Journal of immunological methods最新文献

{"title":"Development of a novel and broadly applicable sandwich ELISA assay based on rabbit single-chain variable fragments and a modified Ig-binding domain of protein L fused to a polystyrene-binding peptide","authors":"Yodai Yamamoto ,&nbsp;Haruka Taniguchi ,&nbsp;Ngoc Minh Nguyen ,&nbsp;Fuki Yokoyama ,&nbsp;Kiattawee Choowongkomon ,&nbsp;Alessandro Angelini ,&nbsp;Jun-Ichi Horiuchi ,&nbsp;Yoichi Kumada","doi":"10.1016/j.jim.2024.113771","DOIUrl":"10.1016/j.jim.2024.113771","url":null,"abstract":"<div><div>Most of currently available sandwich-type enzyme-linked immunosorbent assays (ELISA) require the use of full-length animal-derived antibodies which poses welfare criticisms and are often expensive to produce. There is therefore a strong demand for the development of more affordable and animal-free methods to produce antibodies for sandwich ELISA assay. To address these issues, we propose here the development of a new technology based on two complementary rabbit single-chain variable fragments (scFvs) and an Ig-binding domain of protein L (PpL1) fused to a polystyrene-binding peptide (PS-tag) that can be recombinantly produced in bacteria. Toward this goal, we developed a rabbit scFv capable to bind the antigen via its variable regions while engaging protein L through its constant framework domain. To enhance the density of captured scFv and enable a better solvent exposure, we generated multiple PpL1 variants bearing polystyrene-binding peptides (PS) tags fused to its ends. The tandem trimer of PpL1 variant bearing PS-tags located at the N-terminus (PpL1′-T-PSN) revealed increased antigen-binding signal when immobilized on hydrophilic polystyrene (phi-PS) plates. By CDR-grafting different antigen-binding specificities into our engineered protein L-binding scFv we validated our technology against a different antigen. Finally, to further enhance the sensitivity of our assay, we implemented a protein L-based pretreatment to remove potential inhibitory immunoglobulin often present in the blood samples. The ability to rapidly and cost-effectively generate animal-free recombinant antibody fragments that can be adsorbed and specifically oriented on plates while retaining their antigen-binding properties could lead to the development of innovative and widely applicable sandwich ELISA systems for the efficient, versatile and sensitive detection of different types of antigens.</div></div>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":"534 ","pages":"Article 113771"},"PeriodicalIF":1.6,"publicationDate":"2024-11-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142568110","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Decreased variability in the site-specific results during participation in the External Quality Assurance Program Oversight Laboratory (EQAPOL) proficiency program for IFN-gamma enzyme-linked immunospot (IFN-γ ELISpot) assay","authors":"Paul M. Hargarten ,&nbsp;Cassandra G. Porth ,&nbsp;Mark Berrong ,&nbsp;Darin Weed ,&nbsp;Miranda Carper ,&nbsp;Thomas N. Denny ,&nbsp;Guido Ferrari ,&nbsp;Wes Rountree","doi":"10.1016/j.jim.2024.113770","DOIUrl":"10.1016/j.jim.2024.113770","url":null,"abstract":"<div><div>The NIAID DAIDS-sponsored External Quality Assurance Program Oversight Laboratory (EQAPOL) manages an interferon-gamma (IFN-γ) enzyme-linked immunospot (ELISpot) external proficiency program. The ELISpot program evaluates the accuracy and variability of results across laboratories. The variability in the program is quantified via the dispersion, which is the ratio of the variance over the mean of the background-corrected spot-forming cells (SFC) replicates obtained under stimulation with different peptide pools (CMV, CEF). This report includes the longitudinal analysis of the ELISpot program cohort composed of 22 laboratories from 2011 to 2022 to assess whether the within-lab variability has improved over time. Random intercept models of the dispersion over time showed a significant decrease in overall dispersion from an average of approximately 1.8 in 2011 to approximately 1.25 in 2022. Out of the 21 sites, 16 sites (4 being statistically significant) had a negative trend for dispersion over time. Our finding of a reduction of overall within-lab variability demonstrates the need for and benefit of proficiency testing programs.