Recombinant multiepitope proteins expressed in Escherichia coli cells and their potential for immunodiagnosis.

Recombinant multiepitope proteins (RMPs) are a promising alternative for application in diagnostic tests and, given their wide application in the most diverse diseases, this review article aims to survey the use of these antigens for diagnosis, as well as discuss the main points surrounding these antigens. RMPs usually consisting of linear, immunodominant, and phylogenetically conserved epitopes, has been applied in the experimental diagnosis of various human and animal diseases, such as leishmaniasis, brucellosis, cysticercosis, Chagas disease, hepatitis, leptospirosis, leprosy, filariasis, schistosomiasis, dengue, and COVID-19. The synthetic genes for these epitopes are joined to code a single RMP, either with spacers or fused, with different biochemical properties. The epitopes' high density within the RMPs contributes to a high degree of sensitivity and specificity. The RMPs can also sidestep the need for multiple peptide synthesis or multiple recombinant proteins, reducing costs and enhancing the standardization conditions for immunoassays. Methods such as bioinformatics and circular dichroism have been widely applied in the development of new RMPs, helping to guide their construction and better understand their structure. Several RMPs have been expressed, mainly using the Escherichia coli expression system, highlighting the importance of these cells in the biotechnological field. In fact, technological advances in this area, offering a wide range of different strains to be used, make these cells the most widely used expression platform. RMPs have been experimentally used to diagnose a broad range of illnesses in the laboratory, suggesting they could also be useful for accurate diagnoses commercially. On this point, the RMP method offers a tempting substitute for the production of promising antigens used to assemble commercial diagnostic kits.

Development and evaluation of time-resolved rapid immunofluorescence test for detection of TSOL18 specific antibody in porcine cysticercosis infections.

BACKGROUND: Porcine cysticercosis, a serious zoonotic parasitic disease, is caused by the larvae of Taenia solium and has been acknowledged by the World Organization for Animal Health. The current detection methods of Cysticercus cellulosae cannot meet the needs of large-scale and rapid detection in the field. We hypothesized that the immunofluorescence chromatography test strip (ICS) for detecting Cysticercus cellulosae, according to optimization of a series of reaction systems was conducted, and sensitivity, specificity, and stability testing, and was finally compared with ELISA. This method utilizes Eu3+-labeled time-resolved fluorescent microspheres (TRFM) coupled with TSOL18 antigen to detect TSOL18 antibodies in infected pig sera. RESULTS: ICS and autopsy have highly consistent diagnostic results (n = 133), as determined by Cohen's κ analysis (κ = 0.925). And the results showed that the proposed ICS are high sensitivity (0.9459) with specificity (0.9792). The ICS was unable to detect positive samples of other parasites. It can be stored for at least six months at 4℃. CONCLUSIONS: In summary, we established a TRFM-ICS method with higher sensitivity and specificity than indirect ELISA. Results obtained from serum samples can be read within 10 min, indicating a rapid, user-friendly test suitable for large-scale field detection.

Evaluating the diagnostic accuracy of TpN17 and TmpA recombinant proteins in syphilis detection: a phase II study.

