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Acute rheumatic fever (ARF) and chronic rheumatic heart disease (RHD) are autoimmune sequelae of a Group A streptococcal infection with significant global mortality and poorly understood pathogenesis. Immunoglobulin and complement deposition were observed in ARF/RHD valve tissue over 50 years ago, yet contemporary investigations have been lacking. This study applied systems immunology to investigate the relationships between the complement system and immunoglobulin in ARF. Patients were stratified by C‐reactive protein (CRP) concentration into high (≥10 μg mL−1) and low (<10 μg mL−1) groups to distinguish those with clinically significant inflammatory processes from those with abating inflammation. The circulating concentrations of 17 complement factors and six immunoglobulin isotypes and subclasses were measured in ARF patients and highly matched healthy controls using multiplex bead‐based immunoassays. An integrative statistical approach combining feature selection and principal component analysis revealed a linked IgG3–C4 response in ARF patients with high CRP that was absent in controls. Strikingly, both IgG3 and C4 were elevated above clinical reference ranges, suggesting these features are a marker of ARF‐associated inflammation. Humoral immunity in response to M protein, an antigen implicated in ARF pathogenesis, was completely polarized to IgG3 in the patient group. Furthermore, the anti‐M‐protein IgG3 response was correlated with circulating IgG3 concentration, highlighting a potential role for this potent immunoglobulin subclass in disease. In conclusion, a linked IgG3–C4 response appears important in the initial, inflammatory stage of ARF and may have immediate utility as a clinical biomarker given the lack of specific diagnostic tests currently available. [ABSTRACT FROM AUTHOR]

Any single-enzyme directed evolution strategy has two fundamental requirements: the need to efficiently introduce variation into a gene of interest and the need to create an effective library from those variants. Generation of a maximally diverse gene library is particularly important when employing nontargeted mutagenesis strategies such as error-prone PCR (epPCR), which seek to explore very large areas of sequence space. Here we present comprehensive protocols and tips for using epPCR to generate gene variants that exhibit a relatively balanced spectrum of mutations and for capturing as much diversity as possible through effective cloning of those variants. The detailed library preparation methods that we describe are generally applicable to any directed evolution strategy that uses restriction enzymes to clone gene variants into an expression plasmid.

In contrast to site-directed mutagenesis and rational design, directed evolution harnesses Darwinian principles to identify proteins with new or improved properties. The critical first steps in a directed evolution experiment are as follows: (a) to introduce random diversity into the gene of interest and (b) to capture that diversity by cloning the resulting population of molecules into a suitable expression vector, en bloc. Error-prone PCR (epPCR) is a common method for introducing random mutations into a gene. In this chapter, we describe detailed protocols for epPCR and for the construction of large, maximally diverse libraries of cloned variants. We also describe the utility of an online program, PEDEL-AA, for analyzing the compositions of epPCR libraries. The methods described here were used to construct several libraries in our laboratory. A side-by-side comparison of the results is used to show that, ultimately, epPCR is a highly stochastic process.