Pub Date : 2024-10-17DOI: 10.1016/j.jim.2024.113766
Robert Movérare , Peter Lind , Åsa Marknell DeWitt
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.
{"title":"The mouse monoclonal antibody 4E3 is specific for the G1m17 allotype of human IgG1","authors":"Robert Movérare , Peter Lind , 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}
Pub Date : 2024-10-13DOI: 10.1016/j.jim.2024.113764
Wesley Wu , Sasha Gupta , Sharon A. Sagan , Carson E. Moseley , Scott S. Zamvil , John E. Pak
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 E. coli strain engineered to facilitate disulfide bond formation in the cytoplasm. SHuffle cells can produce a soluble fraction of rhMOG yielding >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 in vitro 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.
实验性自身免疫性脑脊髓炎（EAE）是中枢神经系统（CNS）自身免疫性脱髓鞘疾病（如多发性硬化症（MS）和 MOG 抗体相关疾病（MOGAD））的一种模型。重组人 MOG（rhMOG）细胞外结构域含有致病抗体和 T 细胞表位，用它进行免疫可诱导 B 细胞依赖性 EAE，用于小鼠研究。然而，由于 rhMOG 在细菌细胞中过度表达时不溶解，而且需要低效变性和重折叠，这些研究一直受到 rhMOG 可用性的阻碍。在这里，我们介绍了一种在 SHuffle 细胞中高产生产可溶性 rhMOG 的新方案。SHuffle 细胞可生产可溶性 rhMOG 部分，产量大于 100 毫克/升。分析纯化的 rhMOG 的尺寸排阻色谱多角度光散射（SEC-MALS）和差示扫描荧光测定法发现，其单体均匀，熔点高，表明蛋白质折叠良好。体外增殖试验证明，纯化的 rhMOG 可被表达特异性 T 细胞受体（TCR）的 T 细胞处理和识别，而 T 细胞受体是免疫显性 MOG35-55 多肽表位的特异性 T 细胞受体。最后，用 rhMOG 免疫野生型小鼠（而非 B 细胞缺陷型小鼠）可强效诱导 EAE，这表明诱导作用依赖于 B 细胞。我们的SHuffle细胞法结合了细菌细胞表达的高产率和速度以及增强的二硫键形成和折叠，大大简化了rhMOG的生产，这将有助于进一步研究B细胞依赖性EAE，并拓展人类对中枢神经系统脱髓鞘疾病中MOG的研究。
{"title":"High-yield production of recombinant human myelin oligodendrocyte glycoprotein in SHuffle bacteria without a refolding step","authors":"Wesley Wu , Sasha Gupta , Sharon A. Sagan , Carson E. Moseley , Scott S. Zamvil , 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 >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 MOG<sub>35</sub><sub>–</sub><sub>55</sub> 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}
Pub Date : 2024-10-13DOI: 10.1016/j.jim.2024.113765
Angie Tatiana Murillo Casas , Paula Andrea Castro Martinez , Fernando Borda Rojas , Luz Angela Vega , Anna Cláudia Alves de Sousa , Juliana Lopes Rangel Fietto , Natalie Hell-Mor , Gabriel Andres Tafur-Gómez
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 Leishmania 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(p < 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 (p < 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.
{"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 , Paula Andrea Castro Martinez , Fernando Borda Rojas , Luz Angela Vega , Anna Cláudia Alves de Sousa , Juliana Lopes Rangel Fietto , Natalie Hell-Mor , 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> < 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> < 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}
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.
{"title":"Development of sandwich enzyme-linked immunosorbent assays quantifying mouse urinary megalin, a novel proximal tubular biomarker","authors":"Rina Sofuku , Sayaka Miyazaki , Michihiro Hosojima , Sawako Goto , Kazuya Takemoto , Hideyuki Kabasawa , Taeko Endo , Koichi Komochi , Nanako Sugita , Hiroyuki Aoki , Ryota Kobayashi , Ichiei Narita , 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}
Pub Date : 2024-09-29DOI: 10.1016/j.jim.2024.113758
Marta Valente Pinto , Alex-Mikael Barkoff , Sagida Bibi , Aapo Knuutila , Johanna Teräsjärvi , Elizabeth Clutterbuck , Sophie Gimenez-Fourage , Anke Pagnon , Jacqueline A.M. van Gaans-van den Brink , Veronique Corbiere , Aymeric De Montfort , Anja Saso , Haddijatou Jobe , Sophie Roetynck , Beate Kampmann , Elles Simonetti , Dimitri Diavatopoulos , Eleonora E. Lambert , Jussi Mertsola , Pascal Blanc , Ronald de Groot
Background
Bordetella pertussis 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.
