Technical progress and industrial development of in vitro diagnostic / 中华检验医学杂志

In vitro diagnosis (IVD) is an important source of clinical diagnostic information, and provides an important decision basis for disease prevention, diagnosis and treatment. IVD is a necessary tool for promoting graded diagnosis and treatment, realizing precision medicine, constructing a "Healthy China" and responding to major public health emergencies. Combining the great progress made in the development of in vitro diagnostics in China and the shortcomings and weaknesses faced by it, this article analyzed the demand for IVD, policy support, technical and industrial development trends, and the ways to accelerate the industrialization development, aiming to promote the development and improvement of IVD in China.

Advances in immortalization of human B cells / 生物工程学报

With the advantages of low immunogenicity and long half-life, human monoclonal antibody has become an indispensable biological agent in vivo. Immortalization of human B cells is a potential and effective method to obtain natural human antibody library, which can provide a rich source for the preparation of human monoclonal antibodies. As there are urgent problems to be solved in each platform, the preparation of antibodies based on human B cell immortalization is still limited to the laboratory research stage. At present, there is a lack of a systematic review to clarify the advantages and disadvantages of the existing human B cell immortalization antibody preparation platform and its feasibility analysis. This paper reviews the research on the preparation of human monoclonal antibody based on human B cells immortalization, and describes an in vitro cell culture method, in which hCD40L vesicles are used instead of feeder cells, in order to provide references for the further development of human monoclonal antibody preparation technology.

Progress in interactions between rotavirus infection and innate immunity / 中华微生物学和免疫学杂志

Rotavirus (RV) is one of the leading causes of acute gastroenteritis in infants and young animals worldwide. Rotavirus infection has obvious species specificity and mainly causes diarrhea in infants and young animals. The host innate responses suppress the infection and replication of rotavirus through activating multiple signaling pathways. Meanwhile, rotavirus also antagonizes the innate immune responses in various ways. This article reviewed the mechanisms of host innate immune responses to rotavirus infection and the antagonistic mechanism of rotavirus against host innate immunity with a view to providing reference for the development of therapeutic drugs and the prevention of rotavirus infection.

Advances of targeted protein degradation technology and its applications in diseases therapy / 生物工程学报

Targeted protein degradation (TPD) technology facilitates specific and efficient degradation of disease-related proteins through hijacking the two major protein degradation systems in mammalian cells: ubiquitin-proteasome system and lysosome pathway. Compared with traditional small molecule-inhibitors, TPD-based drugs exhibit the characteristics of a broader target spectrum. Compared with techniques interfere with protein expression on the gene and mRNA level, TPD-based drugs are target-specific, efficaciously rapid, and not constrained by post-translational modification of proteins. In the past 20 years, various TPD-based technologies have been developed. Most excitingly, two TPD-based therapeutic drugs have been approved by FDA for phase Ⅰ clinical trials in 2019. Despite of the early stage characteristics and various obstructions of the TPD technology, it could serve as a powerful tool for the development of novel drugs. This review summarizes the advances of different degradation systems based on TPD technologies and their applications in disease therapy. Moreover, the advantages and challenges of various technologies were discussed systematically, with the aim to provide theoretical guidance for further application of TPD technologies in scientific research and drug development.

Application of paper-based microfluidics in point-of-care testing / 生物工程学报

Point-of-care testing (POCT) is a test method performed on the sampling site or patient bedside. Accurate results can be achieved rapidly by the application of portable analytical instruments and compatible reagents. It has been widely used in the field of in vitro diagnosis (IVD). Paper-based microfluidics technology has great potential in developing POCT due to its advantages in low cost, simple operation, rapid detection, portable equipment, and unrestricted application conditions. In recent years, the development of paper-based microfluidic technology and its integration with various new technologies and methods have promoted the substantial development of POCT technology and methods. The classification and characteristic of the paper are summarized in this review. Paper-based microfluidic sample pretreatment methods, the flow control in the process of reaction and the signal detecting and analyzing methods for the testing results are introduced. The research progress of various kinds of microfluidic paper-based analytical devices (μPADs) toward POCT in recent years is reviewed. Finally, remaining problems and the future prospects in POCT application of paper-based microfluidics are discussed.

Research progress in signal amplification for immunoassays / 中华微生物学和免疫学杂志

With the requirements of early diagnosis, biomarker development and functional definition, the challenge of sensitivity of immunoassay has become increasingly prominent. How to improve it to break the bottleneck has become a major challenge in the field of bioassays. Amplifying the immunosignal is the most direct method to improve detection sensitivity. Biotin-avidin system (BAS), tyramide signal amplification (TSA) and immuno-polymerase chain reaction (Im-PCR) are the most classic signal amplification techniques which significantly improved the sensitivity of immunoassays. In recent years, studies have confirmed that the sensitivity of immunoassays can be further increased by approximately three orders of magnitude with the invention of techniques including catalyzed reporter deposition-based signal amplification, nanotechnologies-based signal amplification and hybridization chain reaction-based signal amplification. Herein, we will summarize the techniques that have been developed in recent years for amplifying the signals of immunodetection and comparatively analyze their advantages and disadvantages in order to provide reference for the developed techniques transformed to clinical application and further research on ultrasensitive immunoassays.

