The titration of tetanus antitoxin V. Effect of formalization method for the fixation of sheep erythrocytes on the indirect haemagglutination test

Two methods of fixation of sheep erythrocytes with formaldehyde for the titration of tetanus antitoxin by the indirect haemagglutination (IHA) test have been compared. The cells fixed with 3% formaldehyde at 4-8 degrees C for 24 h (formaldehyde (I) fixed cells) were less sensitive than the cells fixed with 3% formaldehyde at 4-8 degrees C for 24 h and subsequently treated with 40% formaldehyde at 4-8 degrees C for a further 24 h (formaldehyde (II) fixed cells). The correlation between the toxin neutralization (TN) and IHA titres using formaldehyde (I) fixed cells was better than that obtained with formaldehyde (II) fixed cells. There was no statistically significant difference between TN and IHA titres after treatment of the sera with 2-Mercaptoethanol using formaldehyde (I) fixed cells. Formaldehyde (I) fixed cells can be used for two months with adequate sensitivity to detect the minimum protective level of tetanus antitoxin in the sera.     

The titration of tetanus antitoxin II. A comparative evaluation of the indirect haemagglutination and toxin neutralization tests

Serum samples from 77 guinea pigs immunized against tetanus have been titrated for tetanus antitoxin by a standardized indirect haemagglutination (IHA) test and the conventional toxin neutralization (TN) test. These sera were titrated before and after treatment of the sera with 2-mercaptoethanol (2-ME) by the IHA test using unfixed sheep erythrocytes and erythrocytes fixed with glutaraldehyde, formaldehyde and pyruvic aldehyde. The titres of these sera obtained by IHA using unfixed and glutaraldehyde-fixed sheep erythrocytes before treatment of the sera with 2-ME were two to six times higher than the TN titres, whereas the IHA-titres using formaldehyde- and pyruvic aldehyde-fixed sheep erythrocytes were 10 times higher than the TN titres in some of the sera. There was no statistically significant difference between TN and IHA titres using unfixed and glutaraldehyde-fixed sheep erythrocytes after the treatment of the sera with 2-ME.     

The titration of tetanus antitoxin III. A comparative evaluation of indirect haemagglutination and toxin neutralization titres of human sera

The tetanus antitoxin titres of 174 serum samples from healthy adults were determined by a standardization indirect haemagglutination test (IHA) and the conventional toxin neutralization (TN) test. The serum samples were titrated by the IHA test using glutaraldehyde-fixed and toxoid sensitized sheep erythrocytes before and after the treatment of the sera with 2-mercaptoethanol (2-ME). The IHA method has been found to be very sensitive and specific for the estimation of tetanus antitoxin in human sera. The IHA titres before the treatment of the sera with 2-ME were generally about four times higher than the TN titres and the correlation coefficient between these titres was 0.94. The IHA titres after the treatment of the sera with 2-ME were in good agreement with the TN titres and there was no statistically significant differences between the titres by the two methods. The tetanus antitoxin titres of 50% of the sera were below the minimum protective titres of tetanus antitoxin (0.01 IU/ml). In 19.5% of the sera the antitoxin level (IU/ml) ranged from 0.01 to 0.1, in 20.1% from 0.1 to 1.0 and in 10.4% from 1.0 to 10.0.     

The titration of tetanus antitoxin IV. Studies on the sensitivity and reproducibility of the toxin neutralization test

The quality of tetanus toxin affected the sensitivity of the toxin neutralization (TN) test greatly. By using purified toxin a minimum level of 0.001 IU/ml of tetanus antitoxin could be detected whereas with crude toxin a level of 0.025 IU/ml only could be detected. The TN test described in this report permitted titration of tetanus antitoxin in twofold dilution steps from levels as low as 0.001 IU/ml using 0.6 ml of serum only at the L+/5000 level of purified tetanus toxin. Treatment of the sera with 2-mercaptoethanol (2-ME) did not affect the TN titres showing that the TN test detects the neutralizing antibodies (IgG) which are not affected by 2-ME. The TN test was found to be a highly sensitive and reproducible test.     

The quantitation of rabies-specific antibodies. II. Factors affecting the sensitivity of the haemagglutination inhibition test

Various factors affecting the HAI test for the quantitation of rabies-specific antibodies have been evaluated with a view to obtaining maximum sensitivity and reproducibility in tests using tissue culture antigens prepared in vero cells and concentrated by dialysis. Goose erythrocytes treated with proteolytic enzyme bromelian at a concentration of 0.025% were much more susceptible to HA than those that were untreated or erythrocytes treated with neuraminidase. In addition, other parameters like the use of a phosphate buffered saline (PBS) as a diluent at pH 6.2, incubation at 0-4 degrees C for 1.5-3 h were found to be most critical for achieving maximum HA activity. To remove non-specific inhibitors, serum samples were treated with aerosil, acetone in combination or alone. Of the 73 serum samples tested, removal of non-specific inhibitors by aerosil alone occurred in up to 54.79% of the samples, whereas using acetone-aerosil treatment followed by adsorption with goose erythrocytes, the inhibitors were removed in 98.67% of the samples to a level that was undetectable at the 1:4 starting dilution in the HAI test.     

