Monoclonal Antibody Technology Research Articles

Overcoming challenges in the diagnosis of Schistosoma mansoni infections using POC tests, recombinant protein and monoclonal antibody technology

Control constraints of Schistosomiasis include the lack of diagnostic methods with high sensitivity. We develop a prospective study in southeast Brazil to standardize new sensitive and rapid diagnostic methods for Schistosoma mansoni infection. Currently, we are investigating 6 endemic areas (>1000 individuals with chronic infection) and 84 travelers infected in a fresh water pool (with acute infection). Sera, urine, feces and saliva samples were used for the standardization/validation of innovative methods, including acute, chronic and post‐treatment patients. Comparisons are performed with eggs in feces by 24 Kato‐Katz slides and 2 analysis of Saline Gardient and clinical symptoms. With our new methods using a selected recombinant protein called CCAr, we were able to detect the disease early as 10 days post‐infection and more than 95% of positive cases from chronic and low endemicity areas were obtained. Plus, POC‐CCA® test was improved with a urine concentration step that turned its sensibility from 6% to 56%. Monoclonal antibody and recombinant protein technologies allowed a superior detection method when comparing it to the conventional methods. In conclusion, data showed 100% of sensitivity of chronic patients and 98% of acute patients.Support or Funding InformationFapemig, CNPq, FIOCRUZ, PDTIS (Brazil). Fulbright, NIH, University of Georgia (USA). Schistosomiasis diagnosis data of the individuals from Estreito de Miralta. Brazil, based on the POC‐CCA results and epg obtained by parasitological methods. POC‐CCA (1–2 cassettes) Kato‐Katz Saline Gradient (1 g of feces) (2 slides) (24 slides, 1 g of feces) 49 negative 0 epg 46 42 43 ≤10 epg 3 5 4 11–30 epg 0 0 1 31–60 epg 0 2 1 61–60 epg 0 0 0 33 trace 0 epg 24 17 16 ≥ <10 epg 9 12 15 11–30 epg 0 1 1 31–60 epg 0 2 1 61–80 epg 0 1 0 2 positive 0 epg 0 0 0 <10 epg 2 1 1 11–30 epg 0 0 1 31–60 epg 0 1 0 61–80 epg 0 0 0 Total 84 84 84 84 doi: 10.1371/journal.pntd.0004778.t001 Schistosomiasis positivity based on POC‐CCA, Kato‐Katz and Saline Gradient among 84 individuals, Brazil. Prevalence (%) POC‐CCA Before lyophilization 2 After lyophilization 32 Kato‐Katz 30 Saline Gradient 30 doi: 10.1371/journal.pntd.0004778.t002 Sensitivity and specificity obtained by POC‐CCA evaluated against a combined reference techniques of 24 Kato‐Katz slides and Saline Gradient (1 g of feces) before and after lyophilization. Sensitivity (%), 95% CI Specificity (%), 95% CI Before lyophilization 6 100 After lyophilization 56 83 doi: 10.1371/journal.pntd.0004778.t003 POC‐CCA performance on schistosomiasis diagnosis before and after lyophilization against parasitological methods. POC‐CCA (1–2 cassettes) POC‐CCA after lyophilisation (1–2 cassettes) Kato‐Katz/Saline Gradient Negative 49 32 52 Positive 2 27 32 Trace 33 25 ‐ Total 84 84 84 doi. 10.1371/journal.pntd.0004778.t004 Descriptive changes on the POC‐CCA results before and after lyophilization process and comparison against parasitological results. N Description Negatives to traces 13 10 patients had no eggs in stool, although 1 presented hookworms eggs. The other 3 patients had 3, 8 and 52 epg of feces Negatives to positives 11 4 patients with 1, 2, 3 and 55 epg and 7 individuals with no eggs in stool Traces to positives 15 13 patients showed 1–55 epg while 2 individuals had no eggs in stool, but both presented hookworms eggs doi: 10.1371/journal.pntd.0004778.t005 Cross reactivity analysis of duplicate POC‐CCA before and after lyophilization of urine samples detected by the combined reference parasitological diagnosis for individuals negative for schistosome eggs. Results POC‐CCA Before lyophilization After lyophilization Hookworms Individual 1 Negative Negative Individual 2 Negative Negative Individual 3 Trace Trace Individual 4 Trace Trace Individual 5 Trace Trace Individual 6 Negative Trace Individual 7 Trace Positive (1+) Hymenolepis nana Individual 1 Negative Negative Individual 2 Trace Trace Individual 3 Trace Trace Enterobius vermicularis Individual 1 Trace Trace Individual 2 Trace Trace Individual 3 Trace Trace Individual 4 Trace Negative Ascaris lumbricoides Individual 1 Trace Trace doi: 10.1371/journal.pntd.0004778.t006

