Development Of Diagnostic Reagents Research Articles

A New Strategy for Efficient Screening and Identification of Monoclonal Antibodies against Oncogenic Avian Herpesvirus Utilizing CRISPR/Cas9-Based Gene-Editing Technology.

Marek’s disease virus (MDV) is an important oncogenic α-herpesvirus that induces Marek’s disease (MD), characterized by severe immunosuppression and rapid-onset T-cell lymphomas in its natural chicken hosts. Historically, MD is regarded as an ideal biomedical model for studying virally induced cancers. Monoclonal antibodies (mAbs) against viral or host antigenic epitopes are crucial for virology research, especially in the exploration of gene functions, clinical therapy, and the development of diagnostic reagents. Utilizing the CRISPR/Cas9-based gene-editing technology, we produced a pp38-deleted MDV-1 mutant—GX0101Δpp38—and used it for the rapid screening and identification of pp38-specific mAbs from a pool of MDV-specific antibodies from 34 hybridomas. The cross-staining of parental and mutated MDV plaques with hybridoma supernatants was first performed by immunofluorescence assay (IFA). Four monoclonal hybridomas—namely, 4F9, 31G7, 34F2, and 35G9—were demonstrated to secrete specific antibodies against MDV-1’s pp38 protein, which was further confirmed by IFA staining and confocal analysis. Further experiments using Western blotting, immunoprecipitation (IP), liquid chromatography–tandem mass spectrometry (LC–MS/MS), and immunohistochemistry (IHC) analysis demonstrated that the pp38-specific mAb 31G7 has high specificity and wide application potential for further research in MD biology. To the best of our knowledge, this is the first demonstration of the use of CRISPR/Cas9-based gene-editing technology for efficient screening and identification of mAbs against a specific viral protein, and provides a meaningful reference for the future production of antibodies against other viruses—especially for large DNA viruses such as herpesviruses.

Characterization of the monoclonal antibody specific to the ORF72 protein of koi herpesvirus and cellular distribution analysis of the viral protein.

Koi herpesvirus (KHV) is an emerging pathogen of koi and common carp that causes a severe disease and mass mortality of infected fish. The KHV ORF72 protein is an important capsid protein that has been suggested to be a candidate for the development of diagnostic reagents and KHV vaccines. The purpose of this study was to clone and express the KHV ORF72 gene for further preparation of a specific monoclonal antibody (mAb) and to analyse cellular distribution of the viral protein. The mAb 3E1 could specifically recognize the expressed ORF72 protein of transfected cells by indirect immunofluorescence, and the antigenic site recognized by the mAb 3E1 was mapped to the region of N-terminal 124 residues of KHV ORF72. This mAb was further demonstrated to specifically detect the KHV-infected fish tissue by immunohistochemistry, thereby suggesting its high diagnostic potential. In addition, the cellular distribution analysis of the KHV ORF72 protein revealed that the region of amino acid residues 125-247 was related to mitochondrial localization and proliferation. Furthermore, a putative nuclear export signal (NES) of ORF72 at the residues 201-212 was confirmed on the basis of its function associated with NES activity.

Development and Characterization of Canine Distemper Virus Monoclonal Antibodies.

Five canine distemper virus monoclonal antibodies were developed by immunizing BALB/c mice with a traditional vaccine strain Snyder Hill. Among these monoclonal antibodies, four antibodies recognized both field and vaccine strains of canine distemper virus without neutralizing ability. One monoclonal antibody, 1A4, against hemagglutinin protein of canine distemper virus was found to react only with vaccine strain virus but not field isolates, and showed neutralizing activity to vaccine strain virus. These monoclonal antibodies could be very useful tools in the study of the pathogenesis of canine distemper virus and the development of diagnostic reagents.

Immunogenicity and immunoreactivity of Tp0821 recombinant protein from Treponema pallidum

Treponemapallidum (Tp) is responsible for invading reproductive organs and the skin in early stages, and involves almost all organs/systems at advanced stages. In the present study, screening of the dominant epitope fragment of the Tp outer membrane protein, Tp0821, was performed and the prokaryotic expression vector pQE32/Tp0821 was constructed. The denaturation and dialysis of rTp0821 were achieved through ultrasound, inclusion body washing and dissolution. Experiments in purified rTp0821‑immune New Zealand rabbits indicated that the recombinant proteins were of high immunogenicity, and the irritation led a marked humoral immune response in the New Zealand rabbits. Western blot analysis showed that the purified recombinant proteins reacted with the Tp‑positive infected serum, confirming the high level of immunoreactivity. The delayed type hypersensitivity of rTp0821 recombinant proteins was positive, indicating that rTp0821 induced a specific cell immune response and was selected as a Tp vaccine candidate protein. The findings of the present study provided novel evidence, which provided information for further investigations on the pathogenic mechanism of Tp and the development of diagnostic reagents.