Polymerization and evaluation of the protective efficacy of rotavirus VP4* proteins / 生物工程学报

In previous studies, we found that truncated rotavirus VP4* (aa 26-476) could be expressed in soluble form in Escherichia coli and confer high protection against rotavirus in the mouse mode. In this study, we further improved the immunogenicity of VP4* by polymerization. The purified VP4* was polymerized through incubation at 37 ℃ for 24 h, and then the homogeneity of the particles was analyzed by HPLC, TEM and AUC, while the thermal stability and antigenicity was analyzed by DSC and ELISA, respectively. Finally, the immunogenicity and protective efficacy of the polymers analyzed by a mouse maternal antibody model. The results showed that VP4* aggregated into homogeneous polymers, with high thermostability and neutralizing antibody binding activity. In addition, VP4* polymers (endotoxin <20 EU/dose) stimulated higher neutralizing antibodies and confer higher protection against rotavirus-induced diarrhoea compared with the VP4* trimers when immunized with aluminium adjuvant. In summary, the study in VP4* polymers provides a new strategy for the development of recombinant rotavirus vaccines.

PSO+: nonlinear fitting fluorescence data based on particle swarm optimizing combine with other iteration algorithm / 生物医学工程学杂志

The convective polymerase chain reaction (CCPCR) uses the principle of thermal convection to allow the reagent to flow in the test tube and achieve the purpose of amplification by the temperature difference between the upper and lower portions of the test tube. In order to detect the amplification effect in real time, we added a fluorophore to the reagent system to reflect the amplification in real time through the intensity of fluorescence. The experimental results show that the fluorescence curve conforms to the S-type trend of the amplification curve, but there is a certain jitter condition due to the instability of the thermal convection, which is not conducive to the calculation of the cycle threshold (CT value). In order to solve this problem, this paper uses the dynamic method, using the double S-type function model to fit the curve, so that the fluorescence curve is smooth and the initial concentration of the nucleic acid can be deduced better to achieve the quantitative purpose based on the curve. At the same time, the PSO+ algorithm is used to solve the double s-type function parameters, that is, particle swarm optimization (PSO) algorithm combined with Levenberg-Marquardt, Newton-CG and other algorithms for curve fitting. The proposed method effectively overcoms PSO randomness and the shortcoming of traditional algorithms such as Levenberg-Marquardt and Newton-CG which are easy to fall into the local optimal solution. The of the data fitting result can reach 0.999 8. This study is of guiding significance for the future quantitative detection of real-time fluorescent heat convection amplification.

Advances in reverse genetics systems for rotavirus / 中华微生物学和免疫学杂志

Reverse genetics approaches can directly manipulate the genome of virus at the gene level, making it possible to quickly, directly and thoroughly study the mechanisms of virus replication and pathogenesis. At present, many viruses of the family Reoviridae, such as mammalian orthoreovirus ( MRV) and bluetongue virus ( BTV) , have made great progress in basic viral research using the powerful tool of re-verse genetics. However, for members of the genus Rotavirus in the family Reoviridae, progress in the con-struction of reverse genetic systems has been slow. The remarkable reverse genetics system based on helper-viruses was established in 2006, and it was not until 2017 that the entirely plasmid-based reverse genetics system was successfully established. This paper briefly reviewed the development of reverse genetics systems for rotavirus and prospected the direction for future research in order to provide technical support for acceler-ating the basic research on mechanisms of rotavirus infection.

Development and application of CK-MB specific monoclonal antibodies / 生物工程学报

The aim of this study is to develop creatine kinase isoenzyme MB (CK-MB) specific monoclonal antibodies (mAb), and characterize the monoclonal antibody and further development of quantitative detection assay for CK-MB. The BALB/c mice were immunized with purchased CK-MB antigen, then monoclonal antibodies were prepared according to conventional hybridoma technique and screened by indirect and capture ELISA method. To identify the epitopes and evaluate the classification, purchased creatine kinase isoenzyme MB (CK-MM/BB/MB) antigen was used to identify the epitopes, with immunoblotting and synthetic CK-MM and CK-BB in different linear epitope. A double antibody sandwich ELISA was applied to screen the mAb pairs for CK-MB detection, and the quantitative detection assay for CK-MB was developed. We used 74 cases of clinical specimens for comparison of our assay with Roche's CK-MB assay. We successfully developed 22 strains of hybridoms against CK-MB, these mAbs can be divided into linear, partial conformational CK-MB, CK-MM or CK-BB cross monoclonal antibody and CK-MB specific reaction with partial conformational monoclonal antibody, and CK-MB quantitative detection assay was developed by using partial conformational monoclonal antibody. The correlation coefficient factor r of our reagent and Roche's was 0.930 9. This study established a screening method for CK-MB partial conformational specific monoclonal antibody, and these monoclonal antibodies were analyzed and an established quantitative detection assay was developed. The new assay had a high concordance with Roche's.