B Cell-Based Seamless Engineering of Antibody Fc Domains.

Engineering of monoclonal antibodies (mAbs) enables us to obtain mAbs with additional functions. In particular, modifications in antibody’s Fc (fragment, crystallizable) region can provide multiple benefits such as added toxicity by drug conjugation, higher affinity to Fc receptors on immunocytes, or the addition of functional modules. However, the generation of recombinant antibodies requires multiple laborious bioengineering steps. We previously developed a technology that enables rapid in vitro screening and isolation of specific mAb-expressing cells from the libraries constructed with chicken B-cell line DT40 (referred to as the ‘ADLib system’). To upgrade this ADLib system with the ability to generate customized mAbs, we developed a novel and rapid engineering technology that enables seamless exchanges of mAbs’ Fc domains after initial selections of mAb-producing clones by the ADLib system, using a gene-replacement unit for recombinase-mediated cassette exchange (RMCE). In this system, Cre-recombinase recognition sites were inserted into the Fc region of the active DT40 IgM allele, allowing the replacement of the Fc domain by the sequences of interest upon co-transfection of a Cre recombinase and a donor DNA, enabling the rapid exchange of Fc regions. Combining this method with the ADLib system, we demonstrate rapid Fc engineering to generate fluorescent antibodies and to enhance affinity to Fc receptors.

Preparation and Biological Activity of the Monoclonal Antibody against the Second Extracellular Loop of the Angiotensin II Type 1 Receptor.

The current study was to prepare a mouse-derived antibody against the angiotensin II type 1 receptor (AT1-mAb) based on monoclonal antibody technology, to provide a foundation for research on AT1-AA-positive diseases. Balb/C mice were actively immunized with the second extracellular loop of the angiotensin II type 1 receptor (AT1R-ECII). Then, mouse spleen lymphocytes were fused with myeloma cells and monoclonal hybridomas that secreted AT1-mAb were generated and cultured, after which those in logarithmic-phase were injected into the abdominal cavity of mice to retrieve the ascites. Highly purified AT1-mAb was isolated from mouse ascites after injection with 1 × 107 hybridomas. A greater amount of AT1-mAb was purified from mouse ascites compared to the cell supernatant of hybridomas. AT1-mAb purified from mouse ascites constricted the thoracic aorta of mice and increased the beat frequency of neonatal rat myocardial cells via the AT1R, identical to the effects of AT1-AA extracted from patients' sera. Murine blood pressure increased after intravenous injection of AT1-mAb via the tail vein. High purity and good biological activity of AT1-mAb can be obtained from mouse ascites after intraperitoneal injection of monoclonal hybridomas that secrete AT1-mAb. These data provide a simple tool for studying AT1-AA-positive diseases.