Syphilis is a sexually transmitted infection (STI) caused by the spiral bacterium Treponema pallidum. Diagnosis is based on epidemiology, clinical and serology, but serodiagnosis is challenging because distinct clinical forms of the infection may influence serological performance. Several recombinant Treponema pallidum-proteins have already been tested for syphilis diagnosis and they are critical to achieve high accuracy in serological testing. A total of 647 samples were included in the study: 180 T. pallidum-positive samples, 191 T. pallidum-negative samples and 276 sera from individuals infected with unrelated diseases. The diagnostic potential was validated by analysis of ROC curves. For the indirect ELISA, TpN17 (100%) and TmpA (99%) showed excellent AUC values. Sensitivity values were 97.2% for TpN17 and 90.6% for TmpA, while specificity was 100% for both molecules. According to the clinical phase, TmpA ranged from 84% to 97%, with the highest value for secondary syphilis. TpN17 was 100% sensitive for the primary and secondary stages and 93.2% for recent latent syphilis. All clinical phases achieved 100% specificity. Accuracy values showed that TmpA (> 95%) and TpN17 (> 98%) presented high diagnostic accuracy for all clinical stages of syphilis. Cross-reactivity was only observed in one sample positive for Chagas disease (1.5%), when TpN17 was evaluated. On the other hand, TmpA showed reactivity for two samples positive for Chagas disease (3.1%), one sample positive for HBV (1.25%), two samples positive for HIV (9.5%) and one sample positive for HTLV (1.6%). The TmpA antigen's performance was evaluated in multiple studies for syphilis diagnosis, corroborating our findings. However, TpN17 sensitivity values have ranged in other studies. According to clinical stages of the infection, our findings obtained close performance values.

Immunodiagnosis of Fascioliasis in Ruminants by ELISA Method: A Mini-Review.

Fascioliasis is an important zoonotic disease prevalent in domestic animals and it leads to socioeconomic impact in rural farming communities of the developing world. The gold standard diagnosis of ruminant fascioliasis involves coprological detection of Fasciola spp. eggs or recovery of flukes in infected livers. Coprological analysis is unreliable in the patent period of chronic infection, and even then, its sensitivity is relatively low. Robust diagnostic tools that can promptly and accurately detect an active infection are crucial to avoid complications and further losses in ruminant livestock productivity, as well as to preserve the livelihood of communities at risk. Immunodiagnosis determined by antibody and antigen detection in the sera and faeces of infected ruminants provides a valuable alternative to the parasitological diagnostic approach. This review discusses current developments in immunological techniques by enzyme-linked immunosorbent assay (ELISA) in the detection of ruminant fascioliasis and summarises the performance of various ELISAs in studies conducted to date. Indirect ELISAs demonstrated effective immunodiagnostic performance with high sensitivities and specificities. Cathepsin L ELISA is the most favourable antigen in serodiagnosis, among other recombinant and native proteins evaluated. Sandwich ELISA provides excellent sensitivity and specificity, which correlates well with the fluke burden. Utilising monoclonal antibodies in sandwich ELISA reduces the detection time and performance variations that commonly occur in polyclonal antibody ELISA.

Erratum: Editorial: Immuno-diagnosis of active tuberculosis; are we making progress?

[This corrects the article DOI: 10.3389/fimmu.2023.1092651.].

Immunodiagnosis - the promise of personalized immunotherapy.

Immunotherapy showed remarkable efficacy in several cancer types. However, the majority of patients do not benefit from immunotherapy. Evaluating tumor heterogeneity and immune status before treatment is key to identifying patients that are more likely to respond to immunotherapy. Demographic characteristics (such as sex, age, and race), immune status, and specific biomarkers all contribute to response to immunotherapy. A comprehensive immunodiagnostic model integrating all these three dimensions by artificial intelligence would provide valuable information for predicting treatment response. Here, we coined the term "immunodiagnosis" to describe the blueprint of the immunodiagnostic model. We illustrated the features that should be included in immunodiagnostic model and the strategy of constructing the immunodiagnostic model. Lastly, we discussed the incorporation of this immunodiagnosis model in clinical practice in hopes of improving the prognosis of tumor immunotherapy.

Comparative Analysis of Different ELISA Methods for the Serodiagnosis of Przhevalskiana silenus Infestation in Goats.