In vitro titration of tetanus antitoxin

In vitro methods are used as an alternative to the expensive and time-consuming official method in mice for the titration of tetanus antibodies. Numerous techniques have been developed and used for this purpose, but with mixed results. The difficulty is to get good correlation with in vivo units whatever the vaccinal status of the individual. Today, some of these serological techniques have been virtually abandoned in favor of others which are becoming widespread. RIA is still used, but only in laboratories which already have the necessary equipment and skill. It is gradually replaced by ELISA, a rather delicate technique which however has kept its promises and is amenable to further progress. Passive haemagglutination has been greatly improved by the use of turkey erythrocytes. With this modification, this technique becomes reliable and sensitive and is apparently able to give good results for a large number of sera in case of mass investigation as well as for a quick estimate of the protective level in an individual.     

The use of an immunoenzymatic assay for the estimation of tetanus antitoxin in human sera: a comparison with seroneutralization and indirect haemagglutination

The ability of an enzyme-linked immunosorbent assay (ELISA) to detect tetanus antitoxin in human sera has been evaluated in comparison with the in vivo seroneutralization test. The results of this study, carried out on 171 serum samples, show that ELISA is a sensitive and specific in vitro test for immunity to tetanus in man; it reveals the minimum protective level of 0.01 IU/ml and is well correlated with seroneutralization. A comparison has also been made with indirect haemagglutination. Differences in specificity in low titered sera, although not statistically significant, have been observed. Reported data suggest that the ELISA may be used for the estimation of tetanus antitoxin in sero-epidemiological surveys and for clinical purposes with reliability equal--and perhaps superior--to that of IHA.     

A comparison of the indirect haemagglutination test with the toxin neutralization test for the estimation of diphtheria antitoxin in mouse sera

Serum samples from 42 groups of mice immunized for different immunization periods with various doses of Adsorbed Diphtheria-Tetanus Vaccine, Adsorbed Diphtheria-Tetanus and Pertussis Vaccine and a standard diphtheria toxoid were assayed for their diphtheria antitoxin content by indirect haemagglutination (IHA) and by toxin neutralization (TN) tests. A very good correlation of 0.91 was obtained between the results of the two methods. There was no statistically significant difference between the IHA and the TN titres obtained. Adsorption with sheep red cells and treatment of the sera with 2-mercaptoethanol had no effect on the IHA titres. The minimum level of antitoxin detectable by the IHA test was 0.00039 IU ml-1. IHA proved to be a sensitive, specific and reproducible method which can be used reliably for the assay of diphtheria antitoxin in mouse sera.     

The British Reference preparation for tetanus antitoxin for the flocculation test

To facilitate the quality control of tetanus toxoids used in the formulation of adsorbed vaccines, an equine antitetanus serum has been extensively assayed and established as the British Reference preparation for tetanus antitoxin for the flocculation test.     

Evaluation of the indirect haemagglutination test in the diagnosis of amoebiasis

The IHA test was evaluated in the diagnosis of amoebiasis. Axenically-grown E. histolytica was used as an antigen. A total of 427 sera from symptomatic (intestinal and extra-intestinal) amoebiasis patients. asymptomatic carriers, patients with parasitic intestinal infections other than E. histolytica, and healthy controls were tested. From 288 symptomatic cases of amoebiasis, 232 (80.6 per cent) gave positive reactions. In 93 asymptomatic cases of amoebiasis, 55 (59.1 percent) were seropositive. In testing of sera from 16 subjects with parasitic intestinal infections other than E. histolytica, a low-level positive IHA titres occurred in 2 (12.5 per cent). The test was also positive with a low titre in 3 (10.0 per cent) of the 30 subjects of the healthy control group. The results indicate that the IHA test is of value in the confirmation of intestinal and extra-intestinal amoebic infections especially in cases where the parasite is difficult to demonstrate.     

Use of the toxin-binding inhibition test for estimation of tetanus antitoxin in serum

The usefulness of the toxin binding inhibition test (ToBI) for the titration of tetanus antibody in human sera was assessed. Sera from 80 healthy people with different vaccination histories that had been previously tested by the in vivo toxin neutralization (TN) test were retested by the ToBI test. The lowest tetanus antibody titre which could be detected by using 0.1 Lf/ml tetanus toxin was 0.01 IU/ml. Comparison between the estimates obtained by the ToBI test and those obtained by the TN test (r = 0.93 and r = 0.7 for titration low titre sera) showed good correlation. No overestimation of antibody content was seen in titrating low titre sera by the ToBI test. It is concluded that the ToBI-test is a reliable alternative to toxin neutralization test in mice.