphate-mine settling ponds and a natural wetland in northern Florida between April 1981 and March 1982. Substrate samples were collected monthly (winter) and semi-monthly (summer) from 16 locations on seven ponds. Selected environmental parameters were measured at each location at the time of sampling. Mouse inoculation tests and toxin neutralization tests using enrichment culture filtrates were conducted to identify C. botulinum type C in the samples. The bacteria were identified in 26 (5.6%) of 467 sediment samples. Occurrences were distributed over four of the seven ponds and included nine of the 16 sample locations, but were restricted to the months April through October. The organism occurred over a wide range of ecological conditions found on the ponds during these months. If the presence of C. botulinum type C in the substrate is a prerequisite for botulism to occur, the prevalence and fairly wide distribution of this organism on settling ponds makes it difficult to predict where future outbreaks may occur.

Monoclonal antibodies against tetanus toxin and toxoid.

Monoclonal antibodies against tetanus toxin and its toxoid were produced by immunizing mice with toxoid or toxin. They were measured by an enzyme-linked immunosorbent assay (ELISA), by a toxin neutralization test in mice (in vivo prevention test), and by their ability to prevent binding of 125I-toxin to brain membranes or gangliosides (in vitro prevention test). Six monoclonal antibodies obtained by immunization with toxoid (anti-toxoid 1-6) were investigated in more detail. They belonged to IgG class 1. Three of them (anti-toxoid 1, 2 and 3) recognized both toxoid and toxin as well as fragment B and the light chain of toxin, but not fragment C. Two other antibodies (anti-toxoid 4 and 5) were directed against toxoid only. Neither of them prevented toxin action in vitro or in vivo. Anti-toxoid 6 recognized toxin, toxoid and fragment C, but not light chain, and prevented toxin action in vitro and in vivo. Immunization against toxin was initiated with a toxin-antitoxin complex and boosted with toxin. We studied six antibodies in more detail, all of IgG type 2. Their KD against 125I-tetanus toxin varied from 10(-9) to 10(-10) M. Anti-toxin 2 recognized toxin, toxoid, light chain and fragment B, but not fragment C. The others reacted with toxin, toxoid and fragment C, but not with light chain or fragment B. All of them prevented toxin action in vitro and in vivo. As calculated from the maximal extinction achieved in the ELISA, tetanus toxin combined with a maximum of two different antibody molecules from our set. Gel filtration data indicate that tetanus toxin reacts with monoclonal antibodies one by one. Compared with polyclonal antiserum, monoclonal antibodies yield flatter slopes in both in vitro and in vivo prevention tests. Thus, they cannot substitute for the polyclonal antibodies in clinical situations, and cannot be calibrated in international units.

A comparison of enzyme-linked immunosorbent assay (ELISA) with the toxin neutralization test in mice as a method for the estimation of tetanus antitoxin in human sera

An enzyme-linked immunosorbent assay (ELISA) has been developed for the measurement of tetanus antitoxin in human sera as an alternative to the toxin neutralization test in mice, the currently accepted method of assay. The ELISA was found to be simple and quick to perform and required only small amounts of materials. In addition, the assay was found to give reproducible estimates of antitoxin levels and to measure antitoxin at levels as low as 0.01 IU per ml, a sensitivity similar to that of the neutralization test. Furthermore, a comparison of the results of the ELISA and the neutralization test involving 80 human sera, including sera with both high and low antitoxin levels, showed close agreement in antitoxin levels obtained by the two methods. It was concluded that ELISA was an acceptable alternative to the toxin neutralization test in mice for the measurement of tetanus antitoxin levels in human sera.

Quantification of tetanus antitoxin by toxin neutralization test in mice. A comparison between lethal and paralytic techniques

Tetanus antitoxin may be assayed by toxin neutralization (TN) tests in mice using either the death of mice or early symptoms of tetanic paralysis as the titration endpoint. The present investigation presents a statistical comparison of the two techniques. The correlation between the two techniques is very good. The paralytic technique reduces animal suffering considerably, but the inspection of the animals needs to be carried out more carefully and the method is therefore more time consuming than the lethal technique.