The Relationship between B-cell Epitope and Mimotope Sequences.

B-cell epitope is a group of residues which is on the surface of an antigen. It invokes humoral responses. Locating B-cell epitope is important for effective vaccine design, and the development of diagnostic reagents. Mimotope-based B-cell epitope prediction method is a kind of conformational B-cell epitope prediction, and the core idea of the method is mapping the mimotope sequences which are obtained from a random phage display library. However, current mimotope-based B-cell epitope prediction methods cannot maintain a high degree of satisfaction in the circumstances of employing only mimotope sequences. In this study, we did a multi-perspective analysis on parameters for conformational B-cell epitopes and characteristics between epitope and mimotope on a benchmark datasets which contains 67 mimotope sets, corresponding to 40 unique complex structures. In these 67 cases, there are 25 antigen-antibody complexes and 42 protein-protein interactions. We analyzed the two parts separately. The results showed the mimotope sequences do have some epitope features, but there are also some epitope properties that mimotope sequences do not contain. In addition, the numbers of epitope segments with different lengths were obviously different between the antigen-antibody complexes and the protein-protein interactions. This study reflects how similar do mimotope sequence and genuine epitopes have; and evaluates existing mimotope-based B-cell epitope prediction methods from a novel viewpoint.

Definition of CD4 Immunosignatures Associated with MTB

We have recently described the first true genome-wide screen for CD4+ T-cell reactivity directed against Mycobacterium tuberculosis (MTB) in latent TB-infected individuals. The approach relied on predictions of HLA-binding capacity for a panel of DR, DP, and DQ alleles representative of those most commonly expressed in the general population, coupled with high throughput ELISPOT assays. The results identified hundreds of novel epitopes and antigens, and documented the novel observation that T cells in latent MTB infection are confined to the CXCR3+CCR6+ phenotype and largely directed against three antigenic “islands” within the MTB genome. In parallel, we have made generally available to the scientific community the technical approaches and reagents developed in the process, such as motifs, algorithms, and binding assays for several common HLA class II alleles, and a panel of single allele HLA class II transfected cell lines representative of the most frequent specificities in the general population. Recent efforts have been focused on characterization of epitopes and antigens recognized by patients with active TB and individuals vaccinated with BCG, with the aim of providing the first systematic evaluation of the overlap between latent, active, and BCG cohorts. The definition of a broad range of epitopes restricted by common HLA molecules, will facilitate development of diagnostic reagents, allow a rigorous evaluation of T-cell responses associated with TB infection in humans, and enable the evaluation of the immunogenicity of different vaccine candidates. Furthermore, it might suggest new candidates for vaccine and diagnostic development.

Chicken astrovirus capsid proteins produced by recombinant baculoviruses: potential use for diagnosis and vaccination

Chicken astroviruses (CAstVs) have been characterized recently. Due to their relatively poor growth in cell culture, virus-specific antigens are not readily available for the development of diagnostic reagents and vaccines. For this purpose two capsid protein antigens, specified by the 11672 isolate of CAstV, were produced in insect cells following infection with recombinant baculoviruses. The GST-11672 capsid protein, a fusion protein comprising the capsid protein and glutathione-S-transferase (GST) as an N-terminal affinity tag, and the 11672 capsid protein alone were detected by western blotting as proteins of ~100 and 70 kDa, respectively. Immunization with the affinity-purified GST-11672 capsid protein produced a polyclonal rabbit antiserum, which reacted by indirect immunofluorescence with Group B CAstVs but which showed no reactivity with the Group A CAstV isolate, 612. When used as part of an immunoperoxidase-based immunohistochemical procedure, this rabbit antiserum facilitated the detection of CAstV antigen in formalin-fixed, paraffin-embedded kidney tissue at the sites of histopathology characteristic of nephritis. Although further evaluation with sera from commercial chickens is required, a prototype indirect antibody-detecting enzyme-linked immunosorbent assay (ELISA) based on affinity-purified GST-11672 capsid protein as coating antigen demonstrated considerable potential with low ELISA absorbance values being generated with sera from specific pathogen free (SPF) chickens, and high absorbance values being generated with serum samples from experimentally infected chickens. Immunization experiments of SPF chickens showed that, when administered as mixtures with oil adjuvant, crude cell lysates containing the GST-11672 capsid protein or the 11672 capsid protein elicited virus-specific antibody responses that were detectable by indirect immunofluorescence and by virus neutralization assays.