Progress in preparation of small monoclonal antibodies of knock out technique

With the application of monoclonal antibody technology more and more widely, its production technology is becoming more and more perfect. Small molecule monoclonal antibody technology is becoming a hot research topic for people. The application of traditional Chinese medicine small molecule monoclonal antibody technology has been more and more widely, the technology for effective Chinese medicine component knockout provide strong technical support. The preparation of monoclonal antibodies and small molecule knockout technology are reviewed in this paper. The preparation of several steps, such as: in the process of preparation of antigen, hapten carrier coupling, coupling ratio determination and identification of artificial antigen and establishment of animal immunization and hybridoma cell lines of monoclonal antibody, the large-scale preparation; small molecule monoclonal antibody on Immune in affinity chromatography column method is discussed in detail. The author believes that this technology will make the traditional Chinese medicine research on a higher level, and improve the level of internationalization of Chinese medicine research.

New and Emerging Agents for the Treatment of Hemophilia: Focus on Extended Half-Life Recombinant Clotting Proteins.

Hemophilia A and B are X-linked disorders caused by deficient or defective clotting factor VIII (FVIII) or IX factor (FIX) proteins, and characterized by spontaneous or traumatic bleeding into joints and muscles. Previous use of plasma and plasma-derived clotting factors that lacked appropriate viral inactivation steps in manufacturing led to significant morbidity associated with transfusion-transmitted HIV and hepatitis C virus (HCV). The development of recombinant proteins revolutionized their treatment, and, with no new HIV or HCV infection via clotting proteins for nearly 30 years, greatly improved their lifespan, which now approaches that of the general population, and with the same risks for aging complications. Novel long-acting factor proteins are being licensed to extend FVIII and FIX half-life, thereby reducing infusion frequency and potentially bleed frequency and associated morbidity. Further, novel therapeutics which take advantage of new technologies, including siRNA, monoclonal antibody, and small peptide inhibition technologies, have the potential to simplify treatment and improve outcomes for those with inhibitors.

Abstract 656: Discovery of MAbs against difficult GPCRs, ion channels, and transporters using the MPS Discovery Engine®

Abstract Integral membrane proteins are important drug targets and monoclonal antibodies (MAbs) directed against them are highly sought for therapeutic purposes. However, the complex structure of membrane protein targets makes the discovery of these MAbs especially challenging. To address this need, we have developed the MPS Discovery Engine® to enable the isolation, characterization, and engineering of monoclonal antibodies for challenging membrane protein targets, including GPCRs, ion channels, and transporters. MPS harnesses the strength of Integral's 10+ years of expertise in membrane protein expression and analysis, as well as proprietary technologies including Lipoparticles for high concentration membrane protein presentation and Shotgun Mutagenesis for comprehensive epitope mapping and MAb optimization. Integral Molecular currently has a pipeline of several dozen MAbs against complex membrane proteins that are involved in a variety of diseases, including cancer, inflammation, pain, and infectious diseases. There are currently no FDA-approved MAbs that target GPCRs, ion channels, or transporters, and MPS provides a pathway to these new and previously intractable targets. Citation Format: Sharon H. Willis, Kimberly Mattia, Riley Payne, Moniquetta Hall, Manu Mabila, Christine Rettew, Joseph Couto, Rohan Keshwara, Cheryl Paes, Benjamin J. Doranz, Joseph Rucker. Discovery of MAbs against difficult GPCRs, ion channels, and transporters using the MPS Discovery Engine®. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 656. doi:10.1158/1538-7445.AM2015-656

A Diverse Repertoire of Human Immunoglobulin Variable Genes in a Chicken B Cell Line is Generated by Both Gene Conversion and Somatic Hypermutation.