A reversed rocket electrophoresis method for the determination of high tetanus antitoxin levels

A quantitative reversed rocket electrophoresis method using a non-barbital buffer is described for the identification of plasmas with tetanus antitoxin levels ⩾ 10 i.u. ml −1 and therefore suitable for the production of human tetanus immune globulin. The method is simple, highly reproducible and easily standardized. Excellent agreement between the results obtained by this method and by the in vivo toxin neutralization test was found.

Epidemic Typhus in the United States Associated With Flying Squirrels

Between July 1977 and January 1980, seven cases of sporadic, nonepidemic "epidemic" typhus (Rickettsia prowazekii) were discovered in Virginia, West Virginia, and North Carolina. The reservoir seemed to be the southern flying squirrel (Glaucomys volans), an animal indigenous to the eastern United States; however, the vector or mode of acquisition was not evident. Diagnosis was established principally through complement fixation, indirect immunofluorescence, and toxin neutralization tests. Patients' ages were 11 to 81 years. Most were white women. Six had abrupt onset of illness. Headaches, fever, myalgias, and exanthems were among the presenting complaints. The disease seemed milder than classic louse-born epidemic typhus, but in some instances, it was life-threatening. All patients responded to tetracycline or chloramphenicol. This entity probably is more common than reported, is difficult to recognize, and is produced by an organism seemingly identical to that producing louse-born epidemic typhus.

Immunogenicity of Staphylococcus aureus delta-toxin.

Studies were conducted to determine the immunogenicity of purified Staphylococcus aureus delta-toxin. Rabbits and guinea pigs immunized with delta-toxin incorporated into a multiple antibody, whereas animals given toxin in saline or toxin in saline with Tween 80 did not produce antibody. The immunoglobulin G (IgG) fraction isolated by chromatography on protein A-Sepharose was examined for the presence of anti-delta-toxin antibody by immunoelectrophoresis, immunodiffusion, quantitative precipitation tests, affinity chromatography, and toxin neutralization tests. Although delta-toxin-specific IgG precipitated the toxin in agar gels, the antibody did not neutralize the toxin's hemolytic activity. Delta-toxin binding to human erythrocyte membranes was demonstrated by indirect immunofluorescent staining of toxin-treated erythrocytes.

Influence of anti-fragment A of diphtheria toxin on passive hemagglutination with antitoxin.

Hemagglutination (HA) and toxin neutralization (TN) tests were used to titrate human and guinea-pig serum specimens taken at various stages of immunization for diphtheria antitoxin. The ratio of HA to TN titers varied significantly depending on the immune status. The ratios and the range of their variations became larger and high values exceeding five were often obtained after repeated booster immunization. Such a high value was proved to be related to the antibody against fragment A (FrA) of diphtheria toxin, since the ratio reduced significantly when anti-FrA was absorbed. Anti-FrA was not produced in children after the basic immunization with diphtheria toxoid and was detected in only one-third of vaccines after the booster injection given after 1 year. It was produced abundantly, however, when booster immunization was repeated. The pattern of production of anti-FrA in guinea pigs was similar to that in humans, when immunized with diphtheria toxoid with adjuvant. No anti-FrA was produced even when the animals were immunized repeatedly with plain diphtheria toxoid.

Quantification of Tetanus Antitoxin in Human Sera

We described recently an improved counter-immunoelectrophoretic method for quantification of tetanus antitoxin. The toxin neutralization test in mice is considered to correlate well with protection in humans. In the present study, the correlation coefficient between the two methods was 0.89. Sera containing more than 7.0 I.U./ml could be quantified directly by counter-immunoelectrophoresis, while sera containing less tetanus antitoxin had to be concentrated prior to quantification. The passive haemagglutination test was also compared with the toxin neutralization test in mice. The correlation coefficient between the two methods was 0.76.

Quantification of tetanus antitoxin in human sera. I. Counter-immunoelectrophoresis.