Prediction of Antigen Epitopes on Protein Surfaces Based on Support Vector Machine

B-cell epitope prediction is important for vaccine design, development of diagnostic reagents and for studies to elucidate the interactions between antigen and antibody on a molecular level. Here, we present a new epitope prediction method based on six different scoring functions and exploited LibSVM to predict the antigenic epitopes in protein surface. Using bound structures of the testing dataset, the method was able to predict antigenic epitopes with 50.6% sensitivity, 62.9% specificity, 19% precision and an AUC value of 0.616. While using unbounded structures of the testing dataset, the performance of the method was nearly the same. Compared with another epitope prediction method EPCES, the performance of the method is statistically similar. The results suggest that more effective features that discriminate epitopes from non-epitopes may further improve the performance of the prediction method. Also, the new algorithms for predicting the epitopes are desired and the construction of large with non-redundant datasets is strongly needed.

Effect of Chemotherapy on Whole-Blood Cytokine Responses to Mycobacterium tuberculosis Antigens in a Small Cohort of Patients with Pulmonary Tuberculosis

The development of genomic and proteomic tools has enabled studies that begin to characterize the molecular targets of an effective host immune response to Mycobacterium tuberculosis, including understanding the specific immune responses associated with tuberculosis (TB) disease progression, disease resolution, and the development of latency. One application of such tools is the development of diagnostic reagents and assays useful as a test of cure. Such a test could be of considerable importance for the evaluation of new therapeutics. We and others have previously described immunodominant proteins of M. tuberculosis, including both vaccine and diagnostic candidates. In the present study, we describe the changes in immune responses to a panel of 71 M. tuberculosis antigens in six patients during the course of therapy. The levels of six cytokines were measured in 24-h whole-blood assays with these antigens, revealing that gamma interferon (IFN-γ), tumor necrosis factor (TNF), and interleukin-10 (IL-10) were differentially regulated in response to a subset of antigens. Therefore, measuring the production of these three cytokines in response to a panel of carefully selected M. tuberculosis proteins during the course of TB therapy might be a promising path toward the development of a test of cure and warrants further validation in larger cohorts of pulmonary TB patients.

Anti-Ebola MAb 17A3 reacts with bovine and human α-2-macroglobulin proteins

Monoclonal antibodies (MAbs) were developed against soluble Ebola virus (EBOV) envelope glycoprotein (GP) for the study of the diversity of EBOV envelope and development of diagnostic reagents. Of the three anti-EBOV GP mouse MAbs produced, MAb 15H10 recognized all human EBOV GP species tested (Zaire, Sudan, Ivory Coast), and as well as reacted with the Reston nonhuman primate EBOV GPs. A second MAb, 6D11 recognized EBOV GP species of Sudan and Sudan-Gulu. The third MAb, 17A3, was reported originally in the same article to be EBOV GP specific has now been found to be specific for bovine and human α-2-macroglobulin (α-2M) proteins which were contaminants in the Ebola envelope protein preparation. Thus, while MAbs 15H10 and 6D11 are indeed EBOV GP specific, MAb 17A3 is an α-2-macroglobulin MAb.

