Neutralización de la actividad letal del veneno de serpiente Bothrops atrox por suero hiperinmune de llama (Lama glama) / Neutralization of the lethal activity from Bothrops atrox venom by hyperimune llama serum (Lama glama)

RESUMEN Objetivos: Evaluar la capacidad del suero hiperinmune de llama (Lama glama) para neutralizar la letalidad del veneno de la serpiente Bothrops atrox en ratones de laboratorio. Materiales y métodos: Se calculó la dosis letal media (DL50) de un pool de venenos de serpientes de Bothrops atrox de Perú, y se midieron los títulos de anticuerpos por ensayo ELISA; así como la potencia de neutralización del suero inmune por el cálculo de la dosis efectiva media (DE50) durante el periodo de inmunización. Resultados: La DL50 del veneno fue de 3,96 µg/g, similar a otros trabajos realizados en Bothrops atrox en Perú. Los títulos de anticuerpos contra el veneno se incrementan rápidamente en la llama mostrando una rápida respuesta inmune; sin embargo, la capacidad de neutralización se incrementa más lentamente y requiere de varias dosis y refuerzos de las inmunizaciones alcanzado una DE50 de 3,30 µL/g ratón y una potencia de neutralización 3,6 mg/mL después de 15 inmunizaciones. Conclusiones: El suero hiperinmune de llama es capaz de neutralizar la letalidad del veneno de la serpiente Bothrops atrox de Perú en ratones de laboratorio.

ABSTRACT Objectives: To evaluate the capacity of the hyperimmune llama serum (Lama glama) to neutralize the lethal activity of Bothrops atrox venom in laboratory mice. Materials and methods: Mean lethal dose (LD50) was calculated from a Bothrops atrox venom sample pool from Peru. The antibody titers were measured by ELISA assay; and the immune serum neutralization potency was measured by calculating the mean effective dose (ED50) during the immunization period. Results: The venom's LD50 was 3.96 μg/g; similar to what was found in other studies about Bothrops atrox carried out in Peru. The titers of antibodies against the venom increased rapidly in the llama, demonstrating a fast immune response; however, the neutralization capacity increased slowly and required several doses and immunization reinforcements, obtaining a ED50 of 3.30 μL/g mouse and a neutralization potency of 3.6 mg/mL after 15 immunizations. Conclusions: The hyperimmune llama serum is able to neutralize the lethality of the Bothrops atrox venom from Peru in laboratory mice.

Prokaryotic expression, purification and antiserum preparation of severe acute respiratory syndrome coronavirus 2 nucleocapsid protein / 第二军医大学学报

Objective To express and purify the recombinant nucleocapsid (N) protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and prepare antiserum from immunized mice. Methods The prokaryotic plasmid pET28a-N containing SARS-CoV-2 N gene was transformed into Escherichia coli BL21 (DE3). The expression of recombinant SARS-CoV-2 N protein was induced by isopropyl-β-D-thiogalactopyranoside. The Ni-NTA affinity chromatography column was used to purify the recombinant SARS-CoV-2 N protein, and antiserum was obtained from the BALB/c mice immunized with recombinant SARS-CoV-2 N protein combined with manganese adjuvant through intramuscular and subcutaneous injections. The reactions of recombinant SARS-CoV-2 N protein with SARS-CoV-2 N monoclonal antibodies and severe acute respiratory syndrome coronavirus (SARS-CoV) N polyclonal antibodies were detected by Western blotting. The reaction of mouse antiserum with the recombinant SARS-CoV-2 N protein expressed in the cells transfected with eukaryotic expression plasmid was examined by indirect immunofluorescence assay. Results The recombinant SARS-CoV-2 N protein was successfully induced and expressed as a soluble protein with a molecular weight of about 55 000. High concentration of purified protein was obtained. The results of Western blotting showed that the recombinant SARS-CoV-2 N protein could be specifically recognized by the SARS-CoV-2 N monoclonal antibodies and the SARS-CoV N polyclonal antibodies. The prepared mouse antiserum could also correctly recognize the recombinant SARS-CoV-2 N protein expressed in mammalian cells by indirect immunofluorescence assay. Conclusion Recombinant SARS-CoV-2 N protein has been successfully expressed and purified from the prokaryotic expression system, and mouse antiserum has been prepared, which lays a foundation for establishing a rapid SARS-CoV-2 diagnostic tool and further studying the function of SARS-CoV-2 N protein..