Material and methods
Longitudinal WB samples were collected from a small set of subjects (n = 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 B. pertussis 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.
Results
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.
Conclusions
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.
{"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 , Alex-Mikael Barkoff , Sagida Bibi , Aapo Knuutila , Johanna Teräsjärvi , Elizabeth Clutterbuck , Sophie Gimenez-Fourage , Anke Pagnon , Jacqueline A.M. van Gaans-van den Brink , Veronique Corbiere , Aymeric De Montfort , Anja Saso , Haddijatou Jobe , Sophie Roetynck , Beate Kampmann , Elles Simonetti , Dimitri Diavatopoulos , Eleonora E. Lambert , Jussi Mertsola , Pascal Blanc , 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}
Pub Date : 2024-09-27DOI: 10.1016/j.jim.2024.113762
Guangzhao Li , Yunyan Zhao , Rongzhi Liu , Yabin Zhang , Yong Zhang , Wei Du , Yu Zhang
Background aims
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.
Methods
We enhanced the CBMC recovery rate from cryopreserved cord blood using an optimized dilution buffer and a modified isolation procedure.
Results
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.
Discussion
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.
{"title":"Highly effective strategy for isolation of mononuclear cells from frozen cord blood","authors":"Guangzhao Li , Yunyan Zhao , Rongzhi Liu , Yabin Zhang , Yong Zhang , Wei Du , 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}
Pub Date : 2024-09-26DOI: 10.1016/j.jim.2024.113760
Thais Agostinho Martins , Luiz Daniel de Barros , Beatriz de Souza Lima Nino , Juliana Correa Bernardes , Ana Clécia dos Santos Silva , Ana Flávia Minutti , Sergio Tosi Cardim , Milena Patzer Rose , Valentina Martinez , João Luis Garcia
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 Neospora caninum 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 N. caninum, 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.
新孢子虫病是导致牛流产的主要原因之一，给这些动物造成了巨大的经济损失。因此，本研究旨在评估使用通过蔗糖梯度分离获得的犬新孢子虫亚细胞分馏物进行间接 ELISA 检测的效果。研究使用了 85 份奶牛血清，这些血清之前曾使用间接免疫荧光测定法（IFA）进行过检测。在 1.0 M、1.4 M、1.6 M 和 1.8 M 下分离出三个不同的条带，分别称为馏分一（F1）、馏分二（F2）、馏分三（F3）和馏分四（F4）。这些馏分在 F1 中显示寄生虫膜，在 F2 中显示跳虫和锥体，在 F3 中显示线粒体，在 F4 中显示速虫幽灵。对 IgM 和 IgG 进行了间接 ELISA 检测。此外，将 IFA 作为金标准，确定了灵敏度、特异性和卡帕值。F1（76%）和F3（16%）的特异性分别最高和最低。F2 和 F4 分别显示出最高的灵敏度（93.3%）、卡帕一致性（0.46）和阴性预防值（73%）。与 F1 和 F3 相比，F2 和 F4 的灵敏度（93.3%）、卡帕（0.41）和 NPV 值（75%）都更高，可用于流行病学研究，如筛查。
{"title":"Indirect ELISAs with sucrose subcellular fractions of Neospora caninum as antigens for diagnosis of neosporosis in cattle","authors":"Thais Agostinho Martins , Luiz Daniel de Barros , Beatriz de Souza Lima Nino , Juliana Correa Bernardes , Ana Clécia dos Santos Silva , Ana Flávia Minutti , Sergio Tosi Cardim , Milena Patzer Rose , Valentina Martinez , 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}
Pub Date : 2024-09-24DOI: 10.1016/j.jim.2024.113761
Filomeno Coelho da Silva , Gathoni Kamuyu , Birgitta Michels , Jessica Edney , Laura Hassall , Paul Stickings , Sunil Maharjan , Tim Waterboer , Simon Beddows
Chlamydia trachomatis (Ct) serology is an important tool for monitoring infection and disease burden but there are currently no formal reference reagents to harmonize results reporting. Our objective was to develop a panel of candidate reference reagents with reactivity against the major outer membrane protein (MOMP) and virulence factor (pgp3) antigens. Plasma packs from females (20–40 years old) were screened against MOMP and pgp3 antigens and selected positive and negative samples pooled to create a panel of candidate antibody reference reagents that were tested in two laboratories. Antigen specificity and internal quality assurance were also evaluated. Suitable candidate materials have been selected to produce Ct reference reagents.