</div></div>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":"534 ","pages":"Article 113770"},"PeriodicalIF":1.6,"publicationDate":"2024-10-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142502035","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Human monoclonal antibody cloning and expression with overlap extension PCR and short DNA fragments","authors":"Zachary Ende ,&nbsp;Margarita Mishina ,&nbsp;Robert C. Kauffman ,&nbsp;Amrita Kumar ,&nbsp;Rashmi Kumari ,&nbsp;Paul R. Knight ,&nbsp;Suryaprakash Sambhara","doi":"10.1016/j.jim.2024.113768","DOIUrl":"10.1016/j.jim.2024.113768","url":null,"abstract":"<div><div>Monoclonal antibodies are powerful therapeutic, diagnostic, and research tools. Methods utilized to generate monoclonal antibodies are evolving rapidly. We created a transfectable linear antibody expression cassette from a 2-h high-fidelity overlapping PCR reaction from synthesized DNA fragments. We coupled heavy and light chains into a single linear sequence with a promoter, self-cleaving peptide, and poly(A) signal to increase the flexibility of swapping variable regions from any sequence available <em>in silico</em>. Transfection of the linear cassette tended to generate similar levels to the two-plasmid system and generated an average of 47 μg (14–98 μg) after 5 days in 2 ml cultures with 15 unique antibody sequences. The levels of antibodies produced were sufficient for most downstream applications in less than a week. The method presented here reduces the time, cost, and complexity of cloning steps.</div></div>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":"534 ","pages":"Article 113768"},"PeriodicalIF":1.6,"publicationDate":"2024-10-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142502037","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"The mouse monoclonal antibody 4E3 is specific for the G1m17 allotype of human IgG1","authors":"Robert Movérare ,&nbsp;Peter Lind ,&nbsp;Åsa Marknell DeWitt","doi":"10.1016/j.jim.2024.113766","DOIUrl":"10.1016/j.jim.2024.113766","url":null,"abstract":"<div><div>Allotype is an amino acid variation within the immunoglobulin isotypes. Four allotypes have been described for human IgG1 and two of them (G1m3 and G1m17) are located at position 214 in the CH1 region of the gamma chain. Various diseases have been associated with allotype expression, making the allotype research an interesting field in immunology. However, allotype-specific reagents are rare. In the present study the specificity of a widely used and commercially available IgG1-specific monoclonal antibody named 4E3, described as binding to the hinge region of IgG1, was evaluated. Using the ImmunoCAP™ assay technology, surprisingly no IgG1 was measured in 13 of 23 human serum and plasma samples when 4E3 was used in an antibody-enzyme conjugate as detection reagent. Further evaluation of 4E3 using eight IgG1 myeloma paraproteins revealed that 4E3 did not bind to three of them. No association was seen between the binding pattern and myeloma light chain glycosylation or the kappa or light chain use. By comparing the myeloma paraprotein binding patterns of 4E3 and TM14 (a monoclonal antibody with known G1m3 specificity), it was indicated that 4E3 only bound to myeloma paraproteins expressing the G1m17 allotype. This was confirmed using recombinant human antibodies expressing either the G1m3 or G1m17 allotype. The G1m17 bias of 4E3 seen using ImmunoCAP was also observed in microtiter plate-based enzyme-linked immunosorbent assays. The antibody 4E3 has a G1m17 bias limiting its use in assays to measure IgG1 antibodies. However, it may be a valuable allotype-specific reagent.</div></div>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":"534 ","pages":"Article 113766"},"PeriodicalIF":1.6,"publicationDate":"2024-10-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142467510","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"High-yield production of recombinant human myelin oligodendrocyte glycoprotein in SHuffle bacteria without a refolding step","authors":"Wesley Wu ,&nbsp;Sasha Gupta ,&nbsp;Sharon A. Sagan ,&nbsp;Carson E. Moseley ,&nbsp;Scott S. Zamvil ,&nbsp;John E. Pak","doi":"10.1016/j.jim.2024.113764","DOIUrl":"10.1016/j.jim.2024.113764","url":null,"abstract":"<div><div>Experimental autoimmune encephalomyelitis (EAE) is a model for central nervous system (CNS) autoimmune demyelinating diseases such as multiple sclerosis (MS) and MOG antibody-associated disease (MOGAD). Immunization with the extracellular domain of recombinant human MOG (rhMOG), which contains pathogenic antibody and T cell epitopes, induces B cell-dependent EAE for studies in mice. However, these studies have been hampered by rhMOG availability due to its insolubility when overexpressed in bacterial cells, and the requirement for inefficient denaturation and refolding. Here, we describe a new protocol for the high-yield production of soluble rhMOG in SHuffle cells, a commercially available <em>E. coli</em> strain engineered to facilitate disulfide bond formation in the cytoplasm. SHuffle cells can produce a soluble fraction of rhMOG yielding &gt;100 mg/L. Analytical size exclusion chromatography multi-angle