Objective To develop and verify a sulfosalicylic acid spectrophotometric method for the determination of trace iron ions in diphtheria toxin medium,and apply it preliminarily.Methods The maximum absorbance of the complex of iron and sulfosalicylic acid was scanned by full spectrum;A method for the determination of iron ion in culture medium was developed by linear regression between the absorbance of the complex and the content of iron ion,and the stability,accuracy and precision of the method were verified.The effects of Ca~(2+),Mg~(2+),K~+,Na~+ and reactants on the method were investigated.Spectrophotometric method with sulfosalicylic acid was used to determine trace iron in the self-made and commercial medium for diphtheria toxin production.Sulfosalicylic acid spectrophotometry,ferrizine colorimetry and o-phenanthroline spectrophotometry were used to detect iron content in two kinds of culture media(beef trypsin digestion liquid and 5% polypeptone),and the detection results of the three methods were compared.Results The complex of iron and sulfosalicylic acid showed the maximum absorbance at the wavelength of 425 nm;There was a good linear relationship between the absorbance and concentration of iron ion in the range of 1～0.05 μg/mL,the detection limit was 0.05 μg/mL,and the standard equation was:Y=0.027 9 X+0.046 1,R~2> 0.99;The coefficients of variation(CVs) of A_(425) value of each concentration of standards measured every 5 min were less than 5%;Low(0.05 μg/mL),medium(0.5 μg/mL)and high concentration(1 μg/mL) of Fe~(3+) standard solutions were continuously determined for 3 times.The CVs of 9groups of each concentration measured in parallel were all less than 5% and the recovery rates were higher than 95%;Ca~(2+),Mg~(2+),K~+,Na~+,15 μL of sulfo salicylic acid(20%) and 50 μL of ammonia hydroxide(1:1) showed no interference in the method;The results of toxin-producing medium were consistent with those of diphtheria bacteria;The results of the three detection methods were consistent.Conclusion The developed spectrophotometric method with sulfo salicylic acid can determine the content of trace iron ions in diphtheria toxin medium accurately and effectively.

Objective To explore the possibility of using trypsin digested-polypeptone medium to prepare diphtheria vaccine adsorbed, so as to improve the quality and safety of the vaccine.Methods Corynebacterium diphtheriae(C.diphtheriae) was cultured in trypsin digested-polypeptone medium(polypeptone group) and modified Linggood's medium(modified Linggood's group), and three batches were cultured. The culture solution of C.diphtheriae was collected, and the toxin production detection, SDS-PAGE analysis and viable bacteria count were performed. Diphtherotoxin(DT) was extracted, and the 50% tissue culture infective dose(TCID50) was measured, and the logarithm was taken. After formaldehyde detoxification treatment, DT was tested for purity and analyzed by SDS-PAGE. Diphtheria vaccine adsorbed was prepared according to the corresponding method of Chinese Pharmacopoeia(VolumeⅢ, 2020 edition), and the vaccine titer was detected.Results The mean toxin production of three batches of culture solution of C.diphtheriae in polypeptone group was 177 Lf/m L, slightly lower than that of modified Linggood′s group(183 Lf/m L), and there was no statistically significant difference between them(t = 2. 00, P > 0. 05).The relative molecular weights of toxin protein and diphtheria toxoid obtained from two kinds of culture media were about 60 000. The mean values of A600and viable bacteria, lg TCID50of DT, purity of diphtheria toxoid and titer of diphtheria vaccine adsorbed in modified Linggood's group were significantly higher than those in polypeptone group(t = 9. 390, 31. 180,4. 914, 44. 540 and 4. 510, respectively, each P < 0. 05), while all the indicators in polypeptone group met the relevant requirements of Chinese Pharmacopoeia(VolumeⅢ, 2020 edition). Conclusion Trypsin digested-polypeptone medium is expected to replace the traditional modified Linggood's medium for the preparation of diphtheria vaccine adsorbed, while the preparation technology needs to be further optimized to improve the vaccine quality.

Objective:To evaluate the effects of a booster immunization with a candidate tetanus toxoid, reduced diphtheria toxoid and acellular pertussis combined vaccine (Tdap) in a rat model after primary vaccination with diphtheria, tetanus, acellular pertussis and Sabin strain inactivated poliovirus combined vaccine (DTacP-sIPV) or diphtheria, tetanus, acellular pertussis, inactivated poliovirus and haemophilus type b combined vaccine (DTacP-IPV/Hib) for further preclinical study.Methods:Wistar rats were randomly divided into three groups and respectively immunized with a self-developed DTacP-sIPV, a marketed DTacP-IPV/Hib and normal saline at 0, 1, and 2 months of age. Serum levels of antibody against each component in each group were detected before immunization and after each dose. A booster dose of the candidate Tdap was given 10 months after primary immunization. Serum levels of antibody against each component in each group were detected before, 1 month and 6 months after the booster immunization.Results:One month after three doses of primary immunization, the geometric mean titers (GMT, Log2) of antibodies against diphtheria toxoid (DT), tetanus toxoid (TT), pertussis toxin (PT), filamentous hemagglutinin (FHA) and pertactin (PRN) in the DTacP-sIPV group were 17.41, 18.34, 18.11, 19.93 and 13.91, respectively, and the seroconversion rates of these components all reached 100%. Ten months after primary immunization, the GMTs of antibodies against DT, TT, PT, FHA and PRN decreased to 15.17, 14.26, 13.60, 14.51 and 10.39, respectively, and the seroconversion rates remained above 89%. One month after booster immunization, the GMTs of antibodies against DT, TT, PT and FHA in the DTacP-sIPV and DTacP-IPV/Hib groups were 16.49/17.26, 16.80/17.63, 16.70/17.74 and 18.48/19.26, respectively, and the seroconversion rates of these components all reached 100% with no significant difference between the two groups ( P>0.05). The GMTs of anti-PRN antibody in the DTacP-sIPV and DTacP-IPV/Hib groups were 13.07 and 11.00, and the seroconversion rates were 100% and 88%, which were higher in the DTacP-sIPV group than in the DTacP-IPV/Hib group ( P<0.05). Six months after booster immunization, the GMTs of antibodies against DT, TT, PT, FHA and PRN in the DTacP-sIPV and DTacP-IPV/Hib groups decreased to 15.74/14.87, 15.07/15.14, 14.84/15.73, 16.62/16.37 and 11.44/9.96, respectively, and the seroconversion rates remained above 88%. Conclusions:Booster vaccination with the candidate Tdap vaccine induces humoral immune response following primary immunization with DTacP-sIPV or DTacP-IPV/Hib in the Wistar rat model, while the antibody titer decreases with time.