{"title":"Candidate antibody reference reagents for Chlamydia trachomatis serology","authors":"Filomeno Coelho da Silva , Gathoni Kamuyu , Birgitta Michels , Jessica Edney , Laura Hassall , Paul Stickings , Sunil Maharjan , Tim Waterboer , Simon Beddows","doi":"10.1016/j.jim.2024.113761","DOIUrl":"10.1016/j.jim.2024.113761","url":null,"abstract":"<div><div><em>Chlamydia trachomatis</em> (Ct) serology is an important tool for monitoring infection and disease burden but there are currently no formal reference reagents to harmonize results reporting. Our objective was to develop a panel of candidate reference reagents with reactivity against the major outer membrane protein (MOMP) and virulence factor (pgp3) antigens. Plasma packs from females (20–40 years old) were screened against MOMP and pgp3 antigens and selected positive and negative samples pooled to create a panel of candidate antibody reference reagents that were tested in two laboratories. Antigen specificity and internal quality assurance were also evaluated. Suitable candidate materials have been selected to produce Ct reference reagents.</div></div>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":"534 ","pages":"Article 113761"},"PeriodicalIF":1.6,"publicationDate":"2024-09-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142323085","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}
Pub Date : 2024-09-24DOI: 10.1016/j.jim.2024.113759
Mary T. Angani , Jonathan P. Owen , Ben C. Maddison , Kevin C. Gough
Eimeria is one of the most economically important pathogens in poultry production. Diagnosis of infection has the potential to inform treatment and prevention strategies. Here, phage display technology was used to isolate single chain antibodies (scFvs) that had a broad specificity against oocysts from the seven pathogenic species of Eimeria found in poultry. Three such scFvs, representing 2 scFv HCDR3 motifs, were isolated by random picks of clones isolated after five rounds of iterative enrichment (panning) of phage against the seven Eimeria species. Phage-antibody binding to Eimeria oocysts was also interrogated using next generation sequencing of the HCDR3 region of scFv genes contained with phage particles. This analysis demonstrated that the most abundant scFv found after 5 rounds of panning accounted for over >90 % of scFvs. Furthermore, the three scFvs isolated from random picks of clones were the only antibodies that were enriched through each round of panning. They were also seen to be enriched through the stages of phage panning that included binding to the Eimeria oocysts (selection phase) and to be selected against during the stages that consisted solely of phage propagation (growth only phase). The NGS data was further analysed to identify an additional scFv that demonstrated specific enrichment against 3 Eimeria species at the third round of panning and had the same pattern of enrichment during the selection and growth phases of panning. Rescue and analysis of this phage-scFv demonstrated a binder with broad specificity for Eimeria species. The four antibodies with broad specificity detected all seven Eimeria species in immunoassays. The binding of one such scFv that recognised all species was further validated by fluorescent microscopy.
{"title":"Isolation of phage-antibodies against Eimeria species that infect chickens","authors":"Mary T. Angani , Jonathan P. Owen , Ben C. Maddison , Kevin C. Gough","doi":"10.1016/j.jim.2024.113759","DOIUrl":"10.1016/j.jim.2024.113759","url":null,"abstract":"<div><div><em>Eimeria</em> is one of the most economically important pathogens in poultry production. Diagnosis of infection has the potential to inform treatment and prevention strategies. Here, phage display technology was used to isolate single chain antibodies (scFvs) that had a broad specificity against oocysts from the seven pathogenic species of <em>Eimeria</em> found in poultry. Three such scFvs, representing 2 scFv HCDR3 motifs, were isolated by random picks of clones isolated after five rounds of iterative enrichment (panning) of phage against the seven <em>Eimeria</em> species. Phage-antibody binding to <em>Eimeria</em> oocysts was also interrogated using next generation sequencing of the HCDR3 region of scFv genes contained with phage particles. This analysis demonstrated that the most abundant scFv found after 5 rounds of panning accounted for over >90 % of scFvs. Furthermore, the three scFvs isolated from random picks of clones were the only antibodies that were enriched through each round of panning. They were also seen to be enriched through the stages of phage panning that included binding to the <em>Eimeria</em> oocysts (selection phase) and to be selected against during the stages that consisted solely of phage propagation (growth only phase). The NGS data was further analysed to identify an additional scFv that demonstrated specific enrichment against 3 <em>Eimeria</em> species at the third round of panning and had the same pattern of enrichment during the selection and growth phases of panning. Rescue and analysis of this phage-scFv demonstrated a binder with broad specificity for Eimeria species. The four antibodies with broad specificity detected all seven <em>Eimeria</em> species in immunoassays. The binding of one such scFv that recognised all species was further validated by fluorescent microscopy.</div></div>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":"534 ","pages":"Article 113759"},"PeriodicalIF":1.6,"publicationDate":"2024-09-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142348170","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}