An extensive sero-epidemiological survey of immunity to tetanus is to be performed in Norway during 1978 and 1979 and thus a simple and reliable method for screening sera for content of tetanus antitoxin is needed. An improved counter-immunoelectrophoretic method for quantification of tetanus antitoxin is described. The toxin neutralization test in mice is considered to correlate well with protection in humans. Counter-immunoelectrophoresis has the advantage of using tetanus toxoid instead of toxin as well as being more rapid and less expensive. Specific antibodies of the IgG, IgM, and IgA classes could be read simultaneously for many sera. This may be of importance, since it is reported in the literature that only antibody of the IgG class is capable of neutralizing tetanus toxin in mice. Counter-immunoelectrophoresis is limited by being less sensitive than the toxin neutralization test in mice.

Neutralization of murine typhus toxin by serum of normal human beings and monkeys.

The neutralization of Rickettsia typhi toxin observed in preliminary studies with normal human serum prompted further investigations on the prevalence and nature of the neutralizing activity. As shown by toxin neutralization (TN) tests in mice with toxic suspensions of rickettsiae, serum of 50% of 207 normal persons had titers of 1:32 or greater against R. typhi toxin, regardless of age or residence in the United States. Within this group were 35 persons less than 20 years old who had never received typhus vaccine, and who resided in a typhus-free area (Montana). Serum from all of 25 normal monkeys had titers of 1:512 or greater. With three exceptions, serum specimens positive in murine TN tests did not neutralize toxin of epidemic typhus or react in complement-fixation or microagglutination tests with murine or epidemic typhus antigens. Serum of normal burros, horses, sheep, goats, rabbits, and chickens did not neutralize toxins of either murine or epidemic typhus. The neutralizing factor in serum from normal monkeys was not affected by treatment with potassium periodate or trypsin, but could be completely precipitated by 50% saturation with ammonium sulfate or by acetone. These findings suggest that the murine TN substance detected in the primate serum was of nonspecific origin, not the result of exposure to R. typhi or Rickettsia prowazekii, and must be considered in evaluating the significance of the TN test as a diagnostic procedure.

Rickettsia canada: a new member of the typhus group of rickettsiae isolated from Haemaphysalis leporispalustris ticks in Canada.

A third species has been added to the typhus group of rickettsiae. This new rickettsia was isolated from Haemaphysalis leporispaluslris ticks removed from indicator rabbits in the vicinity of Richmond, Ontario. Inoculation of tick suspensions into embryonated hens' eggs was the method of culture. Results of complement fixation tests with guinea pig, hamster, and rabbit antisera showed that it is a member of the typhus group while results with antiserum prepared in mice suggested that it is different from the two known species, Rickettsia typhi and Rickettsia prowazeki, in the group. Results of toxin neutralization tests furnished the final proof that this is a new species of rickettsia. The name Rickettsia canada is proposed for it.

CLOSTRIDIUM BOTULINUM FOOD POISONING

SummaryThe outbreaks of botulism in the United States during 1963 stimulated renewed interest in this food-borne disease, primarily because commercially prepared foods were involved. Three of the outbreaks were caused by Clostridium botulinum type E in fishery products. Two of these resulted from the consumption of smoked fish from the Great Lakes.A survey has been started to see if C. botulinum type E is common on fish from the Great Lakes. Toxin neutralization tests have shown the organism to be present in cultures from nine of ten locations sampled in Lake Michigan. The organism was found more frequently in the intestinal tract than on gills, livers or the external surfaces of the fish. Over 75% of the cultures prepared from the intestines of fish caught in one large bay of Lake Michigan proved to contain type E toxin. The incidence of the organism in fish from the main body of the lake has been much lower than this.