light scattering (SEC-MALS) and differential scanning fluorimetry of purified rhMOG reveals a homogeneous monomer with a high melting temperature, indicative of a well-folded protein. An <em>in vitro</em> proliferation assay establishes that purified rhMOG can be processed and recognized by T cells expressing a T cell receptor (TCR) specific for the immunodominant MOG₃₅_–₅₅ peptide epitope. Lastly, immunization of wild-type, but not B cell deficient, mice with rhMOG resulted in robust induction of EAE, indicating a B cell-dependent induction. Our SHuffle cell method greatly simplifies rhMOG production by combining the high yield and speed of bacterial cell expression with enhanced disulfide bond formation and folding, which will enable further investigation of B cell-dependent EAE and expand human research of MOG in CNS demyelinating diseases.</div></div>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":"534 ","pages":"Article 113764"},"PeriodicalIF":1.6,"publicationDate":"2024-10-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142467509","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Preliminary field evaluation of indirect ELISA test using the recombinant antigen rLicNTPDase-2 for serodiagnosis of canine leishmaniasis in Colombia","authors":"Angie Tatiana Murillo Casas ,&nbsp;Paula Andrea Castro Martinez ,&nbsp;Fernando Borda Rojas ,&nbsp;Luz Angela Vega ,&nbsp;Anna Cláudia Alves de Sousa ,&nbsp;Juliana Lopes Rangel Fietto ,&nbsp;Natalie Hell-Mor ,&nbsp;Gabriel Andres Tafur-Gómez","doi":"10.1016/j.jim.2024.113765","DOIUrl":"10.1016/j.jim.2024.113765","url":null,"abstract":"<div><div>Leishmaniasis is a significant public health concern, with dogs as the primary reservoir in urban scenarios and facilitating transmission. Diagnosing infected dogs is a crucial step for public health interventions, and the development of new diagnostic platforms can significantly enhance efforts in various regions worldwide. Given the limited availability of diagnostic methods in Colombia, this study evaluates the effectiveness of an Indirect Enzyme-Linked Immunosorbent Assay (ELISA) based on the recombinant protein rLicNTPDase-2 to detect <em>Leishmania</em> in infected dogs. Serum samples were collected from dogs in both endemic and non-endemic areas and classified as natural standards based on prior parasitological diagnoses. The results revealed 24 true positives (TP) and 9 true negatives (TN). Subsequently, the test was then validated with samples from symptomatic and asymptomatic animals, alongside the standards, yielding a specificity of 96 %, a sensitivity of 81 %, efficiency of 90.6 %, a positive predictive value of 92.8 %, and a negative predictive value of 89.6 %. The positive likelihood ratio (RV+) was 20, while the negative likelihood ratio (RV-) was 0.19, indicating high relevance and a robust clinical utility. The area under the curve (AUC) was 1.00, suggesting that the test has excellent discriminatory ability, significantly deviating from the reference diagonal. This is further supported by the significant difference(<em>p</em> &lt; 0.0001) between TN and TP results determined by Fisher's exact test. Involving 163 animals showed 47 % positive and 46 % negative results with a significant difference (<em>p</em> &lt; 0.05) in the mean optical density (OD) values between positive and negative samples. These findings indicate that the ELISA test effectively differentiates between positive and negative samples based on OD values. This study suggests that ELISA based on the recombinant antigen rLicNTPDase-2 could serve as a viable alternative for the serodiagnosis of leishmaniasis in canines in Colombia.</div></div>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":"534 ","pages":"Article 113765"},"PeriodicalIF":1.6,"publicationDate":"2024-10-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142442405","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Development of sandwich enzyme-linked immunosorbent assays quantifying mouse urinary megalin, a novel proximal tubular biomarker","authors":"Rina Sofuku ,&nbsp;Sayaka Miyazaki ,&nbsp;Michihiro Hosojima ,&nbsp;Sawako Goto ,&nbsp;Kazuya Takemoto ,&nbsp;Hideyuki Kabasawa ,&nbsp;Taeko Endo ,&nbsp;Koichi Komochi ,&nbsp;Nanako Sugita ,&nbsp;Hiroyuki Aoki ,&nbsp;Ryota Kobayashi ,&nbsp;Ichiei Narita ,&nbsp;Akihiko Saito","doi":"10.1016/j.jim.2024.113763","DOIUrl":"10.1016/j.jim.2024.113763","url":null,"abstract":"<div><div>Megalin, a type I transmembrane protein, serves as a multi-ligand endocytic receptor in the apical membrane of proximal tubules. Its ectodomain and full-length forms are excreted into human urine, with the former being more abundant. We previously developed two types of sandwich enzyme-linked immunosorbent assays (ELISAs) utilizing