Przhevalskiana silenus (warble fly) grubs cause myiasis in goats, in mountainous and semi-mountainous areas and different regions in Pakistan, and cause substantial losses to livestock. The palpation method for detecting warble flies generally neglects the infestation intensity; therefore, the development of a reliable and efficient diagnostic technique is extremely necessary. This study compared three indirect enzyme-linked immunosorbent assay (ELISA) methods for detecting anti-P. silenus antibodies using the hypodermin C (HyC) purified from Hypoderma spp. Larvae collected in cattle (local isolate, Microbiology Laboratory, PMAS-Arid Agriculture University, Rawalpindi), the crude antigen from the first instar stage of P. silenus, and a commercial Bovine Hypodermosis Antibody ELISA kit (IDEXX Laboratory), for accurately estimating the seroprevalence of goat warble fly infestation (GWFI) in the Pothwar plateau, Punjab, Pakistan. The ELISA with the crude antigen of P. silenus proved very sensitive and specific, 91% and 93%, respectively. The optical density exhibited a monthly variation, and the antibody titer began increasing from June, continually increased from July to December, and gradually decreased thereafter until March. The study confirmed the endemic status of GWFI in the Pothwar region and identified that ELISA based on the crude antigen of P. silenus was a more sensitive and specific immunodiagnostic method for determining seroprevalence, and could be employed for initiating nationwide eradication campaigns.

Production and Characterization of Two Specific ZIKV Antigens Based on Bioinformatic Analysis and Serological Screening.

BACKGROUND: The high structural similarity between the Zika virus (ZIKV) and other flaviviruses, such as Dengue Virus (DENV), complicates the identification of the infecting virus due to the occurrence of cross-reactions in serological assays. This phenomenon has increased the demand for more specific antigens for immunodiagnostic applications. METHODS: The present work aimed to identify specific regions of ZIKV and produce unique antigens through computational methods, molecular and microbiological techniques. RESULTS: Based on the computational analysis we successfully expressed two recombinant proteins derived from specific regions of the ZIKV. Through serological assays using characterized sera, we observed that the region 146-182 of ZIKV's E protein, expressed in tandem, was not reactive despite the predictive sensitivity and specificity observed by computer analyses. On the other hand, the non-denatured fraction 220-352 of ZIKV's NS1 showed greater specificity to IgG+ sera of ZIKV by dot blot and western blot, which highlights its properties as a possible tool in the diagnosis of ZIKV. CONCLUSION: These findings demonstrate that ZIKV NS1 fraction 220-352 is a potential tool that may be applied in the development of serological diagnosis. We also provided data that suggest the non-applicability of the region 146-182 of ZIKV's protein E in serological assays despite previous indications about its potential based on computational analysis.

Increasing temperature denatures canine IgG reducing its ability to inhibit heartworm antigen detection.

BACKGROUND: Immune complexing of target antigen to high affinity host antibody is recognized to impact the sensitivity of commercial heartworm antigen tests. Published information describing the effect of heat on interfering canine host antibodies is lacking. Immune complex dissociation (ICD) by heat treatment of serum for samples initially testing negative for heartworm antigen increases sensitivity of commercial antigen tests, particularly for single sex or low adult infection intensities. In this study the stability and nature of the targeted epitope and mechanism of heat ICD were examined. METHODS: Canine IgG was isolated using protein-A columns from serum originating from four dogs evaluated after necropsy: one dog with evidence of previously cleared infection and three dogs with confirmed heartworm infections. These dogs were expected to have an excess of antibodies based on negative antigen test and to have no or low antigen optical density, respectively, following heat treatment. Interference of antigen detection on (non-heated) positive serum was evaluated, following 1:1 mixing of antibody/PBS solutions previously heated at 25 °C, 65 °C, 75 °C, 85 °C, 95 °C and 104 °C, compared to positive serum/PBS control measured by optical density using a commercial heartworm antigen ELISA and protein quantification. Live heartworms incubated in media for 72 h provided excretory/secretory antigen for antigen stability studies following heat, endopeptidase digestion and disulfide bond reduction. RESULTS: Mixing antigen-positive heartworm serum with antibody solutions demonstrated a significant inhibition of antigen detection for antibody solutions previously heated at 25 °C and 65 °C relative to positive serum/PBS control. Antigen detection optical density was restored at or above the control when positive serum was mixed with solutions previously heated at 75 °C, 85 °C, 95 °C and 104 °C. Significant changes occurred in protein levels for antibody solutions heated at 75 °C, 85 °C, 95 °C and 104 °C. Relative stability of antigen from live heartworms in culture was demonstrated following heat, chemical and enzymatic treatment. CONCLUSIONS: Significant changes in protein levels and antigen binding ability occurred in IgG solutions heated above 65 °C. The findings confirm heat denaturation of antibodies as the suspected mechanism of heat ICD at 104 °C for antigen diagnosis of heartworm. No significant change occurred in antigen detection following heat, chemical or enzymatic digestions supporting a heat-stable linear nature of the epitope.