Immunologic Relationships Among the Spotted Fever Group of Rickettsias Determined by Toxin Neutralization Tests in Mice with Convalescent Animal Serums

Summary Tests of certain variables inherent in the spotted fever toxin neutralization test in mice revealed that a) spotted fever group toxins remained stable for a period of at least 3 hr at either room temperature (26°C) or at 2°C, b) serum antitoxin titers were maximum 15 min after mixing antiserum with toxin and they did not change significantly during 2 hr further incubation at room temperature, c) a relationship existed between the serum neutralization titer and the concentration of toxin neutralized: a 2-fold increase in quantity of toxin reduced the serum titer 4-fold. Three strains of Rickettsia rickettsii associated with spotted fever disease in man behaved alike in reciprocal cross neutralization tests. A strain of R. rickettsii from the rabbit tick Haemaphysalis leporis-palustris, showed a significant serologic difference from one but not from the other two of these strains of R. rickettsii. The rickettsias of boutonneuse fever, South African tick bite fever, Kenya tick typhus and Indian tick typhus, were indistinguishable from one another by the mouse toxin test. These studies with Indian tick typhus confirm recent information concerning its serologic relationship with this group of rickettsias. R. rickettsii and Rickettsia conorii were clearly differentiated from one another by the mouse toxin test. Neither of these two rickettsias produced more than a slight degree of cross neutralization with the other. The rickettsia of Siberian tick typhus could be distinguished by the mouse toxin technique from R. rickettsii and R. conorii as well as from other spotted fever group strains studied. However, a relatively high one-way cross neutralization occurred between the Siberian agent and certain strains of R. rickettsii and the rickettsia from the tick Amblyomma maculatum, but not R. conorii. Two strains of Siberian tick typhus rickettsias, one from the tick Dermacentor nuttalli, and the other of human origin, gave complete cross neutralization with one another and were essentially alike in their cross reactions with other members of the spotted fever group. The maculatum rickettsia exhibited distinct serologic differences from all other spotted fever strains studied. This rickettsia did, however, give notably high nonreciprocal cross reactions with R. conorii strains and with the rickettsia of Siberian tick typhus. Guinea pig antisera against Rickettsia australis and Rickettsia akari showed little or no cross neutralization of toxins of 10 other strains of spotted fever group rickettsias.

Resistance of Mice with Experimental Histoplasmosis to Infection with Rickettsia Typhi

Discussion and Summary Evidence is presented that Histoplasma capsulatum exerted a striking modifying effect upon the course of infection with Rickettsia typhi in mice. This antagonistic action of one agent upon another apparently was not due to specific antibodies since immune serum from guinea pigs with experimental histoplasmosis failed to show any neutralizing effect in the mouse toxin neutralization test, or any fixation of complement in the presence of typhus antigen. Also antityphus guinea pig sera did not show complement-fixing, precipitating, or neutralizing antibodies against H. capsulatum (10). Of particular interest was the fact that the modifying effect occurred only when both agents were injected by the intraperitoneal route. Intravenous, intracerebral, or subcutaneous injection of either one or both agents resulted in slight, if any, inhibitory effect on the course of typhus infection. The necessity for administering both agents via the intraperitoneal route was also indicated in the experiments with R. typhi toxin. This importance of the inoculation route was also emphasized by Horsfall and McCarty (5) who noted that the modifying effect of a non-hemolytic streptococcus on the pneumonia virus of mice (PVM virus) occurred only when both agents were administered by the intranasal route. The time and dose relationships between H. capsulatum and R. typhi were of importance in demonstrating the inhibitory effect. Inhibition occurred only when the interval between injections was sufficient to permit propagation of the fungus to a necessary level. This interval could be shortened or lengthened according to the number of fungus cells in the initial injection. With doses of 109 cells of the fungus, maximum resistance occurred after a 2-day interval. Were doses of H. capsulatum higher than 109 employed, a blocking effect following simultaneous injection of the agents might have occurred. The modifying effect of H. capsulatum on R. typhi infections was not exhibited by six other fungi tested. On the other hand, H. capsulatum was capable of modifying infection not only by R. typhi but also by R. tsutsugamushi. Inhibition of typhus infection was not dependent on active growth of H. capsulatum, since a definite modifying action was produced by killed fungus cells. However, the latter effect was not as great as that following injection of living cells.

Immunochemical studies of antitoxin produced in normal and allergic individuals hyperimmunized with diphtheria toxoid. IV. Differences between human precipitating and non-precipitating skin-sensitizing diphtheria antitoxin as shown by electrophoresis.

