The method transfer and validation of freeze-dried rabies vaccine pyrogen detection in HL-60 monocyte activation assay / 中国药理学通报

Aim To study the application of HL-60 monocyte activation test in the pyrogen detection of freeze-dried rabies vaccine for human use.Methods The established HL-60 mononuclear cell activation test (MAT) was transferred between laboratories and the method was verified; referring to the interference test in the photometric method of the bacterial endotoxin test method of the Chinese Pharmacopoeia, HL-60- IL- 6 MAT was used to detect the recovery and pyrogen content of 13 batches of freeze-dried rabies vaccine for human use.Results The linearity of the amount of 1L-6 secreted by HL-60 cells, which stimulated by dif¬ferent concentrations of endotoxin standards was above 0.95; the calculated minimum detection limit was not more than 0.125 EU • mL"1 ; the recovery experiment with a solution containing 0.5 and 1.0 EU • mL"1 of endotoxin was performed to cheek the accuracy of the method.HL-60-IL-6 was used to detect 13 hatches of Freeze-dried rahies vaccine for human use, and the re¬covery of endotoxin was between 50 % to 200%.It was consistent that HL-60-IL-6 with pyrogens and en¬dotoxin test for 4 batches of freeze-dried rabies vaccine for human use which pyrogens and endotoxin test failed and the 3 batches of water for injection.Conclusion The HL-60 MAT using IL-6 as a detection indicator is suitable for the detection of pyrogenic substances in freeze-dried rabies vaccine for human use.

Study on equivalence between micro kinetic chromogenic assay and kinetic chromogenic assay / 中国药理学通报

Aim To evaluate the equivalence between micro kinetic chromogenic assay anrl kinetic chromogenic assay in order to provide data support for the use of alternative methods.Methods Detection conditions; micro kinetic chromogenic assay and kinetic chromogenic assay limulus reagent were used, sample amount of each well and limulus reagent was 25 jxL ( kinetic chromogenic assay was 100 jxL) , detection wavelength was 405 nm, ONSET OD value was 0.03, and half- well elisa plate was used for detection ( kinetic chromogenic assay was ordinary ELISA plate).The equivalence of the two methods was evaluated by various statistical methods, such as equivalence test, in collaboration with four laboratories in China.Results The results of one-way an OVA, paired T test and equivalence test were consistent, indicating that there were some differences between the existing kinetic chromogenic assay of different manufacturers, while there was no significant difference between the trace or conventional amount of reagent used by each manufacturer.Conclusions Micro kinetic chromogenic assay is e- quivalent to existing reagents in terms of accuracy and recoverv.J.

Advances in Toll-like Receptor Signaling Pathway Induced by Pyrogens / 中国药师

Lipopolysaccharide(LPS),lipteichoic acid(LTA) and zymosan can be recognized by Toll-like receptors (TLRs) ex-pressing in the inflammatory cells membrane,such as macrophages,monocytes and endothelial cells,and then induce the secretion of pyrogenic cytokines followed by triggering inflammatory reaction. The signaling pathway mechanisms were summarized, including the extracellular signaling cascades from pyrogens to individual TLRs,and intracellular signaling cascades from TLRs to nucleus.

Monocyte Activation Test (MAT) as a possibility of replacement for the rabbit pyrogen test in hyperimmune sera

ABSTRACT The use of a commercial kit for the monocyte-activation test (MAT) was evaluated for assessing pyrogenic contamination of hyperimmune sera . Three batches of sera, two pyrogen free and one pyrogenic, were tested. Endotoxin spike recover indicated that sample dilutions from 1/2 to 1/10 are suitable. Kit transport and storage conditions were also evaluated, proving that an adequate cold chain must be assured to achieve good results. Furthermore, the commercial MAT kit seemed suitable to replace the rabbit pyrogen test (RPT) for pyrogen testing of hyperimmune sera, although further tests are needed to a full validation.

Optimization and Preliminary Methodology Study of in vitro Fresh Human Whole Blood Detection Method for Pyrogen-related Factor IL-1β / 中国药房