New immunodiagnostic methods for human tegumentary leishmaniasis in the last 10 years: Technological prospecting.

Leishmaniasis is a neglected disease and more than 1 billion people live in endemic areas with the risk of infection worldwide. Although it is an important epidemiological issue, the gold standard method of diagnosis requires invasive sample collection and is accompanied by a high level of sensitivity variation in results. The present study aims to conduct a patent prospection of immunodiagnostic methods for human tegumentary leishmaniasis in the last 10 years, focused on those with high sensitivity and specificity, and simple usability. We searched seven patent databases: The LENS, WIPO, EPO, USPTO, Patent Inspiration, Google patents, and INPI. Eleven patents were found that satisfy our search criteria, with six of them being registered in 2017. Most patents were registered in Brazil. The information obtained here covers the main characteristics of the immunodiagnostic methods evaluated. Moreover, our prospective study reveals the latest biotechnological advancements achieved in the immunodiagnosis of tegumentary leishmaniasis, especially in Brazil, which holds the majority of patents in this subject. However, no patent for immunodiagnostic methods was found in the last three years, which raises concerns about the present and future trends of leishmaniasis diagnosis.

Diagnosis of Toxoplasmosis Using Surface Antigen Grade 1 Detection by ELISA, Nano-Gold ELISA, and PCR in Pregnant Women.

Introduction: The accurate diagnosis of toxoplasmosis has critical importance in pregnant women. Nanotechnology and molecular biology are making possible opportunities for accurate and rapid diagnosis of many infectious diseases. Aim and Methods: The aim of our study was to compare nano-gold ELISA with ELISA and PCR for diagnosis of toxoplasmosis using Toxoplasma surface antigen grade 1 (SAG1) in pregnant women seeking antenatal care in outpatient clinics. Results: PCR showed the highest diagnostic values than nano-gold ELISA and ELISA regarding sensitivity (97.3% versus 89.2% and 83.8%); specificity (100% versus 94% and 88%); and diagnostic accuracy (98.9% versus 91.95% and 86.2%), respectively. There is no statistical difference between PCR and nanogold ELISA results. Discussion: Nano-gold ELISA had a significant improvement in diagnosis than the traditional ELISA method. Most likely with the assistance of nanoparticles, more antibodies enter the antigen-antibody complex because of the considerable improvement in the surface area of nano-gold particles. Conclusion: Although PCR had higher diagnostic values than nano ELISA, nano ELISA is cheaper and easier than PCR. We recommend nano-gold ELISA with SAG1 as a promising technique in the diagnosis of toxoplasmosis and survey studies.

IgG4 and IgE anti-Strongyloides stercoralis as additional parameters in characterizing patients with diabetes from a hyperendemic area.

We report the detection of IgG, IgG1, IgG4 and IgE anti-Strongyloides stercoralis as complementary tool for screening in patients with diabetes in hyperendemic areas for strongyloidiasis. A panel of 119 serum samples were analyzed: 76 from patients with DM2 and 43 patients with other endocrine diseases and a positive correlation for total IgG levels with IgG4 (rs = 0.559; P = 0.024; n = 16) and IgG and IgE (rs = 0.585; P < 0.0001; n = 76) was found in the diabetes group.

Proof of Concept of a Novel Multiepitope Recombinant Protein for the Serodiagnosis of Patients with Chagas Disease.

