Preparation of Affinity Purified Antibodies against ε-Glutaryl-Lysine Residues in Proteins for Investigation of Glutarylated Proteins in Animal Tissues

The glutarylation of lysine residues in proteins attracts attention as a possible mechanism of metabolic regulation, perturbed in pathologies. The visualization of protein glutarylation by antibodies specific to ε-glutaryl-lysine residues may be particularly useful to reveal pathogenic mutations in the relevant enzymes. We purified such antibodies from the rabbit antiserum, obtained after sequential immunization with two artificially glutarylated proteins, using affinity chromatography on ε-glutaryl-lysine-containing sorbents. Employing these anti(ε-glutaryl-lysine)-antibodies for the immunoblotting analysis of rat tissues and mitochondria has demonstrated the sample-specific patterns of protein glutarylation. The study of the protein glutarylation in rat tissue homogenates revealed a time-dependent fragmentation of glutarylated proteins in these preparations. The process may complicate the investigation of potential changes in the acylation level of specific protein bands when studying time-dependent effects of the acylation regulators. In the rat brain, the protein glutarylation, succinylation and acetylation patterns obtained upon the immunoblotting of the same sample with the corresponding antibodies are shown to differ. Specific combinations of molecular masses of major protein bands in the different acylation patterns confirm the selectivity of the anti(ε-glutaryl-lysine)-antibodies obtained in this work. Hence, our affinity-purified anti(ε-glutaryllysine)-antibodies provide an effective tool to characterize protein glutarylation, revealing its specific pattern, compared to acetylation and succinylation, in complex protein mixtures.

Effect of GP19 Peptide Hyperimmune Antiserum on Activated Macrophage during Ehrlichia canis Infection in Canine Macrophage-like Cells

In terms of its veterinary importance, vaccine development against Ehrlichia canis is needed. However, the effect of developing vaccines on humoral immune response against E. canis infection is still unknown. Novel GP194-43 was synthesized according to E. canis GP19 epitope prediction. To restrict any loss and/or illness in the host animal, rabbits were used in this study to produce GP194-43 hyperimmune sera. The effect of GP194-43 hyperimmune sera on neutralization was examined in vitro by determining the inhibition of E. canis infection of the macrophage-like cell line (DH82) in the presence of the sera. Four groups of DH82 cells received differing treatments. These included E. canis experimentally infected DH82 cells, E. canis-infected DH82 cells with control rabbit serum (untreated group), E. canis-infected DH82 cells with GP194-43 rabbit antiserum (treated group) and uninfected cells (negative control group), respectively. The treated group developed a decrease (p < 0.01) in the percentage of E. canis infected cells after 3 days post-infection at 48.57 ± 1.28. In addition, real-time PCR analyses of cytokine mRNA expression involved with the macrophage, humoral, and cellular immune responses were conducted. The findings revealed an upregulated expression of IFNG in the treated group during the infection. This study demonstrated neutralization in the GP194-43 peptide hyperimmune sera of immunized rabbits. Notably, IFN-γ production could be effectively promoted in canine macrophages in relation to the activation of macrophages and adaptive immune responses. The results of this study indicate the potential for the use of this immunogen in further investigations involving immunized and infected dogs as E. canis host species.

Structural and immunological characterization of an epitope within the PAN motif of ectodomain I in Babesia bovis apical membrane antigen 1 for vaccine development