OBJECTIVE:To establish the detection method for pyrogen-related factor interleukin 1β(IL-1β)through optimiz-ing detection condition,and to conduct preliminary methodology study. METHODS:The in vitro fresh human whole blood detec-tion method was used. The bacterial endotoxin standard solution(5,2,0.8,0.32 EU/ml)were added into diluted blood;using di-luted RPMI 1640 as blank control,the content of IL-1β in blood sample was determined by ELISA after incubation. The relation-ship of the addition of different attenuants(RPMI 1640 culture,sterilized normal saline)and fetal bovine serum,final dilution vol-ume fraction(40%,20%,10%and 8.3%)and storage duration(2,5,6,8,26 h)with the contents of endotoxin IL-1βwere in-vestigated,and related coefficient and detection limits were calculated. Different dilution times of Qingkailing injection and Ginaton injection samples and interference solutions were added into diluted blood to detect their recovery. RESULTS:The results indicated that RPMI 1640 media and 40% diluted blood was more sensitive(detection limit was 0.128 EU/ml,r=0.993);while the addition of fetal bovine serum didn’t influence the results. The detection limits of blood sample storied at 4 ℃ for 26 h were 0.128 EU/ml (r>0.990). When Qingkailing injection and Ginaton injection were diluted 10,32 and more times,the detection method was not interfered and the recovery ranged 68%-118%. CONCLUSIONS:Established in vitro fresh human whole blood detection method can be used for the detection of pyrogen,and provides trial evidence for the pyrogen detection of TCM injection.

Application of Monocyte Activation Test in the Pyrogen Detection of Vaccine / 中国药师

Objective:To study the application of HL-60 monocyte activation test in the pyrogen detection of vaccines. Methods:HL-60 cells were incubated with the standard pyrogen(endotoxin,zymosan and lipoteichoic acid)solution and the sample solution at different concentrations,and then IL-6 level was detected using ELISA kit. According to the concentration of standard pyrogen solution and the level of IL-6,the standard curve and linear relationship were obtained. Based on the IL-6 value from the sample solution,the pyrogen content of the sample was calculated. The standard pyrogen solution at middle concentration of the standard curve was used as the interference sample,and the interference assay was performed referring to the bacterial endotoxin test in Chinese pharmacopoeia. The pyrogen recovery rate and the content were detected using HL-60 / IL-6 assay in seven kinds of vaccines such as influenza vaccine(Split Virion),inactivated;hepatitis A(live)vaccine,freeze-dried;rabies vaccine(Vero Cell)for human use,freeze-dried;lyophilized human measles and rubella combined vaccine,live;measles and mumps combined vaccine, live; haemorrhagic fever with renal syndrome bivalent vaccine, inactived; group A and C meningococcal polysaccharide vaccine. Results:① The linear relationship between the pyrogen concentration and the IL-6 level was good,and R2 value was 0. 996,0. 993 and 0. 997,respectively. ② The recovery rate of pyrogen in the above vaccines ranged from 50% to 200% . ③ The endotoxin concentration of 10 batches of group A and C meningococcal polysaccharide vaccine was detected using HL-60 / IL-6 assay,and the average value of 6 batches from factory A was 134. 73 EU·ml-1 ,and that of 4 batches from factory B was 40. 75 EU·ml -1 . The pyrogen in the other vaccines was all negative. Conclusion:The HL-60 / IL-6 assay is suitable for the pyrogen detection of the seven kinds of vaccines.

Verification of the new proposed version of pyrogen result judgment algorithm / 中国药学杂志

OBJECTIVE: To verify the new proposd version of pyrogen result judgment algorithm (NPRJA) and validate practicality, accuracy and reliability. METHODS: Six hundred and fifty-four batches of pyrogen test data from our laboratory in near three years were used; 3 648 rabbit body temperature fluctuations qualified in preview were statistically analyzed. Only single rabbit having lever was used to judge the pyrogen qualified or not; using logic anlysis method determined the NPRJA. RESULTS: According to the existing Chinese Pharmacopoeia volume III, suspicious rate of pyrogenic results was 28.0%, but the suspicious rate was only 10.4% in NPRJA. The new model can reduce over 60% of the repeat test. However, all failed samples were not leaked in new determiner. In preflight warm qualified rabbits, the temperature fluctuations were rather more than reach 0.4℃ within the first 3 h, they had more than 8% in whole rabbits. CONCLUSION: In pyrogen test rabbit own body temperature appears fluctuation, single rabbit warming≥ 0.6℃ serves as one of the criteria for re-examination of the sample, its results will lead to raising re-examination rate! The new proposed version of pyrogen result judgment algorithm was used to threshold of the sum of temperature rise of all tested rabbits, that not only guarantees the no changing of accuracy in existing present pyrogen test results, also significantly reduces the amount of animals and experimental repetitions. Thereby reducing the amount of experimental and labor costs. Meanwhile, it might be convenient and practical, as will as a worthy utilization and promoting patterns.

Suitability of human whole blood in vitro pyrogen test for meningococcal groups ACYW135 polysaccharide vaccine / 中国药学杂志

OBJECTIVE: To investigate the suitability of human whole blood in vitro pyrogen test (IL-1β and IL-6) for meningococcal groups ACYW135 polysaccharide vaccine. METHODS: Fourteen batches of vaccines from different manufacturers were collected and spiked with bacterial endotoxin. The recovery was determined with human WB/ IL-1β and IL-6 in vitro pyrogen test. RESULTS: The recoveries of 14 batches of vaccines were between 50%-200% at a dilution less than the maximum valid dilution (MVD). All of the 14 batches of vaccines were qualified products as shown by the pyrogen test. CONCLUSION: The human whole blood in vitro pyrogen test is suitable to control the pyrogenin meningococcal groups ACYW135 polysaccharide vaccine.