Chagas disease remains a neglected disease that is considered to be a public health problem. The early diagnosis of cases is important to improve the prognosis of infected patients and prevent transmission. Serological tests are the method of choice for diagnosis. However, two serological tests are currently recommended to confirm positive cases. In this sense, more sensitive and specific serological tests need to be developed to overcome these current diagnosis problems. This study aimed to develop a new recombinant multiepitope protein for the diagnosis of Chagas disease, hereafter named rTC. The rTC was constructed based on amino acid sequences from different combinations of Trypanosoma cruzi antigens in the same polypeptide and tested using an enzyme-linked immunosorbent assay (ELISA) to detect different types of Chagas disease. rTC was able to discriminate between indeterminate (IND) and cardiac (CARD) cases and cross-reactive diseases, as well as healthy samples, with 98.28% sensitivity and 96.67% specificity, respectively. These data suggest that rTC has the potential to be tested in future studies against a larger serological panel for the diagnosis of Chagas disease.

P22 protein complex in the serodiagnosis of animal tuberculosis: Antigenic stability and cross-reactivity with Corynebacterium pseudotuberculosis infection.

The P22 ELISA was recently developed for the serodiagnosis of animal tuberculosis. Herein, the stability of the P22 antigen in different presentations and storage conditions, and the cross-reactivity with Corynebacterium pseudotuberculosis infection in small ruminants were evaluated. For the stability assay, serum samples from cows, sheep, goats, alpacas, badgers, and wild boar were used in the P22 ELISA. The cross-reactivity analysis used sera from sheep and goats with caseous lymphadenitis (CLA). Differences in the immune recognition of P22 were found when the antigen was stored at 40 °C, but without altering the negative or positive status of each sample. P22 ELISA presented 5.71 % cross-reactivity when CLA-positive sheep were evaluated, but no cross-reaction was observed among CLA-positive goat serum samples. This study showed that the P22 protein complex is stable under different formulations and temperatures, and that the assay presents a low cross-reactivity with CLA.

Diagnostic Methods of Common Intestinal Protozoa: Current and Future Immunological and Molecular Methods.

Intestinal protozoan infection is a persisting public health problem affecting the populations of developing countries in the tropical and subtropical regions. The diagnosis of intestinal protozoa remains a challenge especially in developing countries due to a shortage of laboratory facilities, limited health funding, and the remoteness of communities. Despite still being widely used, conventional diagnoses using microscopy and staining methods pose important limitations, particularly due to their low sensitivities and specificities. The selection of diagnostic methods needs to be carefully considered based on the objective of examination, availability of resources, and the expected parasite to be found. In this review, we describe various immunodiagnosis and molecular diagnostic methods for intestinal protozoa infection, including their advantages, disadvantages, and suitability for different settings, with a focus on Entamoeba histolytica, Giardia duodenalis, and Cryptosporidium spp.

Padronização de um teste ELISA indireto para diagnóstico da cinomose canina / Standardization of an indirect ELISA test for canine diagnosis diagnosis

A cinomose é uma enfermidade causada pelo vírus Canine Distemper Virus (CDV). Essa doença afeta principalmente cães, mas também acomete outras espécies domésticas e selvagens. A imunidade do animal está relacionada ao grau que a esse patógeno vai atingir o organismo do indivíduo. Ela afeta a respiração do animal, pode causar vômito, diarreia, convulsões, podendo levar o animal à óbito. O objetivo do presente trabalho foi padronizar um teste ELISA indireto com antígeno de superfície para o diagnostico cinomose utilizando amostras de soro canino. Para padronização da técnica, fez-se necessário o estudo da diluição do antígeno para identificar a melhor concentração para sensibilização da placa. O teste foi aplicado primeiramente com diferentes diluições do antígeno para detecção do melhor desempenho do antígeno. Feito isso, foi testado em um banco de soro de 45 animais comprovadamente positivos no teste ELISA comercial e em soro de 45 animais comprovadamente negativos no teste ELISA comercial, posteriormente foi calculado o ponto de corte, especificidade e sensibilidade do teste. O teste ELISA indireto se mostrou com excelência como um teste de diagnóstico para a cinomose canina, obtendo-se ponto de corte de densidade óptica de 0,229, sensibilidade de 95,5% e especificidade de 84,4%.