Background Bovine babesiosis caused by Babesia bovis (B. bovis) has had a significant effect on the mobility and mortality rates of the cattle industry worldwide. Live-attenuated vaccines are currently being used in many endemic countries, but their wide use has been limited for a number of reasons. Although recombinant vaccines have been proposed as an alternative to live vaccines, such vaccines are not commercially available to date. Apical membrane antigen-1 (AMA-1) is one of the leading candidates in the development of a vaccine against diseases caused by apicomplexan parasite species. In Plasmodium falciparum (P. falciparum) AMA-1 (PfAMA-1), several antibodies against epitopes in the plasminogen, apple, and nematode (PAN) motif of PfAMA-1 domain I significantly inhibited parasite growth. Therefore, the purpose of this study was to predict an epitope from the PAN motif of domain I in the B. bovis AMA-1 (BbAMA-1) using a combination of linear and conformational B-cell epitope prediction software. The selected epitope was then bioinformatically analyzed, synthesized as a peptide (sBbAMA-1), and then used to immunize a rabbit. Subsequently, in vitro growth- and the invasion-inhibitory effects of the rabbit antiserum were immunologically characterized. Results Our results demonstrated that the predicted BbAMA-1 epitope was located on the surface-exposed α-helix of the PAN motif in domain I at the apex area between residues 181 and 230 with six polymorphic sites. Subsequently, sBbAMA-1 elicited antibodies capable of recognizing the native BbAMA-1 in immunoassays. Furthermore, anti-serum against sBbAMA-1 was immunologically evaluated for its growth- and invasion-inhibitory effects on B. bovis merozoites in vitro. Our results demonstrated that the rabbit anti-sBbAMA-1 serum at a dilution of 1:5 significantly inhibited (p < 0.05) the growth of B. bovis merozoites by approximately 50–70% on days 3 and 4 of cultivation, along with the invasion of merozoites by approximately 60% within 4 h of incubation when compared to the control groups. Conclusion Our results indicate that the epitope predicted from the PAN motif of BbAMA-1 domain I is neutralization-sensitive and may serve as a target antigen for vaccine development against bovine babesiosis caused by B. bovis.

New immunoassay systems based on recombinant human lactoferrin

In this work, soluble and solid phase immunoreagents, including recombinant human lactoferrin (rhLF), a complex of rhLF with europium ions, rabbit antiserum to rhLF, anti-rhLF immunoglobulin purified by antigen-affinity chromatography and the conjugates of this immunoglobulin with an Eu3+ chelate or horseradish peroxidase have been obtained by a combination of biochemical and synthetic methods using rhLF as an initial compound. Biospecific interactions of the reagents in four immunochemical systems were assessed by measuring the enzyme activity or time-resolved fluorescence. The study resulted in the development of fast and precise immunoassays for biologically active rhLF in transgenic goat milk and in protein fractions obtained in the course of pure rhLF manufacture, as well as in pharmaceutical preparations and food additives.

Natural autoantibodies to the gonadotropin-releasing hormone receptor in polycystic ovarian syndrome

Context Polycystic ovarian syndrome (PCOS) is a complex disease with different subtypes and unclear etiology. Among the frequent comorbidities are autoimmune diseases, suggesting that autoantibodies (aAb) may be involved in PCOS pathogenesis. Objective As the gonadal axis often is dysregulated, we tested the hypothesis that aAb to the gonadotropin-releasing hormone receptor (GnRH-R) are of diagnostic value in PCOS. Design An in vitro assay for quantifying aAb to the GnRH-R (GnRH-R-aAb) was established by using a recombinant fusion protein of full-length human GnRH-R and firefly luciferase. A commercial rabbit antiserum to human GnRH-R was used for standardization. Serum samples of control subjects and different cohorts of European PCOS patients (n = 1051) were analyzed. Results The novel GnRH-R-aAb assay was sensitive, and signals were linear on dilution when tested with the commercial GnRH-R antiserum. Natural GnRH-R-aAb were detected in one control (0.25%) and two PCOS samples (0.31%), and 12 samples were slightly above the threshold of positivity. The identification of samples with positive GnRH-R-aAb was reproducible and the signals showed no matrix interferences. Conclusion Natural GnRH-R-aAb are present in a very small fraction of adult control and PCOS subjects of European decent. Our results do not support the hypothesis that the GnRH-R constitutes a relevant autoantigen in PCOS.