Anti-peptide antibodies differentiate between plasmodial lactate dehydrogenases

Malaria lactate dehydrogenase, a glycolytic enzyme, is a malaria diagnostic target in lateral flow immunochromatographic rapid diagnostic tests. Recombinant Plasmodium yoelii LDH was cloned into the pET-28a vector, expressed and the expressed protein purified from a Ni-NTA affinity matrix. A pan-malarial LDH antibody directed against a common malaria LDH peptide (APGKSDKEWNRDDLL) and two anti-peptide antibodies, each targeting a unique Plasmodium falciparum (LISDAELEAIFDC) and Plasmodium vivax (KITDEEVEGIFDC) LDH peptide were raised in chickens. The antibodies were affinity purified with the appropriate peptide affinity matrix. The affinity purified anti-peptide antibodies detected recombinant P. falciparum, P. vivax and P. yoelii LDH and native P. falciparum and P. yoelii LDH in western blots and immunofluorescence studies. The pan-malarial antibody detected LDH from the three malaria species in western blots. The species-specific anti-peptide antibodies differentiated between P. falciparum and P. vivax LDH. Affinity purified chicken antibodies against recombinant PfLDH, PvLDH and PyLDH proteins each detected the parent and orthologous proteins with similar titers in an ELISA. The study supports an anti-peptide antibody approach to the development of diagnostic reagents.

Detection of Ebola virus envelope using monoclonal and polyclonal antibodies in ELISA, surface plasmon resonance and a quartz crystal microbalance immunosensor

Ebola virus (EBOV) Zaire, Sudan, as well as Ivory Coast are virulent human EBOV species. Both polyclonal and monoclonal antibodies (MAbs) were developed against soluble EBOV envelope glycoprotein (GP) for the study of EBOV envelope diversity and development of diagnostic reagents. Three EBOV Sudan-Gulu GP peptides, from the N-terminus, mid-GP, and C-terminus regions were used to immunize rabbits for the generation of anti-EBOV polyclonal antibodies. Polyclonal antisera raised against the C-terminus peptide could detect both Sudan-Gulu as well as Zaire GPs, while anti-N and mid-region peptide polyclonal sera recognized only EBOV Sudan-Gulu GP. Of the three anti-EBOV GP mouse MAbs produced, MAb 15H10 recognized all human EBOV GP species tested (Zaire, Sudan and Ivory Coast), and as well as reacted with the Reston non-human primate EBOV GPs. In addition, MAb 15H10 bound virion-associated GP of all known EBOV species. MAb 17A3 recognized GPs of both EBOV Sudan-Gulu and Zaire, while MAb 6D11 recognized only EBOV Sudan-Gulu GP. To detect EBOV GP, these antibody reagents were used in ELISA, surface plasmon resonance and in a quartz crystal microbalance immunosensor. Thus, polyclonal and monoclonal antibodies can be used in combination to identify and differentiate both human and non-human primate EBOV GPs.

Development of diagnostic reagents: Raising antibodies against synthetic peptides of PfHRP-2 and LDH using microsphere delivery

Malaria causes significant morbidity and mortality worldwide, including countries with mainly imported malaria. In developing nations, scarce resources lead to inadequate diagnostic procedures. Microscopy of Giemsa-stained thick and thin films remains the current gold standard for diagnosis. Although it has good sensitivity and allows species identification, it is time consuming, requires microscopical expertise and maintenance of equipment. Antigen tests are promising tools for the diagnosis of malaria. Two such antigens are Plasmodium falciparum histidine rich protein (pfHRP-2) and lactate dehydrogenase (LDH). The present study was aimed to develop indigenous, rapid and sensitive immunodiagnostic method based on detection of PfHRP-2 and LDH antigen in the blood. Unique peptide sequences of PfHRP-2 (two regions) and LDH (three regions) antigen were synthesized by solid phase technique and purified to homogeneity. The antibodies raised against these sequences were raised in mice as well as rabbit using microspheres (PLGA) to generate high titre and affinity antibodies. The peptide-specific peak titres varied from 25,000 to 50,000 and affinity of the antibodies produced was found to be in order of 0.73–5.3 nM. The antibodies generated using microspheres were able to detect the PfHRP-2 and LDH antigen in the culture supernatant and parasitized RBC lysate of P. falciparum respectively by sandwich ELISA up to 0.002% parasitaemia level. The assay allowed the detection of parasite infections of 0.08–2.68% parasitaemia with a sensitivity of 100% in the whole blood of P. falciparum positive patients. No cross-reactivity was observed with P. vivax infected patients.