Establishment and evaluation of a triple-color human papillomavirus pseudovirion neutralization assay / 中华预防医学杂志

Objective@#To establish a triple-color pseudovirion-based neutralization assay (PBNA) and evaluate its capability of detecting immunogenicity of the sera generated by the immunization of HPV 9-valent vaccine.@*Methods@#HPV pseudovirus (PsVs) 6/11/16/18/31/33/45/52/58 with the encapsidated fluorescence expressing red fluorescent plasmid N31-MCHREEY, green fluorescent N31-EGFP or blue fluorescent N31-mTagBFP were generated. The concentration of HPV PsVs and the infection titers of HPV PsVs were detected by double-antibody sandwich ELISA and TCID50, respectively. The single- and triple color HPV 16/33/45 PsVs were used to detect the neutralization titers of mice sera immunized with HPV 9-valent vaccine and confirmed the accuracy and specificity of the triple-color PBNAs. Then, the single- and triple color HPV 6/11/18/31/33/45/52/58 PsVs were employed to detect the neutralization titers of cynomolgus macaques sera immunized with HPV 9-valent vaccine and determined whether the triple-color PBNAs could be applied to evaluate the immunogenicity of the sera generated by the immunization of HPV9-valent vaccine.@*Results@#The concentration of HPV16 PsVs encapsulating green, red or blue fluorescent plasmid was 5.0 to 6.0 μg/ml and HPV6/11/18/31/33/45/52/59 triple-color HPV PsVs was about 1.0 to 3.0 μg/ml. 9 types HPV PsVs containing EGFP, Mcherry or mTagBFP reporter plasmid were obtained and the concentration can meet the need of neutralization detection. 9 types single-color fluorescent HPV PsVs had similar infectivity against 293FT cells with the infection titer values between 1×104 and 1×105. The results of PBNAs showed that there was no significant difference in the anti-HPV neutralization titers of mice sera induced by HPV 9-valent vaccine between single-color and triple-color HPV16/33/45 PsVs (P>0.05). Similarly, there was also no significant difference in the anti-HPV neutralization titers of cynomolgus macaques sera induced by HPV 9-valent vaccine between single-color and triple-color HPV6/11/18/31/33/45/52/58 PsVs (P>0.05).@*Conclusion@#We successfully established the triple-color PBNAs and verified the accuracy and specificity of triple-color PBNAs consistent with single-color PBNAs. The triple-color PBNAs can be applied to evaluate the immunogenicity of HPV 9-valent vaccine's immune serum.

Experiment study on corneal moist chamber storage with aqueous removed and recipient serum tamponaded / 中国医师杂志

Objective To explore the new preservation method of cornea by evaluating the structure and function of rabbit's endothelia on the condition of whole-eye preservation with aqueous removed and recipient's serum tamponaded. Methods Forty New Zealand big white rabbits (80 eyes) were random divided into two groups, 40 eyes in group A (control group) and 40 eyes in group B (experimental group). The vitality of endothelial cell on the condition of tow corneal preservation methods which were the moist chamber preservation (group A) and the whole-eye preservation with aqueous removed and recipient's serum tamponaded (group B) was compared. At the 2nd day, the 5th day, the 7th day, the 10th day, the vitality of endothelial cell was appraised through ultra-microstructure by scanning electron microscope and the trpan biuealizarin red stain. The corneal thickness was measured, and corneal endothelial cells density was calculated, and cell size was observed by image analysis system. Result In group B, the corneas remained transparent for 7 days , and the rate of vitality was 90% for7 days and that was over 80% for 10 days. In group A, the corneas remained transparent for 2 days, and the endothelial cell losing and dying were found after 5 days. Cell vitality, cell density and cell size had no statistical difference between Group B for 7 days and group A for 2 days. (all P＞0. 05). In group A, at the 5th day, the 7th day ,the 10th day, the average corneal thickness were (0.64 ± 0.04) mm, (0. 79 ± 0. 03) mm , (1.06 ± 0. 03) mm. In the group B, at the 5th day, the 7th day ,the l0th day, the average corneal thickness were (0. 55 ±0.03)mm, (0.65 ±0. 02) mm , (0. 85 ± 0. 05) mm. The average corneal thickness had significant difference between group A and group B (all P ＜ 0. 05). Conclusion Recipient serum had the function with supporting the structure and function of rabbit's corneal endothelial cell, and it could prolong the storage time with the moist chamberstorage at the same time.

The peptide containing the conserved sequence of HPV L1 was used in the detection of polyvalent HPV-positive clinical specimens / 肿瘤

Objective: To confirm that the antiserum induced by short peptide in the region of AA 448-477 at HPV16 L1 C-terminal can respond with many types of HPV L1. Methods: This sutdy synthesized a short peptide containing the conserved sequence of HPV L1 and immunized animal with the synthetic peptide to obtain the polyclonal antiserum. The type of HPV in clinical specimens, such as cervical cancer and condyloma acuminatum tissues, was identified by PCR. ELISA, Western blotting, and inummohistochemistry were used to determine whether the anti-serum of the short peptide could react with different types of HPV-positive specimens. Results: ELISA assay showed that the reaction between antigen and antibody was positive in HPV6, 11, 16, and 18 and other HPV-positive clinical specimens. Western blotting revealed that there was a strip at 56 kDa in all types of HPV-positive clinical specimens, but there was no strip in HPV-negative clinical specimens. Immunohistochemistry analysis demonstrated that the histological reaction had varying features in different clinical specimens. The positive-reaction appeared only in the epithelium tissues, which was regional and had apparent borders. Conclusion: This study confirms that the common conserved sequence really has HPV L1 common epitope. Its anti-serum can respond with many types of HPV L1. These findings provide an important basis for further researching a broad-spectrum HPV L1 vaccine or publicizing broad-spectrum HPV detection kits.

