Development and verification of kinetic chromogenic quantitative detection method for endotoxin content in intermediate of component pertussis antigen / 中国生物制品学杂志

@#Objective To develop a kinetic chromogenic quantitative method for the determination of endotoxin content in intermediate of pertussis antigen,and to verify the method so as to better control the quality of diphtheria,tetanus,and pertussis vaccine(DTP vaccine).Methods A kinetic chromogenic assay[Limulus Amebocyte Lysate(LAL)]was developed to detect the endotoxin content in the intermediate products of pertussis antigens after detoxification,and verified for the linearity,specificity,accuracy,reproducibility and intermediate precision. The quantitative detection results of kinetic chromogenic assay were compared with those of gel method.Results The absolute value of the linear correlation coefficient（|r|）of the kinetic chromogenic assay was more than 0. 99;under the maximum effective multiple dilution,the interference test recovery of the intermediate was within 50% — 200%,and pertussis toxin(PT)diluted to 10,100 and 1 000 times,filamentous hemagglutinin(FHA)diluted to 3 000,5 000 and 10 000 times,and pertussis adhesin(PRN)diluted to 50,75 and 100 times had no interference effect on the experiment after detoxification;the accuracy verification recovery rates of PT,FHA and PRN were 125%,110% and 99% respectively;and the CVs of reproducibility verification were 7. 21%,8. 31% and 5. 84%,and the CVs of intermediate precision verification were 6. 04%,16. 29% and 12. 23%,respectively.The bacterial endotoxin content of the three batches of pertussis antigen intermediates detected by kinetic chromogenic assay was consistent with that verified by gel method,both of which were less than the limit of bacterial endotoxin in the intermediates of pertussis antigen after detoxification.Conclusion The developed kinetic chromogenic assay has good linearity,specificity,accuracy and precision with accurate detection results,which can be used to detect the endotoxin content in intermediate products of component pertussis antigen after detoxification.

Valoración de endotoxinas bacterianas en el inyectable ácido zoledrónico mediante la prueba de lisado del amebocito de Limulus / Assessment of bacterial endotoxins in Zoledronic acid injectable drug by using Limulus amebocyte lysate test

Objetivo: valorar las endotoxinas bacterianas por la técnica del lisado del amebocito de Limulus para el producto inyectable ácido zoledrónico, por el método de gelificación. Métodos: el ensayo se realizó mediante dos pruebas: 1) confirmación de la sensibilidad del lisado etiquetado, para lo cual se preparó una curva estándar con diluciones seriadas dobles de endotoxina por cuadruplicado; y 2) el ensayo del producto de inhibición, en el que se prepararon diluciones seriadas dobles de endotoxina con agua apirogénica y con las muestras de los lotes a ensayar sin sobrepasar la máxima dilución válida. Se determinó el punto final y se calculó la media geométrica. Se definió la dilución de trabajo, la cual se validó por cuadruplicado en tres lotes consecutivos. Resultados: la sensibilidad del lisado resultó 0,03125 UE/mL. La máxima dilución válida fue de 112 UE/mL y la dilución de trabajo 1/100. La cantidad de endotoxinas bacterianas presentes en tres lotes del producto inyectable no sobrepasó el límite establecido, por lo que cumplió con las especificaciones de calidad establecidas para el ensayo. Conclusión: la estandarización de las condiciones del método por gelificación, hace que este resulte eficaz, confiable, rápido y de fácil ejecución, por lo que puede emplearse como ensayo de rutina en el control de la calidad del inyectable analizado

Objective: To asses the presence of bacterial endotoxins in Zoledronic Acid injectable drug by using the Limulus amebocyte lysate test, particularly by the gelling procedure. Methods: The assay was performed in two tests: the first was the confirmation of labeled lysate sensitivity by preparing a standard curve with serial double dilutions of endotoxins four times, and the second was the inhibition product test in which serial double dilutions of endotoxins were prepared with apyrogenic water and with samples from the batches to be tested, without exceeding the maximum valid dilution. The end point was determined and the geometric mean was calculated. Working dilution was defined and then validated four times in three consecutive batches. Results: The lysate sensitivity was 0.03125 EU/mL). The maximum valid dilution and the working dilution were 112 EU/mL and 1/100 EU/mL) respectively. The amount of bacterial endotoxins present in three batches of the injectable drug did not exceed the set limit, so it complied with the quality specifications for this test. Conclusions: The standardization of the gelling method conditions makes it possible to state that this method is effective, reliable, quick and easy-to-perform, so it can be used as a regular test in the quality control of the analyzed parenteral drug

