Journal of immunological methods最新文献

{"title":"Workflow improvement and financial gain after integration of high-throughput sample processing system with flow cytometer in a high-volume pathology laboratory: Results from a prospective comparative study using Lean principles","authors":"","doi":"10.1016/j.jim.2024.113767","DOIUrl":"10.1016/j.jim.2024.113767","url":null,"abstract":"<div><h3>Background</h3><div>Highly efficient clinical laboratories are essential for monitoring many human illnesses. Ampath Laboratory Services, the largest pathology lab in South Africa, analyzes large numbers of peripheral blood samples for CD4 levels yearly.</div></div><div><h3>Objective</h3><div>To assess productivity and quality of a newer integrated automated solution, the BD FACSDuet™ Sample Preparation System/BD FACSLyric™ Flow Cytometer using conventional assessment methods and Lean concepts.</div></div><div><h3>Materials and methods</h3><div>This prospective study compared the performance of the BD FACS™ Sample Preparation Assistant [SPA] III and BD FACSCanto™ II Flow Cytometer with the newly introduced integrated system (BD FACSDuet™/BD FACSLyric™). They were validated for accuracy, precision, and external quality assessment. Process mapping and Lean assessment helped identify steps leading to waste. An economic model was developed to characterize workflow and economic impact associated with total daily hands-on time, processing time, and reworks.</div></div><div><h3>Results</h3><div>Strong linear correlation was present between both systems. Precision and accuracy studies revealed that all coefficient of variation (CV)% values were below 20% of allowable limits. External proficiency assessments were within limits. The fully automated workflow of BD FACSDuet™/BD FACSLyric™ permitted better consistency with significantly shorter processing time and batch processing and reduced operator interventions. Lean assessment identified defects with motion, over-processing, waiting, and non-utilized talent. Significant reductions in hands-on and total daily processing time that could increase daily specimen testing efficiency and fewer reworks were associated with the BD FACSDuet™/BD FACSLyric™. Lean improvements translated to significant economic savings associated with operator costs and unnecessary reagent consumption.</div></div><div><h3>Conclusion</h3><div>BD FACSDuet™/BD FACSLyric™ is an accurate, reliable, and cost-effective fully automated system for high-volume flow cytometry labs that perform T-cell enumeration using a single-platform and single-tube approach.</div></div>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":null,"pages":null},"PeriodicalIF":1.6,"publicationDate":"2024-11-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142467508","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":"Epitope profiling of cow's milk allergen-specific antibodies with determining IgE content in epitopes-ALL, a 14-epitopes mixture.","authors":"Yoshihiro Watanabe, Ikuo Okafuji, Satoko Tamai, Natsuko Hosokawa, Takako Ohbayashi, Shigeki Kato, Kiyoaki Ito, Mitsuhiro Kawano, Yusei Ohshima","doi":"10.1016/j.jim.2024.113773","DOIUrl":"https://doi.org/10.1016/j.jim.2024.113773","url":null,"abstract":"<p><p>Allergen-specific antibodies (Abs), IgE, and IgG4 increase during the early phase of oral immunotherapy (OIT) of allergen food in patients; subsequently, IgE levels decrease and specific IgG4 levels increase after successful OIT treatment. The detailed profile of these Abs during OIT remains largely unclear. We developed a diagnostic tool to assess the OIT efficacy and extent of responsiveness based on a profiling method by identifying epitopes recognized by the Ab classes of IgE or IgG4. A peptide microarray followed by microplate analysis using synthetic peptides was used to identify 14 epitopes widely recognized by IgE and/or IgG4 in the serum samples of patients with OIT among the amino acid sequences of five major cow's milk allergens. The set of defined 14 epitopes clarified different epitope profiles of allergen-specific IgE and IgG4 in each patient's serum samples. Moreover, the total signal of Abs recognizing all 14 epitopes was equal to the sum of all individual epitope-specific Abs. It was further observed that the quantitative value of IgE concentrations of 14 epitopes-ALL correlated with the ImmunoCAP IgE value. These findings strongly imply that the quantity of IgE and IgG4 recognizing epitopes-ALL may easily be used to measure allergy severity. To investigate this potential, we developed an immunochromatographic method that can detect IgE and IgG4 levels in patient samples. This study clearly demonstrated the usefulness of the defined 14 epitopes and their mixture, \"epitopes-ALL,\" and that the simple and reliable methods of immunochromatography and microplate analyses demonstrating the epitope profile of allergen-specific Abs are applicable for diagnostic use at multiple disease stages and the OIT-treatment course in patients with cow's milk allergy.