Standardization of serum free media for cryopreservation of RK13 cells.

Foetal calf serum present in the media used for cryopreservation was replaced by various synthetic polymer such as gelatin, glycerol, carboxymethyl cellulose and dimethyl sulphoxide at various concentration. Growth pattern of cells, % survival and karyological studies have been done in the present study. It was found that optimum concentration of carboxymethyl cellulose was 0.1% in combination with 10% glycerol and 10% DMSO. At this concentration percentage survival of cells was found maximum and karyotype was found normal without any abnormality in the chromosomes. It was concluded from the study that serum free media can be employed for the cryopreservation of these cells which are further used for production of tissue culture vaccines without causing any adverse affects.

Adverse reactions after measles vaccination in India.

BACKGROUND: One hundred and sixty million doses of measles vaccine have been used since the Expanded Programme of Immunization began in 1985. Mild-to-moderate vaccine reactions are not infrequent and are accepted by parents but occasionally severe reactions and even death occur. We studied these severe reactions. METHODS: From 1986 to 1994, 1762 batches of measles vaccine were tested and found to be satisfactory by the World Health Organization criteria. These were released for mass immunization. RESULTS: After 40 reported incidents of severe reactions or deaths in the field, 59 intact samples of vaccine produced by different manufacturers were tested and found to be safe, i.e. they were not toxic and were sterile. However, on testing reconstituted or used vials a few were found to be toxic and many were unsterile. Reactions occurred in 115 vaccinees resulting in death of 79 children. These reactions were characterized by high fever, vomiting and profuse watery diarrhoea resulting in death within 24 hours. CONCLUSIONS: Reactions to the vaccines were more likely to be related to the toxic shock syndrome due to the use of unsterile syringes and needles and perhaps the use of reconstituted vaccines beyond their specified time for administration resulting in contamination.

A rapid test for measuring the infectivity of Yellow Fever vaccine.

An in-house reference preparation of 17D-204 Yellow Fever vaccine was tested 60 times using LD50 mouse assay and CCID50 method. The mean virus titre, standard deviation and coefficient of variation obtained were comparable. Student's t-test indicated that there was a statistically insignificant difference between the two methods (p > 0.05) used for potency test with a correlation coefficient of 0.666 (p < 0.05). CCID50 assay for measuring the infectivity of Yellow Fever vaccine is rapid, sensitive and reproducible.

Transplacental immunity and waning of maternal antibody in measles.

Since transplacental immunity and waning of maternally derived measles specific antibodies play an important role in determining the optimum age for vaccination of infants against measles, a study was carried out in which 150 paired samples and 581 infant serum samples were tested for measles specific antibodies. Out of these paired samples, 132 pairs showed measles antibodies in both mother and cord. HAI antibody was absent in 3 paired samples whereas, 5 mothers could not pass on the antibodies in the cord samples. In the remaining 10 serum samples only cord blood showed the presence of antibodies without the detectable level of antibodies in mother. Statistically no significant difference between the mother and cord blood titers was observed by applying the student 't' test for comparison of the mean (t = 0.01). Analysis of 581 infant serum samples for prevalence of maternal antibodies indicated that 83% of the samples at the age of 3 months or below had measles antibodies but with the increase in age there was tremendous loss with only 19-20% at the age of 6-7 months. After 7 months the percentage of infants which had antibody varied from 11-13%. There was negative correlation between age and seropositivity (r = -0.72) which was highly significant (p < 0.05).

Factors affecting the sensitivity and reproducibility of passive haemagglutination test for the quantitation of measles-specific antibodies.