monoclonal antibodies that target the amino-terminal ligand-binding domain-I and the carboxyl-terminal cytoplasmic region of human megalin, respectively. The former, termed “A-megalin” ELISA, primarily identifies ectodomains of megalin, whereas the latter, “C-megalin” ELISA, specifically recognizes full-length megalin originating from urinary extracellular vesicles. This study developed novel sandwich ELISAs to assess mouse urinary A-megalin and C-megalin, thereby facilitating studies involving these biomarkers in mouse disease models. Immunoblotting and immunohistochemistry of monoclonal antibodies against human megalin were performed to assess their compatibility with mouse megalin in novel sandwich ELISAs, which were constructed and validated using human assay protocols. Immunoblot analysis of megalin in urinary extracellular vesicles and supernatant was performed to investigate the ratio of ectodomain to full-length forms in mouse urine. Stable measurements having a precision and accuracy within 15 % were achieved in the measurement of quality control samples. A-megalin and C-megalin were detectable in the urine of C57BL/6 mice, whereas most urine samples from kidney-specific conditional megalin-knockout mice were below detection limits. Ectodomain forms of megalin were at least approximately 70 times more abundant than the full-length form, even in mouse urine. In conclusion, we successfully developed sandwich ELISAs for assessing mouse urinary A-megalin and C-megalin to evaluate primarily ectodomain and full-length forms of megalin, respectively.</div></div>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":"534 ","pages":"Article 113763"},"PeriodicalIF":1.6,"publicationDate":"2024-10-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142391049","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"A novel whole blood assay to quantify the release of T cell associated cytokines in response to Bordetella pertussis antigens","authors":"Marta Valente Pinto ,&nbsp;Alex-Mikael Barkoff ,&nbsp;Sagida Bibi ,&nbsp;Aapo Knuutila ,&nbsp;Johanna Teräsjärvi ,&nbsp;Elizabeth Clutterbuck ,&nbsp;Sophie Gimenez-Fourage ,&nbsp;Anke Pagnon ,&nbsp;Jacqueline A.M. van Gaans-van den Brink ,&nbsp;Veronique Corbiere ,&nbsp;Aymeric De Montfort ,&nbsp;Anja Saso ,&nbsp;Haddijatou Jobe ,&nbsp;Sophie Roetynck ,&nbsp;Beate Kampmann ,&nbsp;Elles Simonetti ,&nbsp;Dimitri Diavatopoulos ,&nbsp;Eleonora E. Lambert ,&nbsp;Jussi Mertsola ,&nbsp;Pascal Blanc ,&nbsp;Ronald de Groot","doi":"10.1016/j.jim.2024.113758","DOIUrl":"10.1016/j.jim.2024.113758","url":null,"abstract":"<div><h3>Background</h3><div><em>Bordetella pertussis</em> continues to cause whooping cough globally even in countries with high immunisation coverage. Booster vaccinations with acellular pertussis vaccines are thus used in children, adolescents, and adults. T cell immunity is crucial for orchestrating the immune response after vaccination. However, T cell assays can be expensive and difficult to implement in large clinical trials. In this study, a whole blood (WB) stimulation assay was developed to identify secreted T cell associated cytokines in different age groups after acellular pertussis booster vaccination.</div></div><div><h3>Material and methods</h3><div>Longitudinal WB samples were collected from a small set of subjects (<em>n</em> = 38) aged 7–70 years participating in a larger ongoing clinical trial. For assay development, samples were diluted and incubated with purified inactivated pertussis toxin (PT), filamentous haemagglutinin (FHA), inactivated <em>B. pertussis</em> lysate, and complete medium (M) as stimulating conditions, with anti-CD28 and anti-CD49d as co-stimulants. Different timepoints around the vaccination (D0, D7, D14, D28), WB dilution factor (1:2, 1:4) and incubation time (24 h, 48 h, 72 h) were compared. Responses to 15 cytokines were tested with Luminex/multiplex immunoassay.</div></div><div><h3>Results</h3><div>The optimized assay consisted of WB incubation with M, PT, and FHA (including the two co-stimulants). After 48 h incubation, supernatants were collected for measurement of seven selected T cell associated cytokines (IL-2, IL-5, IL-10, IL-13, IL-17 A, IL-17F, and IFN-y) from samples before and 28 days after vaccination. PT stimulation showed a trend for upregulation of IL-2, IL-13, and IL-17 A/F for adult subjects, whereas the responses of all cytokines were downregulated for the paediatric subjects. Furthermore, PT and FHA-stimulated WB showed diverse cytokine producing profiles.</div></div><div><h3>Conclusions</h3><div>The developed WB-based cytokine assay was shown to be less costly, easy to perform, and functional in differently aged individuals. Further, it requires only a small amount of fresh blood, which is beneficial especially for studies including infants. Our results support the use of this assay for other immunological studies in the future.