</p>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":null,"pages":null},"PeriodicalIF":1.6,"publicationDate":"2024-11-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142568170","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":"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":"","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":null,"pages":null},"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":"A note of caution for using calmodulin antibodies.","authors":"Mads Munk, Martin W Berchtold","doi":"10.1016/j.jim.2024.113772","DOIUrl":"10.1016/j.jim.2024.113772","url":null,"abstract":"<p><p>Calmodulin (CaM) is a ubiquitous intracellular calcium receptor that regulates a plethora of cellular functions through interactions with target proteins. In mammals, an identical Calmodulin protein is expressed by 3 independent genes (CALM1, CALM2, CALM3). Therefore, antibodies generated against either of the three products (CaM1, CaM2, CaM3) of these genes cannot be distinguished, and conclusions based on the supposedly specific CaM antibodies claiming functions of one of the 3 genes may be misleading. In this paper we present 44 articles, using such antibodies for Western blot, ELISA assay, immunohistochemistry or which are based on proteomics and the use of databases with incorrect annotations, all potentially reaching misleading conclusions. This should be taken as a note of caution for researchers working with Calmodulin antibodies and misleading databases.</p>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":null,"pages":null},"PeriodicalIF":1.6,"publicationDate":"2024-10-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142567997","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":"","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":null,"pages":null},"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":"Development of an ELISA for an effective potency determination of recombinant rabies human monoclonal antibody.","authors":"Ambika Divase, Sambhaji Pisal, Manjusha Dake, Rajeev Dhere, Pravin Kumar Dakshinamurthy, Peddireddy Srinivas Reddy, Chandrashekhar Kamat, Digamber Singh Chahar, Jayanta Pal, Neelu Nawani","doi":"10.1016/j.jim.2024.113769","DOIUrl":"10.1016/j.jim.2024.113769","url":null,"abstract":"<p><p>Rapid Fluorescence Focus Inhibition Test (RFFIT) is the most widely used cell-based assay to measure the potency of recombinant human rabies monoclonal antibodies. Nonetheless, RFFIT assay is time-consuming and it requires well-equipped biosafety level 2 facility, virulent live rabies virus cultures, permissive cell lines, and well-trained manpower. Therefore, the development of alternative methods to the RFFIT has been encouraged by the World Health Organization (WHO) expert working groups to overcome these barriers. An In-vitro ELISA test has been developed as an alternative to the RFFIT assay, for quantifying the rabies monoclonal antibody (mAb) potency using inactivated rabies virus vaccine (Rabivax-S). It is based on the specific interaction between the antigen and the antibody, that induces neutralizing antibody response to rabies virus. The ELISA was validated involving accuracy and precision within 20 % coefficient of variance. The validation has been done by 4PL standard curve with linearity r² ˃ 0.98 and LLOQ of 0.3 μg/mL indicating high assay sensitivity. The specificity of the assay was ascertained by challenging with another homologous non-rabies humanized mAb, which does not show binding with the rabies virus. The indirect ELISA developed here, is precise, robust, and accurate to quantitate the potency of rabies monoclonal antibody. It is highly sensitive and has a broad range of detection. It is easy to perform, and it has a short turnaround time (results available in few hours). Furthermore, it is cost effective and can be performed with low-cost resource setting, as there is no requirement of handling the live cells and live virus and also BSL-2 Facility.</p>","PeriodicalId":16000,"journal":{"name":"Journal of immunological methods","volume":null,"pages":null},"PeriodicalIF":1.6,"publicationDate":"2024-10-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142502036","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":"Human monoclonal antibody cloning and expression with overlap extension PCR and short DNA fragments","authors":"","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":null,"pages":null},"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":"","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":null,"pages":null},"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":"","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":null,"pages":null},"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":"","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":null,"pages":null},"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}