Various factors affecting the passive haemagglutination test (PHA) for the quantitation of measles-specific antibodies have been evaluated with the aim to obtain maximum sensitivity and reproducibility of the test. The antigen used for sensitization was prepared in Vero cells using Edmonston 245 strain of measles virus. Sheep red blood cells (SRBCs) were found most sensitive for use in PHA test. The optimum dilution of tannic acid was found to be 1:40,000 for tanning of fixed and 1:10,000 for unfixed SRBCs, when the tanning was carried out at 4 degrees C overnight. Fixed and tanned SRBCs sensitized with 32 HA units of measles HA antigen at 56 degrees C for 30 mins were found optimal. SRBCs from different sheep affected the sensitivity of the assay. Stability study of SRBCs showed that storage at -70 degrees C of glutaraldehyde-fixed and sensitized SRBCs gave better results as compared to those stored at -20 degrees C and +4 degrees C. Tanned SRBCs could be stored at -70 degrees C only up to 15 days. Sensitized SRBCs with-stood two cycles of freezing and thawing after removal from -20 degrees C and -70 degrees C. Sensitized SRBCs could be stored for 120 days without any significant loss of titer at -20 degrees C, +4 degrees C or 22 degrees C; when lyophilized with stabilizers, there was a slight decline in the titer after exposure at 37 degrees C for 30 days. The lyophilized sensitized SRBCs after reconstitution were found to be stable at +4 degrees C for 3 days without any loss in the titer.(ABSTRACT TRUNCATED AT 250 WORDS)

Study on the stability of 17D-204 yellow fever vaccine before and after stabilization.

To monitor the parameters controlling yellow fever vaccine production, eight different lots produced without stabilizers were studied. It was found that the freeze-thaw cycle did not have any adverse effect on virus infectivity and the mean loss in virus titre during lyophilization was 0.51 log10 ml-1. A thermodegradation study after storage of vaccine at different temperatures showed that the vaccine did not pass the accelerated stability test. To stabilize the 17D-204 substrain vaccine, four stabilizers with different sugars and amino acids were examined. The optimum time for addition of the stabilizers was found to be during homogenization of infected embryos. An accelerated stability test at 37 degrees C indicated that stabilizers S1, S2 and S3 kept the vaccine stable for up to 4 weeks, whereas the vaccine was only stable for up to 3 weeks in stabilizer S4. Stabilizer S3 was found statistically to be the best. Reproducibility in production methodology was established by preparing and testing more batches of the vaccine using stabilizer S3.

Verocytopathic activity of Escherichia coli O157 & other 'O' serogroups isolated from patients of diarrhoea.

Twenty diarrhoeal isolates identified as Esch. coli O157 were screened for cytopathic activity on the continuous Vero cell line. Of these, seven strains (35%) showed cytopathic effect which was maximum on the 4th day after inoculation, with a maximum titre of 1 in 128. The total loss of cytopathic activity was observed in the positive filtrates subjected to a temperature of 100 degrees C for 15 min, while heating at 65 degrees C for 15 min, resulted in partial loss of this activity. All the verocytopathic strains were obtained from infants and children and were devoid of heat labile (LT) and heat stable (ST) toxins as well as enteroinvasive property.

The quantitation of rabies-specific antibodies. III. A comparative evaluation of modified counter immunoelectrophoresis, haemagglutination inhibition and serum neutralization titres of human sera.

The rabies-specific antibodies of 73 serum samples from vaccinated humans were determined by the modified counter immunoelectrophoresis (MCIE), and the haemagglutination inhibition test (HAI) by using the conventional serum neutralization test (SN) as a yard-stick. Both MCIE and HAI were found to be sensitive and specific for the estimation of rabies antibodies. In general, the unitages obtained by the MCIE and SN showed statistically insignificant differences (P greater than 0.05) and the correlation coefficient between the two methods was 0.697 (P less than 0.05). Although the unitage of the sera detected by HAI tests was lower by a factor of 0.155 from the unitage of SN tests, there was statistically insignificant differences between the two techniques (P greater than 0.05) with a correlation coefficient of 0.556 (P less than 0.05).

The quantitation of rabies-specific antibodies. I. Modified counter immunoelectrophoresis. A rapid and sensitive method.

Modified counter immunoelectrophoresis was standardized with respect to dilution of tissue culture antigen and indicator serum, the incubation time for neutralization and the effect of an electric current. The technique was found to be sensitive enough to detect a minimum level of antibodies (0.5 IU/ml) by using a 16 mA current per slide for 2 h, indicator serum of 15 IU/ml and the use of an antigen at a concentration of 1:35. Above all, the incubation period did not affect the neutralization of the virus. The test was also applied to the detection of rabies-specific antibody levels in 73 human sera. The test was found to be simple, quick and economical for titration of rabies antibodies.

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.