Electrophoresis on a starch-supporting medium was used to fractionate sera containing human diphtheria antitoxin of the following varieties (a) precipitating antitoxin, (b) non-precipitating skin-sensitizing antitoxin, and (c) mixtures containing precipitating and skin-sensitizing antitoxins. Aliquots of the protein fractions thus separated were tested for activity using the rabbit toxin neutralization test, precipitin techniques, and passive transfer tests in human skin. Non-precipitating, skin-sensitizing diphtheria antitoxin migrated largely as a fast moving gamma (gamma(1)) globulin. Passive transfer studies of isolated antitoxic fractions showed that they were as potent as whole serum in the ability to cause immediate wheal reactions. These fractions were not precipitable using appropriate quantities of purified toxoid. Precipitating diphtheria antitoxin migrated largely as a slow moving gamma (gamma(2)) globulin. Isolated antitoxic fractions of appropriate strength obtained from representative sera were precipitable by toxin and were unable to cause immediate wheal reactions upon toxoid challenge in human recipients. Mixtures of skin-sensitizing and precipitating antitoxins were separable by the technique of starch electrophoresis. The individual components removed from mixtures by this method retained the properties by which they could be characterized in whole serum.

The estimation in vitro of small amounts of diphtheria antitoxin by means of a haemagglutination technique.

1. Treatment of washed sheep erythrocytes with dilute solutions of tannic acid rendered them capable of adsorbing diphtheria toxin from solution. Diphtheria toxoid was not adsorbed under similar conditions.2. Cells thus sensitized with diphtheria toxin were agglutinable by a proportion of children's sera and human cord sera; sera of rabbits, mice and horses, immunized with soluble or adsorbed diphtheria toxoid, also agglutinated sensitized cells, as did also the sera of children suffering from diphtheria and treated with antitoxic serum. Clear-cut results were usually obtained, but the end-point of titrations on horse sera was, in some cases, difficult to determine.3. Using intradermal toxin neutralization tests, a highly significant linear relationship was demonstrated between the haemagglutinating titres of children's sera for the sensitized red cells, and the antitoxin content of their sera. The antitoxin content corresponding to the smallest detectable amount of haemagglutinating antibody was 0·008 unit per ml. serum.4. The haemagglutinating titre was an accurate measure of the mean antitoxin level of a group of sera and a close measure of the antitoxin content of most specimens in individual tests.

The toxins of the psittacosis-lymphogranuloma group of agents; differentiation of strains by the toxin neutralization test.

Journal Article The Toxins of the Psittacosis-Lymphogranuloma Group of Agents: III. Differentiation of Strains by the Toxin Neutralization Test Get access G. P. Manire, G. P. Manire From the George Williams Hooper Foundation, University of California, San Francisco 22, California Search for other works by this author on: Oxford Academic PubMed Google Scholar K. F. Meyer K. F. Meyer From the George Williams Hooper Foundation, University of California, San Francisco 22, California Search for other works by this author on: Oxford Academic PubMed Google Scholar The Journal of Infectious Diseases, Volume 86, Issue 3, May 1950, Pages 241–250, https://doi.org/10.1093/infdis/86.3.241 Published: 01 May 1950 Article history Received: 21 December 1949 Published: 01 May 1950

STREPTOCOCCI FROM SCARLET FEVER, ERYSIPELAS AND SEPTIC SORE THROAT

In studying colonies of hemolytic streptococci on chocolate agar, I find that typical cultures from erysipelas produce a vivid green after from twenty-four to forty-eight hours' growth, while those from scarlet fever produce no color change except occasionally a slightly greenish tinge after several days' incubation. Twenty-five cultures from scarlet fever, thirteen from erysipelas and forty from other sources have been so examined. Most of these cultures have been examined immunologically as well, generally by the opsonic method but many also by agglutination and toxin neutralization tests to determine their specificity. The forty strains from sources other than scarlet fever and erysipelas did not belong immunologically to the scarlet fever group; some turned chocolate agar green, others did not. In examining cultures of scarlet fever and erysipelas streptococci isolated a long time, it is necessary to grow them on a blood agar plate to make sure that the original colonies