Research progress in rotavirus VP4 subunit vaccine / 生物工程学报

Rotaviruses are leading causes of worldwide acute diarrhea in children younger than 5 years old, with severe consequence of social and economic burden. Vaccination is the most effective way to control rotavirus infection, however, the licensed rotavirus vaccines are ineffective in some low-income countries of Africa and Asia, where the mortality caused by rotavirus is higher than other areas. In addition, there are also safety concerns such as increased risk of intussusception. Therefore, it is urgent to improve the efficiency and safety of rotavirus vaccine to reduce the morbidity and mortality caused by rotavirus. Till now, many efforts are made to improve the effectiveness of rotavirus vaccines, and the inactive vaccine becomes the main trend in the research of rotavirus vaccine. The developments in recombinant rotavirus vaccines, especially in VP4 subunit vaccines are summarized in this review, and it could be helpful to develop effective recombinant rotavirus vaccines in further studies.

Impact of maternal antibodies on protective efficacy of rotavirus vaccine:a review / 中华微生物学和免疫学杂志

Rotavirus is one of the most common causes of gastroenteritis among children under five years of age worldwide. Most children are infected with rotavirus in their early childhood. Currently, no ef-fective treatment is available for diarrhea caused by rotavirus infection, and vaccination is the most effective way to prevent rotavirus infection. With the introduction of rotavirus vaccines, the morbidity and mortality of severe diarrhea in children caused by rotavirus infection are declined significantly, but these rotavirus vac-cines have significantly lower efficacy in developing countries in Africa and some countries in Asia where the mortality of rotavirus-related diseases is high than in developed countries. High titers of maternal antibodies may be one of the reasons why the efficacy of rotavirus vaccines is low in these countries. Extensive studies have been conducted on the relationship between neonatal response to rotavirus vaccine immunization and ro-tavirus-specific antibodies in the mother′s milk and serum in recent years. However, results of some studies are in conflict. In this review, we summarize the relationship between maternal antibodies and the immune responses after vaccination with rotavirus vaccines in order to provide basis for improving the efficacy of rota-virus vaccines in low-income countries.

Impact of maternal antibodies on protective efficacy of rotavirus vaccine:a review / 中华微生物学和免疫学杂志

Rotavirus is one of the most common causes of gastroenteritis among children under five years of age worldwide. Most children are infected with rotavirus in their early childhood. Currently, no ef-fective treatment is available for diarrhea caused by rotavirus infection, and vaccination is the most effective way to prevent rotavirus infection. With the introduction of rotavirus vaccines, the morbidity and mortality of severe diarrhea in children caused by rotavirus infection are declined significantly, but these rotavirus vac-cines have significantly lower efficacy in developing countries in Africa and some countries in Asia where the mortality of rotavirus-related diseases is high than in developed countries. High titers of maternal antibodies may be one of the reasons why the efficacy of rotavirus vaccines is low in these countries. Extensive studies have been conducted on the relationship between neonatal response to rotavirus vaccine immunization and ro-tavirus-specific antibodies in the mother′s milk and serum in recent years. However, results of some studies are in conflict. In this review, we summarize the relationship between maternal antibodies and the immune responses after vaccination with rotavirus vaccines in order to provide basis for improving the efficacy of rota-virus vaccines in low-income countries.

Development, identification and application of 33 monoclonal antibodies against cardiac troponin T / 生物工程学报