Evaluation of Removing Pyrogen Effect of Activated Carbon in Traditional Chinese Medicine Injection Technics / 中国中医药信息杂志

Objective To establish the pyrogen removing effect of activated carbon in the technics of traditional Chinese medicine (TCM) injections. Methods The content of bacterial endotoxin concentration was detected by kinetic turdimetric assay to evaluate the effect of removing pyrogen before and after using activated carbon in concentrated solution of TCM injection. Results The activated carbon adsorption rate of Shuanghuanglian concentrated solution≥70%and the activated carbon adsorption rate of Danshen concentrated solution>95%. Conclusion Pyrogen manufacturing process is scientific and rational by adding activated carbon adsorption in powder injection of TCM. The bacterial endotoxin of large doses can't be removed fully by activated carbon adsorption.

Tachypleus Amebocyte Lysate Test Using in Transfusion Reaction / 中华医院感染学杂志

OBJECTIVE To discuss the application of tachypleus amebocyte lysate test in transfusion reaction. METHODS Based on agglutination when tachypleus amebocyte lysate test encountered bacterial endotoxin, the quantity of the bacterial endotoxin was tested. RESULTS Twenty-three cases were analyzed with tachypleus amebocyte lysate test, the main reasons were excessive pyrogen, improper combined drug compatibility, excessive agents or unqualified pyrogen of the agent added, pollutions on transfusion devices and air in the treatment room, disobeying axenic operation rules, overlong drug dispensing, too fast transfusion speed, etc. CONCLUSIONS On prevention of transfusion reaction, all the axenic operation rules should be strictly followed, single dose transfusion, to reduce transfusion reaction.

Comparative evaluation of pyrogens tests in pharmaceutical products

A comparison of methodologies for detection of pyrogens in pharmaceutical products was performed. The rabbit pyrogen test was optimized and the dose-response curve was obtained for the 2nd International Standard for bacterial endotoxins, establishing 13.81 EU/mL/kg as the concentration of endotoxin necessary to induce a temperature rise of 0.5ºC. The 0.5ºC cut-off was shown to give results that were more compatible with the pyrogenic doses for humans. The Limulus amoebocyte lysate test (LAL) was standardized with gel-clot and chromogenic endpoints, and used for the comparative evaluation of pharmaceutical products showing good agreement. The use of beta-glucan-reactive and non-reactive LAL reagents identified some products with false-positive results. The interference test was carried out and the specifications validated for some new products as the maximum valid dilution. The results emphasized the importance and limitations of the assays recommended for the evaluation of purity and quality control of parenteral medicinal products, improving the existing methodologies in the context of reduction and replacement in the use of animal models.

Realizou-se a comparação de metodologia para avaliação de pirogênios em produtos farmacêuticos. Otimizou-se o teste da hipertermia em coelhos elaborando a curva dose-resposta com o 2º Padrão Internacional de endotoxinas bacterianas, com base na qual determinou-se a concentração de 13,81 UE/mL por kg de peso corporal, necessária para produzir aumento de temperatura de 0,5ºC. Observou-se que o limite de 0,5ºC forneceu resultados comparáveis com as doses pirogênicas para o homem. Padronizou-se o teste do lisado de amebócitos do Limulus (LAL) com determinação do ponto final cromogênico e por geleificação, que foram utilizados para a avaliação de produtos farmacêuticos obtendo-se resultados concordantes. Avaliaram-se as respostas de reagentes LAL reativos e não-reativos a beta-glicanos, observando diferenças que poderiam reprovar amostras com base em resultados falso-positivos. Executou-se o teste de interferências, validou-se o procedimento e estabeleceu-se a máxima diluição válida para produtos farmacêuticos sem especificações farmacopéicas. Os resultados enfatizam a importância e as limitações dos ensaios preconizados para avaliação da pureza e controle da qualidade de produtos farmacêuticos parenterais, contribuindo para aprimorar as metodologias existentes no contexto da redução e substituição dos modelos animais.

Study on BET in sodium bicarbonate injection with TAL / 中国药学杂志

OBJECTIVE:To determine whether bacterial endotoxins test(BET) can replace the pyrogen test for the sodium bicarbonate injection.METHODS:The comparative experiment of tachypleus amebocyte lysate(TAL) and rabbits were studied to determine the sensitivity.RESULTS:The sensitivity of TAL test was 0.125 EU.ml－1, 12 times higher than that of using rabbits.CONCLUSIONS:BET may replace pyrogen test.

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.