Distemper is a disease or the disease by the CDV virus, Distemper Virus. This disease mainly affects dogs, but also affects other domestic and wild species. The animal's immunity is related to the degree to which it will reach the individual's organism. It affects the animal's breathing, can cause vomiting, diarrhea, convulsions, and can lead to death. The aim of the present work test was to standardize an indirect ELISA for distemper diagnosis in experiments using a surface antigen. For the study of technical identification, it was necessary to specify the antigen for the best concentration of plaque sensitization. The test was initially applied with different dilutions of the antigen to detect the best performance of the antigen. This was tested in a serum bank of 45 animals proven positive in the commercial ELISA test and in the serum of 45 animals proven negative in the commercial ELISA test, later it was tested on the cut-off point, specificity and sensitivity of the test. The indirect ELISA test proved to be excellent as a diagnostic test for canine distemper, with an optical density cut-off of 0.229, sensitivity of 95.5% and specificity of 84.4% being obtained.

Advances in diagnosis of gastrointestinal nematodes in livestock and companion animals.

Diagnosis of gastrointestinal nematodes in livestock and companion animals has been neglected for years and there has been an historical underinvestment in the development and improvement of diagnostic tools, undermining the undoubted utility of surveillance and control programmes. However, a new impetus by the scientific community and the quickening pace of technological innovations, are promoting a renaissance of interest in developing diagnostic capacity for nematode infections in veterinary parasitology. A cross-cutting priority for diagnostic tools is the development of pen-side tests and associated decision support tools that rapidly inform on the levels of infection and morbidity. This includes development of scalable, parasite detection using artificial intelligence for automated counting of parasitic elements and research towards establishing biomarkers using innovative molecular and proteomic methods. The aim of this review is to assess the state-of-the-art in the diagnosis of helminth infections in livestock and companion animals and presents the current advances of diagnostic methods for intestinal parasites harnessing (i) automated methods for copromicroscopy based on artificial intelligence, (ii) immunodiagnosis, and (iii) molecular- and proteome-based approaches. Regardless of the method used, multiple factors need to be considered before diagnostics test results can be interpreted in terms of control decisions. Guidelines on how to apply diagnostics and how to interpret test results in different animal species are increasingly requested and some were recently made available in veterinary parasitology for the different domestic species.

PARASITES OF THE BRAZILIAN ROCK CAVY, KERODON RUPESTRIS: REVEALING THEIR HISTORY IN THE BRAZILIAN SEMIARID REGION.

Using microscopy and/or immunodiagnosis, the authors analyzed 284 fecal samples from the Brazilian rock cavy, Kerodon rupestris, that were collected between 1984 and 2015 in Serra da Capivara National Park for the presence of helminths and protozoa. Fourteen morphospecies of helminth eggs of the following taxa were found: Trematoda, Nematoda, Strongylidae, Lagochilascaris sp., Strongylida, Trichuris (2 species), Oxyuridae (3 species), Ancylostomatidae (2 species), and Ascarididae (2 species), along with 3 protozoan taxa: Coccidia, Cryptosporidium sp., and Balantidium sp. During the last 30 yr, the population of K. rupestris has increased in the region as a consequence of the creation and management of the National Park, and data from this study show a concurrent increase in the diversity of intestinal parasites in this host, including new reports. Some of these species have zoonotic potential, which suggests that K. rupestris may be in contact with domestic farm animals and/or human feces. These results show the importance of integrating different diagnostic approaches for the identification of protozoa in the region and indicate that further methods need to be employed to increase recovery. This work highlights the usefulness of parasite studies in assessing the health of ecosystems, especially in protected areas, which should be considered by park managers and health agencies.