</div></div>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":"534 ","pages":"Article 113758"},"PeriodicalIF":1.6,"publicationDate":"2024-09-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142365506","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Highly effective strategy for isolation of mononuclear cells from frozen cord blood","authors":"Guangzhao Li ,&nbsp;Yunyan Zhao ,&nbsp;Rongzhi Liu ,&nbsp;Yabin Zhang ,&nbsp;Yong Zhang ,&nbsp;Wei Du ,&nbsp;Yu Zhang","doi":"10.1016/j.jim.2024.113762","DOIUrl":"10.1016/j.jim.2024.113762","url":null,"abstract":"<div><h3>Background aims</h3><div>Cord blood mononuclear cells (CBMCs) comprise a variety of single-nucleated cells found in the cord blood, mainly consisting of monocytes and lymphocytes. They also include a smaller proportion of other cell types, such as hematopoietic stem and progenitor cells (HSPCs) and mesenchymal stromal cells (MSCs). CBMCs are vital for acquiring HSPCs, MSCs, and other immune cells, like natural killer cells. These cells are essential for supporting subsequent research and clinical applications. Although automated equipment for CBMC enrichment has shown promise, the high cost of these machines and the expense of disposable consumables limit their routine use. Furthermore, limited information is available on manual strategies for isolating CBMCs from cryopreserved cord blood. Therefore, we aimed to optimize the dilution buffer and refine the isolation procedure for CBMCs.</div></div><div><h3>Methods</h3><div>We enhanced the CBMC recovery rate from cryopreserved cord blood using an optimized dilution buffer and a modified isolation procedure.</div></div><div><h3>Results</h3><div>We achieved average recovery rates of 42.4 % and 54.3 % for CBMCs and CD34+ cells, respectively. Notably, all reagents used in the isolation procedure were of GMP-grade or pharmaceutical preparations, underscoring the potential clinical benefits of our strategy.</div></div><div><h3>Discussion</h3><div>We devised an optimized protocol suitable for routine research and clinical applications for enhanced recovery of CBMCs from cryopreserved cord blood units using an optimized dilution buffer and a modified isolation procedure.</div></div>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":"534 ","pages":"Article 113762"},"PeriodicalIF":1.6,"publicationDate":"2024-09-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142348168","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Indirect ELISAs with sucrose subcellular fractions of Neospora caninum as antigens for diagnosis of neosporosis in cattle","authors":"Thais Agostinho Martins ,&nbsp;Luiz Daniel de Barros ,&nbsp;Beatriz de Souza Lima Nino ,&nbsp;Juliana Correa Bernardes ,&nbsp;Ana Clécia dos Santos Silva ,&nbsp;Ana Flávia Minutti ,&nbsp;Sergio Tosi Cardim ,&nbsp;Milena Patzer Rose ,&nbsp;Valentina Martinez ,&nbsp;João Luis Garcia","doi":"10.1016/j.jim.2024.113760","DOIUrl":"10.1016/j.jim.2024.113760","url":null,"abstract":"<div><div>Neosporosis is one of the major causes of abortion in cattle, and it is responsible for significant economic losses in those animals. Thus, this study aimed to evaluate indirect ELISA using subcellular fractions of <em>Neospora caninum</em> obtained via sucrose gradient separation. Eighty-five sera from dairy cattle previously tested using indirect immunofluorescence assay (IFA) were used. Three distinct bands were separated at 1.0 M, 1.4 M, 1.6 M, and the pellet at 1.8 M, which were identified as fractions one (F1), two (F2), three (F3), and four (F4), respectively. These fractions showed parasite membranes in the F1, rhoptry and conoids in the F2, mitochondria in the F3, and tachyzoite ghosts remain in F4. Indirect ELISAs for IgM, and IgG were performed. Additionally, sensitivity, specificity, and kappa values were defined considering the IFA as the gold standard. The highest and lowest specificities were observed for F1 (76 %) and F3 (16 %), respectively. F2 and F4 showed the highest sensitivity (93.3 %), kappa agreement (0.46), and Negative Preventive Value (NPV) (73 %) respectively. It was possible to standardize indirect ELISAs using whole soluble antigen and subcellular fractions of <em>N. caninum</em>, and F2 and F4 showed higher sensitivity (93.3 %), kappa (0.41), and NPV values (75 %) than F1, and F3, which could be used for epidemiology studies such as screening.</div></div>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":"534 ","pages":"Article 113760"},"PeriodicalIF":1.6,"publicationDate":"2024-09-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142348169","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}