Prokaryotic expression of mouse Stra8 and preparation and identification of rabbit anti-mouse Stra8 polyclonal antibody
Objective To prokaryotically express mouse stimulated by retinoic acid gene 8 (Stra8) and prepare rabbit anti-mouse Stra8 polyclonal antibody. Methods The recombinant plasmid pET28a-Stra8 was constructed by cloning technology and identified by double enzyme digestion and sequencing, and then transformed into E. coli BL21 (DE3).
The expression of Stra8 recombinant protein was induced by IPTG. The prokaryotic protein was purified by His-TAG affinity chromatograph and then used to immune New Zealand white rabbits to obtain Stra8 polyclonal antibody. ELISA, Western blot and immunofluorescence assays were used to determine the antibody titer, validity and specificity, respectively. Results The recombinant plasmid pET28a-Stra8 was constructed successfully as double enzyme digestion and sequencing showed, and the prokaryotic protein was expressed and purified effectively.
The titer of the polyclonal antibody reached 1:106. Western blotting showed that the polyclonal antibody could specifically recognize native Stra8 protein in the testis. Immunofluorescence assay revealed that the polyclonal antibody had good reactivity, and could recognize Stra8 protein in mouse testis. Conclusion Stra8 prokaryotic protein can be effectively induced in E. coli and specific rabbit anti-Stra8 polyclonal antibody has been prepared.
Mining the Antibody Repertoire for Solutions to SARS-CoV-2
In this issue of Cell Host & Microbe, Nielsen and colleagues sequence antibody repertoires of patients with severe COVID-19 to reveal potentially convergent features on the background of a larger, polyclonal response. Their findings suggest that, as databases improve, it may be possible to monitor virus-specific B cells after infection or vaccination using antibody sequencing.
IgG3 collaborates with IgG1 and IgA to recruit effector function in RV144 vaccinees
While the RV144 HIV vaccine trial lead to moderately reduced risk of HIV acquisition, emerging data from the repeat failure of the HVTN702 trial point to the critical need to re-examine the relationships between previously identified correlates of reduced risk of protection in the RV144 study. Specifically, the induction of V2-binding, non-IgA, IgG3 antibody responses with non-neutralizing functions were linked to reduced risk of infection in RV144 vaccinees. While each of these features was individually linked to reduced risk of infection, the relationships and interactions between these humoral immune signatures remain unclear.
Thus, here we comprehensively profiled the humoral immune response in 300 RV144 vaccinees to specifically decipher the relationships between humoral biomarkers of protection and susceptibility. Here, we found that vaccine-specific IgG1, IgG3, and IgA were highly correlated. However, ratios of IgG1:IgG3:IgA provided new insights into subclass/isotype polyclonal functional regulation. For instance, in the absence of high IgG1 levels, IgG3 antibodies exhibited limited functional activity, pointing to IgG3 as a critical contributor, but not sole driver, of more effective antiviral humoral immunity. Moreover, in contrast to previous findings, higher IgA levels were linked to enhanced antibody effector function, including neutrophil phagocytosis (ADNP), complement deposition (ADCD) and NK degranulation (CD107a). Several IgA-associated functions were increased in infected vaccinees in a case:control dataset, suggesting that rather than blocking, IgA may have driven deleterious functions, thereby compromising immunity. These data highlight the interplay between IgG1, IgG3 and IgA, pointing to the critical need to deeply profile the relationships between subclass/isotype selection.
From infections to autoimmunity: Diagnostic challenges in common variable immunodeficiency
Common variable immunodeficiency (CVID) is the most common clinically significant primary antibody deficiency diagnosed in adults. The early symptoms are not specific. They include common infections, mainly of the respiratory tract, caused by typical microorganisms, so cases can be missed in primary care. In the majority of patients increased susceptibility to infections coexists with signs or symptoms of autoimmunity, inflammation or polyclonal lymphoproliferation, which can divert diagnosis from immune deficiency.
The overall incidence of malignancy is increased in CVID and certain cancers are significantly more common. Lymphomas and gastric carcinoma are the most frequently reported malignancies in CVID, so a high index of suspicion is recommended. Diagnostic delay in CVID is seen worldwide. The main goal of this paper is to increase the awareness about CVID among health care professionals. We aim to present features which can be helpful in CVID diagnosis in order to shorten the “latency” of proper management of CVID patients.
We review clinical symptoms, complications and laboratory abnormalities of CVID. Immunoglobulin replacement therapy is regarded as the cornerstone of pharmacological intervention. New modes of Ig application, mainly subcutaneously and via the hyaluronidase-facilitated subcutaneous route, help to adjust therapy to patients’ needs and preferences. Still there remain unmet needs. It remains to be seen whether CVID complications can be avoided by earlier diagnosis, treatment and thorough monitoring in the context of increased risk of malignancy.
Development of patient tailored protocols depending on the clinical phenotype and risk factors might be more appropriate. The most important consideration is to diagnose suspected cases and stratify patients in a precise and timely way. Work is needed to define features predictive of unfavorable prognosis.
Antigenic Variation of the Dengue Virus 2 Genotypes Impacts the Neutralization Activity of Human Antibodies in Vaccinees
Dengue virus (DENV) infects an estimated 390 million people each year worldwide. As tetravalent DENV vaccines have variable efficacy against DENV serotype 2 (DENV2), we evaluated the role of genetic diversity within the pre-membrane (prM) and envelope (E) proteins of DENV2 on vaccine performance. We generated a recombinant DENV2 genotype variant panel with contemporary prM and E isolates that are representative of global genetic diversity.
The DENV2 genotype variants differ in growth kinetics, morphology, and virion stability. Importantly, the DENV2 genotypic variants are differentially neutralized by monoclonal antibodies, polyclonal serum neutralizing antibodies from DENV2-infected human subjects, and vaccine-elicited antibody responses from the TV003 NIH DENV2 monovalent and DENV tetravalent vaccines. We conclude that DENV2 prM and E genetic diversity significantly modulates antibody neutralization activity. These findings have important implications for dengue vaccines, which are being developed under the assumption that intraserotype variation has minimal impact on neutralizing antibodies.
Platinum nanoflowers with peroxidase-like property in a dual immunoassay for dehydroepiandrosterone
Platinum nanoflowers (PtNFs) were utilized in a competitive enzyme-linked immunosorbent assay (ELISA) and in a lateral flow immunoassay (LFIA) for superior peroxidase-like activity and intense brown color, respectively. PtNFs were linked to the polyclonal antibody (pAb) to form the pAb-PtNFs probes for the dual immunoassay. Based on optimized pAb-PtNF probes, both enzyme-linked immunosorbent assay (PtNFs-ELISA) and lateral flow immunoassay (PtNFs-LFIA) perform very well. The absorbance at 450 nm decreases linearly in the DHEA concentration range 2.1 to 118.1 ng mL-1, and the limit of detection is 1.3 ng mL-1 and the IC50 value is 15.7 ng mL-1 of PtNFs-ELISA.
The visual cut-off value of PtNFs-LFIA is 10.0 ng mL-1. The average recoveries from spiked samples range from 95.0 to 108.9% with a coefficient of variation below 12.2%. Excellent recoveries and correlation between the two methods were observed. Furthermore, the designed immunosensors exhibited good selectivity, confirming a broad development prospect in DHEA monitoring. Graphical Abstract.