Purification of Crimean–Congo hemorrhagic fever virus nucleoprotein and its utility for serological diagnosis

Crimean–Congo hemorrhagic fever virus (CCHFV) causes a zoonotic disease, Crimean–Congo hemorrhagic fever (CCHF) endemic in Africa, Asia, the Middle East, and Southeastern Europe. However, the prevalence of CCHF is not monitored in most of the endemic countries due to limited availability of diagnostic assays and biosafety regulations required for handling infectious CCHFV. In this study, we established a protocol to purify the recombinant CCHFV nucleoprotein (NP), which is antigenically highly conserved among multiple lineages/clades of CCHFVs and investigated its utility in an enzyme-linked immunosorbent assay (ELISA) to detect CCHFV-specific antibodies. The NP gene was cloned into the pCAGGS mammalian expression plasmid and human embryonic kidney 293 T cells were transfected with the plasmid. The expressed NP molecule was purified from the cell lysate using cesium-chloride gradient centrifugation. Purified NP was used as the antigen for the ELISA to detect anti-CCHFV IgG. Using the CCHFV NP-based ELISA, we efficiently detected CCHFV-specific IgG in anti-NP rabbit antiserum and CCHFV-infected monkey serum. When compared to the commercially available Blackbox CCHFV IgG ELISA kit, our assay showed equivalent performance in detecting CCHFV-specific IgG in human sera. These results demonstrate the usefulness of our CCHFV NP-based ELISA for seroepidemiological studies.

Characterization and detection of Passiflora mottle virus and other two potyviruses causing passionfruit woodiness disease in Vietnam

Passionfruit plantation in Vietnam increased to 10,000 ha in 2019. However, the outbreaks of passionfruit woodiness disease (PWD) have become a serious threat for the production. In this study, five virus isolates DN1, DN4, NA1, GL1 and GL2 were collected from different areas of Vietnam. Their causal roles for PWD were verified by back inoculation to passionfruit. Analyses of coat protein (CP) and genomic sequences revealed that GL1 isolate is closely related to East Asia Passiflora virus (EAPV) AO strain of Japan (polyprotein nt/aa identities of 98.1% / 98.2%), while GL2 isolate is related to Telosma mosaic virus (TelMV) isolate PasFru, China (polyprotein nt/aa identities of 87.1% / 90.9%). CP comparison, host range and cytological characterization indicated that DN1, DN4 and NA1 are potyviruses, but different from EAPV and TelMV. Phylogenic analyses of their CP and genome sequences indicated that these three isolates and passionfruit severe mottle-associated virus Fujian isolate of China belong to a distinct clade, which does not satisfy the threshold (76% nt identity of polyprotein) to be regarded as any of potyviral species. Thus, a new species name of “Passiflora mottle virus” has been proposed by ICTV. A rabbit antiserum was produced against the CP of DN1 and it can discriminate Passiflora mottle virus (PaMoV) from TelMV and EAPV in western blotting and ELISA without cross reactions. Field surveys of 240 samples by ELISA and RT-PCR disclosed that PWD in Vietnam is mainly caused by PaMoV; followed by EAPV, mixed-infection of PaMoV/EAPV, and rare cases of TelMV.

Characterization of an MLP Homologue from Haemaphysalis longicornis (Acari: Ixodidae) Ticks