In Vitro Bioassay of Endotoxin Using Fluorescein as a pH Indicator in a Macrophage Cell Culture System

Based on the biological activity of endotoxin, we propose a possible new method for detecting endotoxin using a pH- indication system of macrophage culture media. After RAW 264.7 macrophage cells were treated with lipopolysaccharide (LPS), the addition of fluorescein to the LPS-treated media reproductively reduced its absorption and emission spectra (it was a dose-dependent reduction). The advantages of this LPS- detection method were compared with the Limulus Amebocyte Lysate (LAL) test by using purified bacterial LPS (Salmonella minnessota, Escherichia coli, and Pseudomonas aeruginosa). Additionally, the absorption and fluorescence intensity of fluorescein, following treatment of RAW 264.7 cells with a high concentration of Staphylococcus aureus (Gram-positive, lysed bacteria), could not generally be detected by the LAL test, but they were found to be reduced, in a dose-response relationship, with this new system. The macrophage culture system-method might be a good supplement to the LAL assay for detection of LPS, Gram-negative and Gram-positive bacteria.

In vitro study on the neutralizing LPS activity of modeling peptides from the limulus antilipopolysaccharide factor / 第三军医大学学报

Objective To evaluate the endotoxin-neutralizing activity of modeling peptides from the limulus antilipopolysaccharide factor (MPLALFs, Ms) in vitro. Methods The endotoxin-binding activity of Ms was examined by biosensor technique and shown in values of Kon and Kd. The endotoxin-neutralizing effect was analyzed by limulus amebocyte lysate test. Results The biosensor technique results showed that the Kon values of M_0, M_1, M_2, M_3, M_4, M_6, M_8 and M_ 10 binding to LPS 055∶B5 were (840?5.716), (549?6.532), (842?6.530), (627?2.450), (996?5.716), (814?8.982), (556?1.633) and (635?2.449) arc second, of which M_4 and M_1 had the highest and lowest endotoxin-binding activity, respectively. The M_4 reacted to LPS with a Kd of 72.377 ?mol/L. The results obtained by the limulus amebocyte lysate test were the same with those from the biosensor technique. Conclusion M_4 has a potential good endotoxin-neutralizing effect in vitro.

Effects of various pH in testing bacterial endotoxins / 中国药学杂志

OBJECTIVE: To study the effect of samples having different pH values in the bacterial endotoxins test (BET). METHODS: The BET was conducted as an assay of the concentration of endotoxins by using kinetic-turbidimetric technique. Samples having different pH values but containing same concentration reference standard endotoxin (RSE) were tested by TAL/LAL reagents. The average result on the standard curve was calculated and the mean recovery observed. RESULTS: The reagents had three highly sensitivities in pH 5.20, pH 7.83/pH 6.23 and pH 10.55. After adjustment to pH 6 to 8, the mean recovery was 76.5%～115.9% or 85.8%～99.8%. When the sample pH value was less than 3 or more than 12, the test was unsuitable for the inhibition test, and the mean recovery was less than 56.8%. CONCLUSION: It is necessary to adjust the pH of the solution to be tested in the BET, adjust the sample to pH (6.5～7.5) when using TAL reagents, and adjust to pH (6.2±0.1) or pH(6.7～7.8) when using LAL reagents.

ANALYSIS OF THE RESULT OF LAL TEST FOR PYROGEN OF 200 BATCHES OF INJECTIONS / 中国海洋药物

This paper deals with an. analysis of the result of LAL test for pyrogen of 200 batches injection. The result is reliable and shows that the operation of this method is easy, quick and sensitive.

Infusion Reaction: An Analysis of 58 Cases / 中华医院感染学杂志

OBJECTIVE To investigate the reasons of infusion reaction. METHODS The liquid and disposable infusion tubes were tested by limulus amebocyte lysate(LAL).Blood samples were collected when infusion reaction occurred and the bacteria in blood culture were detected. RESULTS A total of 58 cases of infusion reactions within two years were from clinical department.LAL of liquid infusion was positive in 5 cases.Two isolations were identified from liquid infusion respectively.LAL was negative and no isolation for the unsealed bottle of liquid drugs with same batch number.LAL was negative for samples of same lot infusion tubes.The main drugs caused infusion reaction were refined medical herbs,large molecule substance,blood products and the medicine contained potassium ion.53.4% infusion reactions occurred in autumn.The seniles had high incidence.Bacteria were isolated from 5 patients in blood culture after liquid infusion. CONCLUSIONS More than one reason that cause infusion reaction.It is important to reduce incidence of infusion reaction through quality control of infusion tubes and aseptic process in infusion.