Use of superparamagnetic beads for the isolation of a peptide with specificity to cymbidium mosaic virus

A modified method for the rapid isolation of specific ligands to whole virus particles is described. Biopanning against cymbidium mosaic virus was carried out with a commercial 12-mer random peptide display library. A solution phase panning method was devised using streptavidin-coated superparamagnetic beads. The solution based panning method was more efficient than conventional immobilized target panning when using whole viral particles of cymbidium mosaic virus as a target. Enzyme-linked immunosorbent assay of cymbidium mosaic virus-binding peptides isolated from the library identified seven peptides with affinity for cymbidium mosaic virus and one peptide which was specific to cymbidium mosaic virus and had no significant binding to odontoglossum ringspot virus. This method should have broad application for the screening of whole viral particles towards the rapid development of diagnostic reagents without the requirement for cloning and expression of single antigens.

Clinical outcome of patients with lymph node‐negative breast carcinoma who have sentinel lymph node micrometastases detected by immunohistochemistry

We read with great interest the article by Chagpar et al. and congratulate the authors.1 We agree that micrometastases do not affect the outcomes significantly in patients with breast carcinoma. At the same time, a fundamental problem in cancer research is the identification of the cell type capable of sustaining the growth of the neoplastic clone. There is overhelming evidence that virtually all tumors are clonal and represent the progeny of a single cell. Two theories were developed to explain why not every cell within a tumor is capable of regenerating the tumor. The stochastic theory predicts that every tumor cell is potentially tumor initiating but that entry into the cell cycle is governed by low-probability stochastic events. Conversely, the hierarchy theory proposes that the tumor is functionally heterogeneous and that only a limited number of cells are capable of initiating the tumor.2 Al-Hajj et al. provided a major step forward with the identification of human breast carcinoma-initiating cells. The authors found that only a minority of breast carcinoma cells had the ability to form new tumors. They prospectively identified and isolated the tumorigenic cells as CD44(+)CD24(-/low)Lineage(-) in eight of nine patients.3 The ability to seperate tumorigenic and nontumorigenic populations of tumor cells should allow the molecular characterization of these cells and the elucidation of the pathways that account for their tumorigenic potential. Furthermore, the existence of a subset of tumorigenic cells within a tumor would provide an explanation for a number of clinical observations made in breast carcinoma patients. For example, it has been demonstrated that up to 30% of breast carcinoma patients may demonstrate micrometastatic disease in their bone marrow at the time of presentation. However, after 5 years, only 50% of these patients will demonstrate clinically evident metastases. This finding has been speculated to be the result of tumor dormancy. An alternative explanation is that the disseminated tumor cells in this group of patients arose from the spread of nontumorigenic cells, and only when tumor stem cells disseminate and subsequently self-renew will metastatic tumors form. This second explanation suggests that the development of diagnostic reagents that allow for the prospective identification of tumorigenic cells may have prognostic significance for patients with breast carcinoma.3 Mesult Tez M.D.*, Selda Tez M.D.*, * Ataturk Chest Diseases, Research Hospital, Ankara, Turkey

Crustacean primary cell culture: A technical approach.

Crustacean cell culture has gained attention as a potent model to assist in the development of diagnostic reagents and probes for use in the shrimp, crayfish and lobster industries. The availability of such cellular tools is especially important to industries which use intensive aquaculture methods and thus have increased risk of disease problems. Indeed, crustacean cell cultures offer potential for studying the effects of pathogens in vitro and for increasing our knowledge on developmental and sexual maturation processes, or endocrine regulation in crustaceans. Although numerous attempts have been undertaken, no established cell line of marine crustaceans has been reported to date. However, primary cultures obtained from various organ sources are reported with increasing frequency. They represent the first steps towards the establishment of cell lines and they provide useful information concerning the most suitable cell culture conditions involved in the survival and proliferative capacity of the various tissues.

Detection of GB virus C/hepatitis G virus RNA by reverse-transcription polymerase chain reaction.