Comprehensive treatment for patients bitten by venomous snakes / 中华全科医师杂志

Objectives To summarize experiences and improve efficacy of comprehensive treatment for patients with wound bitten by venomous snakes. Methods Totally, 547 patients with wound bitten by venomous snakes were hospitalized during January 1993 to December 2002, with comprehensive treatment focusing on purified antivenom serum and paying attention to intensive care for the lung, brain, kidney, blood and circulatory function to detect and handle with viscera damage earlier. Large-dose corticosteroid and anisodaminie was used in treatment for snake-bitten patients to improve clinical effects.Results Among 547 patients, 501 were cured (91.6%), 28 (5.1%) improved, and limb dysfunction was left in 8 (1.5%), including two needed skin grafting, fingers or toes amputed in three and one hemiplegia, and ten died with a case-fatality ratio of 1.8%. Conclusions Multiple organ failure caused by venomous snake bite is one of high risk factor leading to death. Comprehensive and symptomatic treatment for snake-bitten wound can reduce its case-fatality.

Purificación de IgG1, IgG2 e IgG3 mediante cromatografía de afinidad a partir de intacglobín / Purification of IgG1, IgG2 and IgG3 by affinity chromatography starting from Intacglobin

Se describe un método de purificación de las subclases IgG1, IgG2 e IgG3 a partir de Intacglobin mediante cromatografía de afinidad con el empleo de proteína A Sepharose y la aplicación de un gradiente lineal de pH. Se detectó la presencia de IgG1, IgG2 e IgG3 en las fracciones eluidas mediante doble inmunodifusión e inmunoelectroforesis, utilizando sueros comerciales anti IgG, anti IgG1, anti IgG2 y anti IgG3. El procedimiento desarrollado se caracterizó por su sencillez, elevada resolución y la relativa pureza de las subclases aisladas. Se logró un rendimiento total del 53,7 %, lo que indica la eficiencia del método.

A method for the purification of IgG1, IgG2, and IgG3 subclasses starting from Intacglobin by affinity chromatography with the use of protein A Sepharose and the application of a lineal gradient of pH is described. IgG1, IgG2 and IgG3 were detected in the eluted fractions by doble immunodifussion and immunoelectrophoresis by using anti IgG, anti IgG1, anti IgG2 and anti IgG3 commercial sera. The procedure developed was characterized by its simplicity, high resolution and the relative purity of the isolated subclasses. A total yield of 53.7 % was attained, which demonstrates the efficiency of this method.

Experimental study on the loss of PMN's bactericidal capacity induced by bypass-activated complement / 第三军医大学学报

In order to verify that bypass-activated complement can enable polymorphonuclear neutrophils (PMN) to lose their bactericidal capacity,3 experiments were performed as follows:(DA monolayer culture of human PMN was added with zymosan-activated human serum (ZAHS).then the superoxide ion (O2-,the specific granules,and the intracellular bactericidal activity of the cultured PMNs were signigicantly decreased and reached the lowest point in the 6th hour after the adding of ZAHS.0.05 ml of ZAHS showed the maximal effects on the PMNs.(2)Mice were injected intravenously with 0.5 ml of ZAHS and were killed 6 hours later.All the 3 parameters mentioned above dropped markedly in the PMNs isolated from the blood and the lungs of the mice.Pathologically,the lungs of the rats showed acute interstitial inflammation,focal edema,hemorrhage and atelectasis.and subcellular damages on the pulmonary blood-barrier.(3)ZAHS,after neutralization in vitro with antiserum against human C3 and C5,lost most of its harmful effects mentioned above.The findings suggest that the harmful effects of ZAHS originate from the fragments of C3 and Cs and they are most effective when its dosage is suitable and there must be a sufficient length of time to react before the phagocytosis of PMNs.The possible mechanism of the origination of the harmful effects of ZAHS is as follows:Before PMNs undertake phagocytosis,they releas the bactericidal and inflamnagenic (O2-and specific granules extra-cellularly.So the intracellular bactericidal activity of PMNs is decreased.The accumulation of PMNs in the pulmonary tissues can damage the PMNs themselves and the adjacent pulmonary blood-barrier,which will leads to the occurrence of infection and multiple organ failure.