The aim of this study is to prepare and characterize cardiac troponin T (cTnT) monoclonal antibodies (mAb), and further develop a chemiluminescence quantitative detection assay for cTnT. BALB/c mice were immunized with recombinant cTnT antigen, and specific mAbs were prepared using conventional hybridoma technique and screened by indirect ELISA method. To identify the epitopes, several cTnT peptide fragments were synthesized or expressed by genetic engineering. A double antibody sandwich ELISA method was used to screen the mAb pairs for cTnT detection, and the automatic chemiluminescence detection assay for cTnT was developed. In total 220 clinical specimens were used for system comparison between our assay and Roche cTnT assay; further performance characteristics was evaluated by testing 238 clinical samples and 784 physical examination samples. We successfully screened 33 strains of hybridoms against cTnT, and the mAbs' epitopes were identified. Mab E16H8 and C8G11 with a detection limit of 10 pg/mL cTnT antigen were selected to develop the full automatic chemiluminescence quantitative assay. The correlation coefficient of our reagent with Roche's was 0.959 9, with a coincidence rate of 95%. The assay presented a sensitivity of 97.5%, and a specificity of 99.15% in detection of clinical samples. The cTnT concentration was less than 0.080 6 ng/mL in 99% of general population, which agrees with the definition of WHO on patients with acute myocardial infarction (AMI). In summary, we developed monoclonal antibodies against predominant epitopes for diagnostics of cTnT, and an automatic tubular chemiluminescence quantitative detection assay was further developed, which presents a high coincidence rate with Roche's.

Performance characteristic comparison of two commerical kits for anti-HCV confirmatory test / 中华检验医学杂志

Objective To detect anti-HCV in serum of hepatic disease patients by performing the confirmatory test, and further to confirm HCV infection. Methods Two recombinant immunoblot assays (CWT and CHIRON RIBA HCV 3.0 Strip Immunoblot Assay) were used respectively to detect anti-HCV in 477 human serum samples, which comprised 350 HCV-infected patients' specimens, 7 none-A none-E hepatitis specimens, 30 HBV-infected patients' specimens, 30 hepatitis E virus infected patients'specimens, and 60 specimens drawn from blood donors. The latter three groups served as controls. Results A total of 120 control non-HCV-infected patients' specimens were negative when tested by both assays. Among 350 HCV-infected patients, 341 were positive and 9 were indeterminated by CWT assay; 343 were positive and 7 were indeterminated by CHIRON RIBA HCV 3. 0 SIA. Seven none-A none-E hepatitis specimens tested by both assays turned out to be 2 positive, 4 negative and 1 indeterminate. The consistency rate of these two assays was 99. 16% (Kappa=0.98). Conclusion CWT assay is highly coherent with CHIRON RIBA HCV 3.0 SIA assay in the methodology of anti-HCV antibody detection, which can be applied in the determination of HCV infection among none-A none-E hepatitis patients.

The research of genotype 4 hepatitis E virus(HEV) capsid recombinant protein and the construction of cellular model for the investigation of viral infection early period / 中华微生物学和免疫学杂志

Objective To express the recombinant caspid of genotype 4 hepatitis E virus(HEV) ORF2. Methods HEV recombinant capsid protein D66 was expressed in E. coli, using the ORF2 fragment (aa368-606, obtained from swine bile) of genotype 4 HEV. Results The recombinant capsid proteins D66 self-assemble to be particle with a radius of 13 nm through dimeric form in neutral solution. Coated particles reacted well with sera obtained from patients during acute or recovered phase of HEV infection. Immunofluorescence and immnoblot assay suggested that D66 bound and penetrated HepG2 cell lines, and the process of attachment was blocked by sera collected from patients during acute or recovered phase of HEV infection.Conclusion Recombinant D66 particles simulate the structure at the surface of genotype 4 HEV well and specifically adhere and penetrate the host cells, which lays the foundation for the investigation of the molecular mechanism of genotype 4 HEV infection.

Chemiluminescent immunoassay for high-sensitivity C-reactive protein / 生物工程学报

We developed a high-sensitivity C-reactive protein quantifiable chemiluminescent immunoassay (hs-CRP CLIA). The high-purity native CRP was purified from hepatic cirrhosis patient ascetic fluid by affinity and ion exchange chromatography and used as an immunogen to develop the monoclonal antibodies (mAbs) against CRP. Twenty-two mAbs were identified reactive with CRP in ELISA and 13 of them were reactive in the phosphorycholine ligand capture ELISA. The mAbs 10C5 and 10C11 were selected to develop the hs-CRP CLIA. The linearity and performance of the hs-CRP CLIA was characterized. It was showed not reactive when testing against other serum materials (IgG, hemoglobin and triglyceride). The reliable correlation (R2 > 0.993) was obtained between testing value (RLU/S) and the concentration of human serum CRP calibrator. The linearity fell in the range of 0.04-20.38 mg/L. The assay has good accuracy and reproducibility, the mean recovery was 99% and the precision of the intra- and inter assay was CVs (4.2%-5.8%) and (9.0%-11.5%), respectively. In testing of 90 human sera, this assay performed well and correlated comparably with a commercial hs-CRP ELISA kit. Thus, hs-CRP CLIA is an accurate, reliable, quantifiable assay for detection of high-sensitive C-reactive protein in serum, it may be useful to improve the risk assessment of cardiovascular disease and the prognosis of inflammatory bowel disease.