Recently, a novel RNA virus, designated GB virus C or hepatitis G virus (GBV-C/HGV) has been identified which may possibly be associated with human hepatitis. In this study, the nucleotide sequences of the partial nonstructural 5 (NS5) gene of GBV-C/HGV derived from sera of eight Chinese patients were determined. The overall degree of nucleotide conservation and the existence of regional highly conserved sequences make this part of the genome suitable for the development of diagnostic reagents. On the basis of sequence analysis, two sets of oligonucleotide primers were designed to establish a nested polymerase chain reaction (PCR) for detection of GBV-C/HGV RNA. The efficacy of three PCR methods (first, one stage PCR, second, nested PCR with primers from the NS5 region designed according to the prototype sequence and the third, our newly developed PCR) was compared in 133 Chinese patients with liver disease. The positive rates of these three methods were 8.3%, 11.3% and 18.0% respectively. The specificity of our PCR detecting system was verified by sequencing and restriction fragment length polymorphism (RFLP). In conclusion, because of the heterogeneity and geographic distribution character of GBV-C/HGV, it is necessary to assess the sequence variation among Chinese patients infected with GBV-C/HGV. This may allow to identify GBV-C/HGV RNA with high sensitivity and specificity.

Characterization of the gene for an immunodominant 72 kDa lipoprotein of Mycoplasma mycoides subsp. mycoides small colony type.

With the aim of characterizing specific immunogenic proteins of Mycoplasma mycoides subsp. mycoides small colony (SC) type, the aetiological agent of contagious bovine pleuropneumonia, a gene encoding a major immunogenic protein of 72 kDa named P72 was cloned and expressed in Escherichia coli. The expressed protein was of the same apparent molecular mass as that produced by the parent strain. The predicted molecular mass of P72, based on the DNA-deduced amino acid sequence, was 61.118 kDa, significantly lower than the apparent molecular mass of endogenous or recombinant P72 on SDS-PAGE. Analysis of the amino acid sequence revealed a typical prokaryotic signal peptidase II-membrane lipoprotein lipid attachment site and a transmembrane structure domain in the leader sequence at the amino-terminal end of the protein. P72 was shown to be a lipoprotein and its surface location was confirmed by trypsin treatment of whole cells. An unassigned gene encoding a peptide with some similarity to P72 was found on the genome sequence of M. capricolum subsp. capricolum but not on that of Mycoplasma genitalium. The P72 gene was detected in 11/11 M. mycoides subsp. mycoides SC strains. Antiserum against recombinant P72 reacted strongly with 12/12 strains of M. mycoides subsp. mycoides SC, weakly with Mycoplasma bovine group 7 strain PG50, but not with other members of the 'mycoides cluster' or closely related mycoplasmas. Cows experimentally contact-infected with M. mycoides subsp. mycoides SC developed a humoral response against P72 within 35 d. P72 is a specific antigenic membrane lipoprotein of M. mycoides subsp. mycoides SC with potential for use in development of diagnostic reagents. It seems to belong to a family of lipoproteins of the "mycoides cluster'.

Use of baculovirus expression vectors: development of diagnostic reagents, vaccines and morphological counterparts of bluetongue virus

The productivity and flexibility of insect baculovirus expression vectors and the ability of the baculovirus genome to incorporate (and express) large amounts of foreign DNA allows this system to be used for both single and multiple gene expression. Using the system, bluetongue virus (BTV) genes have been expressed to develop diagnostic reagents and vaccines as well as to understand the basic structures of the virions. BTV which causes disease in ruminants in many parts of the world, consists of 10 double-stranded RNA segments enclosed by double capsids that are composed by 7 structural proteins. Since each protein is encoded by a single RNA species, DNA clones of all 10 RNA species were synthesized and individually expressed in baculovirus vectors at high levels. This has yielded proteins that have been shown to be excellent diagnostic and vaccine reagents. In addition, multiple expression vectors have been used to synthesize morphological structures (viral and subviral) representing BTV.

Use of baculovirus expression vectors: development of diagnostic reagents, vaccines and morphological counterparts of bluetongue virus

The productivity and flexibility of insect baculovirus expression vectors and the ability of the baculovirus genome to incorporate (and express) large amounts of foreign DNA allows this system to be used for both single and multiple gene expression. Using the system, bluetongue virus (BTV) genes have been expressed to develop diagnostic reagents and vaccines as well as to understand the basic structures of virions. BTV which causes disease in ruminants in many parts of the world, consists of 10 double-stranded RNA segments enclosed by double capsids that are composed by 7 structural proteins. Since each protein is encoded by a single RNA species, DNA clones of all 10 RNA species were synthesized and individually expressed in baculovirus vectors at high levels. This has yielded proteins that have been shown to be excellent diagnostic and vaccine reagents. In addition, multiple expression vectors have been used to synthesize morphological structures (viral and subviral) representing BTV.