Pub Date : 2024-09-13DOI: 10.1016/j.jim.2024.113756
Alfredo Toraño, Inmaculada Moreno, José Antonio Infantes, Mercedes Domínguez
We present a time-course saturation ELISA for measuring the equilibrium constant of the monoclonal antibody (mAb) SIM 28 against horse radish peroxidase (HRP). The curves of HRP binding to a series of fixed mAb dilutions were plotted to completion, and the Kt (= Ks) value (time to occupy 50 % of the mAb paratopes) was determined for each mAb dilution and HRP concentration. Analysis of the kinetic mechanism of the reaction by Lineweaver-Burk and Hanes plots showed that the slope and y-intercept were affected, indicating that mAb ligand saturation follows non-competitive inhibition kinetics in this assay format. In this kinetics, the inhibition constant Ki (= Kd) is the time required to double the slope or halve the Vmax of the Lineweaver-Burk plot. The Kt values of the time courses were doubled (2 x Kt) and normalized by dividing by the total reaction time to obtain a unitless factor which, when multiplied by the concentration of HRP, gives the Ki. The affinity constant of mAb SIM 28 was determined from ELISA data (n = 16) by three methods: i) doubling of Kt, ii) Beatty equation (Kaff = (n-1)/2 (n [HRP’]t - [HRP]t), and iii) SPR (Biacore) analysis. The calculated affinities (mean ± 95 % confidence limits) were i) 4.6 ± 0.67 × 10−9 M, ii) Kaff = 0.23 ± 0.03 × 109 M−1 (Kd = 4.8 ± 0.81 × 10−9 M), and iii) 4.3 ± 0.57 × 10−9 M, respectively. The similar results obtained with the three different techniques indicate that this time-course saturation ELISA, combined with the double Kt method, is a repeatable and direct approach to mAb affinity determination.
{"title":"Description of a non-competitive ELISA based on time course analysis of ligand binding at saturation, and a direct method for calculating the affinity of monoclonal antibodies","authors":"Alfredo Toraño, Inmaculada Moreno, José Antonio Infantes, Mercedes Domínguez","doi":"10.1016/j.jim.2024.113756","DOIUrl":"10.1016/j.jim.2024.113756","url":null,"abstract":"<div><p>We present a time-course saturation ELISA for measuring the equilibrium constant of the monoclonal antibody (mAb) SIM 28 against horse radish peroxidase (HRP). The curves of HRP binding to a series of fixed mAb dilutions were plotted to completion, and the K<sub>t</sub> (= K<sub>s</sub>) value (time to occupy 50 % of the mAb paratopes) was determined for each mAb dilution and HRP concentration. Analysis of the kinetic mechanism of the reaction by Lineweaver-Burk and Hanes plots showed that the slope and y-intercept were affected, indicating that mAb ligand saturation follows non-competitive inhibition kinetics in this assay format. In this kinetics, the inhibition constant K<sub>i</sub> (= K<sub>d</sub>) is the time required to double the slope or halve the V<sub>max</sub> of the Lineweaver-Burk plot. The K<sub>t</sub> values of the time courses were doubled (2 x K<sub>t</sub>) and normalized by dividing by the total reaction time to obtain a unitless factor which, when multiplied by the concentration of HRP, gives the K<sub>i</sub>. The affinity constant of mAb SIM 28 was determined from ELISA data (<em>n</em> = 16) by three methods: i) doubling of K<sub>t</sub>, ii) Beatty equation (K<sub>aff</sub> = (n-1)/2 (n [HRP’]<sub>t</sub> - [HRP]<sub>t</sub>), and iii) SPR (Biacore) analysis. The calculated affinities (mean ± 95 % confidence limits) were i) 4.6 ± 0.67 × 10<sup>−9</sup> M, ii) K<sub>aff</sub> = 0.23 ± 0.03 × 10<sup>9</sup> M<sup>−1</sup> (K<sub>d</sub> = 4.8 ± 0.81 × 10<sup>−9</sup> M), and iii) 4.3 ± 0.57 × 10<sup>−9</sup> M, respectively. The similar results obtained with the three different techniques indicate that this time-course saturation ELISA, combined with the double K<sub>t</sub> method, is a repeatable and direct approach to mAb affinity determination.</p></div>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":"534 ","pages":"Article 113756"},"PeriodicalIF":1.6,"publicationDate":"2024-09-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142229820","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}
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