Establishment of a Bacterial Endotoxin Test for Monitoring Pyrogen in Wilsonii Injection / 中国药房

OBJECTIVE:To establish a bacterial endotoxin test for monitoring pyrogen in wilsonii injection METHODS:Bacterial endotoxin test procedure and guiding principle in Chinese Phamacopoeia were used RESULTS:The test showed that the reaction with limulus agent was not interfered by wilsonii injection with a sensitivity of 0 25Eu/ml CONCLUSION:Limulus lysate method can be used to take the place of rabbit method for monitoring pyrogen in wilsonii injection

Effect of naoreqing oral liquid on cAMP contents of hypothalamus and cerebrospinal fluid in endogenous pyrogen-induced febrile rabbits / 中国病理生理杂志

AIM: To investigate the antipyretic mechanism of Naoreqing (NRQ) oral liquid, a Chinese medicine. METHODS: ① Fever models of rabbits were established by intravenous. injection of endogenous pyrogen (EP). ② The antipyretic action of NRQ were observed. ③ cAMP contents in the hypothalamus (HP) and the cerebrospinal fluid (CSF) were determined by radioimmunoassay. RESULTS: ① NRQ obviously reduced body temperature of febrile rabbits induced by EP (P

Comparison of the effect of two heat dissipating prescriptions on leucocytic pyrogen fever and cAMP concentration of cerebrospinal fluid in rabbits / 中国病理生理杂志

0.05). (2) Febrileresponse of LP was significantly inhibited by No.Ⅰ, No.Ⅱ, respectively. Concentrationof cAMP in CSF of LP group, 1 hour after injection of LP, was 140.94?51.74 pmol/ml, while that of the No.Ⅰ+ EP group was (83.48?8.11 pmol/ml) obviously lowerthan that of LP group (P

The febrifugal effect and analysis on its mechanism of electric needling "Shang gen" point after sectioning infraorbital nerves of rabbits with fever / 中国病理生理杂志

In order to observe the febrffugal response and analysis on its mechanism of needling "Shang gen" point bilateral infraorbital nerves of rabbits were sected in the experiment. Fever models were made by leukocytic pyrogen. The experiment findings were as follows:1. It was found that when febrile response of leukocytic pyrogen in rabbits was significant, the cAMP content in CSF was strikingly increase (25.14?2.11pmol/L). During electric needling "Shang gen" point of febrile rabbits, however, fever response was markedly inhibited (△T was 0.27?0.07 at 60min); The cAMP content rise in CSF was markedly inhibited (11.22?0.13 pmol/ml).2. After sectioning bilateral infraorbital nerves of rabbits, the fever response of febrile rabbits was not depressed (△T was 0.71?0.06 at 60min) during electric needling "Shang gen" point. In the mean time, the cAMP content in CSF was significantly increased (28.53?1.12 pmol/ml).3. The authors inferred that the mechanism of febrffugal response of electric needling "Shang gen" point might be closely associated with producing nerves impulse (from the that point) which inhibited probablely the synthesis of cAMP through some modes.

Observations on the changes of cAMP,cGMP levels in CSF during conscious rabbits with LP fever given acupuncture on main points / 中国病理生理杂志

When the fever was induced by leukocytic pyrogen(LP)on rabbits, we observed the effects of acupuncture on the main point of "Du Channel". At the same time measured the changes of cAMP and cGMP levels in CSF of the animals.It was found that cAMP levels increased (cAMP levels of normal group 18.60?1.56 pmol/ml and experimental group 25.30?1.14pmol/ml,P

Effects of gypsum on both the fever and the cyclic nucleotids levels in CSF in rabbits with fever / 中国病理生理杂志

The purpose of this study was to investigate the effects of gypsum on LP-induced fever and the CSF levels of cAMP and cGMP in rabbits. It was found that gypsum could inhibit the rise of body temperature caused by LP injection, (△T of gypsum +LP group is 0.40?0.08℃, TRI_3 2.55; LP group 0.91?0.06℃, TRI_3 4.99 P

An experimental method for endogenous pyrogen preparation from monocytes of rabbits' blood / 中国病理生理杂志

In order to obtain monocytic endogenous pyrogen, Ficoll—Hypaque's separation technique was used in this paper. We obtained mononuclear cells from rabbits' blood in vitro and added endotoxin free saline to make suspension in the concentration of 0.65?10~6/mL. Endogenous pyrogen was produced from monocytes by stimulation of purified endotoxin of colon bacilli and incubation in saline at 37-38℃ stirred and shaked continuously for 10 hr. There is EP in collecting fluid after centrifugation and ultrafiitration.The result obtained shows:1. Employing Ficoll-Hypaque's separation technique, high concentration monocytic endogenous pyrogen can be obtained.2. Monocytic endogenous pyrogen have marked febrile effect.