Members of the cysteine-rich protein (CRP) family are known to participate in muscle development in vertebrates. Muscle LIM protein (MLP) belongs to the CRP family and has an important function in the differentiation and proliferation of muscle cells. In this study, the full-length cDNA encoding MLP from Haemaphysalis longicornis (H. longicornis; HLMLP) ticks was obtained by 5′ rapid amplification of cDNA ends (RACE). To verify the transcriptional status of MLP in ticks, HLMLP gene expression was assessed during various developmental stages by real-time PCR (RT-PCR). Interestingly, HLMLP expression in the integument was significantly (P < 0.01) higher than that observed in other tested tissues of engorged adult ticks. In addition, HLMLP mRNA levels were significantly downregulated in response to thermal stress at 4 °C for 48 h. Furthermore, recombinant HLMLP was expressed in Escherichia coli, and Western blot analysis showed that rabbit antiserum against H. longicornis adults recognized HLMLP and MLPs from different ticks. Ten 3-month-old rabbits that had never been exposed to ticks were used for the immunization and challenge experiments. The rabbits were divided into two groups of five rabbits each, where rabbits in the first group were immunized with HLMLP, while those in the second group were immunized with phosphate-buffered saline (PBS) diluent as controls. The vaccination of rabbits with the recombinant HLMLP conferred partial protective immunity against ticks, resulting in 20.00% mortality and a 17.44% reduction in the engorgement weight of adult ticks. These results suggest that HLMLP is not ideal as a candidate for use in anti-tick vaccines. However, the results of this study generated novel information on the MLP gene in H. longicornis and provide a basis for further investigation of the function of this gene that could potentially lead to a better understanding of the mechanism of myofiber determination and transformation

Construction and expression of a prokaryotic expression vector for the goat sry gene

Goats are economically important animals in the world, and their sex is an important factor in their economic efficiency. Reconstruction of a goat SRY gene expression vector can lay a foundation for studying the immunogenicity and sex determination of SRY protein at the molecular level. In this study, the coding region of the goat SRY gene was used as the target gene fragment for synthesis and optimization, and the cloning vector was used as a template to amplify the target gene and finally ligated to the expression vector pET-SUMO. The recombinant plasmid was then verified by the double restriction enzyme method and transformed into Escherichia coli (DE3). After the induction of expression by Isopropyl â-D-Thiogalactoside (IPTG), the cells were lysed, and SDS-PAGE electrophoresis was performed to observe the expression of the recombinant protein. Additionally, the immunological activity of the recombinant protein was assessed. The target gene was successfully ligated into the prokaryotic expression vector pET-28a; additionally, the prokaryotic expression plasmid pET-SUMO was successfully constructed, and the SRY antigen protein (42 kDa) was expressed. The titer of the rabbit antiserum (PAI-1608012-1) was more than 1:50000, as measured by ELISA, which demonstrated that the titer and the sensitivity of the rabbit serum reached the expected levels. In this study, the prokaryotic expression vector pET-SUMO was successfully constructed. The recombinant protein has high immunopotency and immunoreactivity, which lays a foundation for the preparation of antibodies and the molecular sexing of goats in the future.

Reducing the risk of misdiagnosis of indirect ELISA by normalizing serum-specific background noise: The example of detecting anti-FGFR3 autoantibodies

Indirect enzyme-linked immunosorbent assay (ELISA) is an important diagnostic method as it enables the quantification of the presence of autoantibodies in human blood sera. However, unspecific binding of antibodies to the solid phase causes considerable serum-specific background noise (SSBN), involving the risk of false positive diagnosis. Therefore, we present a simple and concise, yet obvious proof-of-principle of a recently suggested normalization method. The method is based on subtracting SSBN by using non-coated ELISA wells as a control for each serum-of-interest. We performed ELISA to quantify anti-fibroblast growth factor receptor 3 (FGFR3) antibody levels in three positive controls (two anti-FGFR3-positive patients and a rabbit antiserum against FGFR3) and 58 negative controls (healthy blood donors). In all subjects, we found considerable unspecific reactivity which strongly varied among subjects. The conventional normalization method was not able to balance this strong SSBN, as demonstrated by 2/58 false positive healthy controls and one FGFR3-positive patient that was hidden in the noise (false negative). SSBN normalization reduced the frequency of false-positives to 0/58. Further, all three anti-FGFR3-positive sera were successfully detected and even doubled their z-score used to determine positivity. Albeit occupying more space on the ELISA plate, we strongly recommend considering this normalization method when working with blood sera. To better put the idea across to the community, we depict the SSBN issue and its solution in a graphic scheme. We conclude that SSBN normalization increases the sensitivity and specificity of indirect ELISA and thereby reduces the risk of false positive and false negative diagnosis.