Chicken immune responses to human proteins are often more robust than rodent responses because of the phylogenetic relationship between the different species. For discovery of a diverse panel of unique therapeutic antibody candidates, chickens therefore represent an attractive host for human-derived targets. Recent advances in monoclonal antibody technology, specifically new methods for the molecular cloning of antibody genes directly from primary B cells, has ushered in a new era of generating monoclonal antibodies from non-traditional host animals that were previously inaccessible through hybridoma technology. However, such monoclonals still require post-discovery humanization in order to be developed as therapeutics. To obviate the need for humanization, a modified strain of chickens could be engineered to express a human-sequence immunoglobulin variable region repertoire. Here, human variable genes introduced into the chicken immunoglobulin loci through gene targeting were evaluated for their ability to be recognized and diversified by the native chicken recombination machinery that is present in the B-lineage cell line DT40. After expansion in culture the DT40 population accumulated genetic mutants that were detected via deep sequencing. Bioinformatic analysis revealed that the human targeted constructs are performing as expected in the cell culture system, and provide a measure of confidence that they will be functional in transgenic animals.

Lateral Flow Immunoassay.

Lateral flow immunoassays (LFIAs) are a staple in the field of rapid diagnostics. These small handheld devices require no specialized training or equipment to operate, and generate a result within minutes of sample application. They are an ideal format for many types of home test kits, for emergency responders and for food manufacturers and producers looking for a quick evaluation of a given sample. LFIAs rely on high quality monoclonal antibodies that recognize the analyte of interest. As monoclonal antibody technology becomes more accessible to smaller laboratories, there has been increased interest in developing LFIA prototypes for potential commercial manufacture. In this chapter, the basics of designing and building an LFIA prototype are described.

Imaging and Mapping of Tissue Constituents at the Single-Cell Level Using MALDI MSI and Quantitative Laser Scanning Cytometry.

For nearly a century, histopathology involved the laborious morphological analyses of tissues stained with broad-spectrum dyes (i.e., eosin to label proteins). With the advent of antibody-labeling, immunostaining (fluorescein and rhodamine for fluorescent labeling) and immunohistochemistry (DAB and hematoxylin), it became possible to identify specific immunological targets in cells and tissue preparations. Technical advances, including the development of monoclonal antibody technology, led to an ever-increasing palate of dyes, both fluorescent and chromatic. This provides an incredibly rich menu of molecular entities that can be visualized and quantified in cells-giving rise to the new discipline of Molecular Pathology. We describe the evolution of two analytical techniques, cytometry and mass spectrometry, which complement histopathological visual analysis by providing automated, cellular-resolution constituent maps. For the first time, laser scanning cytometry (LSC) and matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) are combined for the analysis of tissue sections. The utility of the marriage of these techniques is demonstrated by analyzing mouse brains with neuron-specific, genetically encoded, fluorescent proteins. We present a workflow that: (1) can be used with or without expensive matrix deposition methods, (2) uses LSC images to reveal the diverse landscape of neural tissue as well as the matrix, and (3) uses a tissue fixation method compatible with a DNA stain. The proposed workflow can be adapted for a variety of sample preparation and matrix deposition methods.

Quantitative Assay of Immunoglobulin Free Light Chains (FLC): Evaluation of Monoclonal Versus Polyclonal Antibody and Immunoturbidimetric Versus Immunonephelometric Detection Technology

Introduction. It has been rarely reported that a laboratory test introduced so rapid and radical changes in diagnostic algorithm as is the case with the quantitative assay of free light chains of immunoglobulins (FLC) in serum and its role in the diagnostic algorithm of monoclonal gammopathies. Since the first description of immunoassay in year 2001 until today, new evidence has continuously been reported in the literature that confirm the clinical usefulness of this test in diagnosis, monitoring and prognosis of monoclonal plasma-proliferative diseases, especially diseases of light chains such as primary amyloidosis, light chain deposition disease (LCDD) or light chain multiple myeloma (LCMM) and nonsecretory multiple myeloma (NSMM). Recently, a commercial test has become available on the market that uses polyclonal antibodies to specific epitopes of free light chains which are hidden in the intact immunoglobulin molecules. In 2011, a commercial immunoassay was launched on the market that uses monoclonal instead of polyclonal antibodies, reducing the variability between different series of reagents and controlling excess antigen in the sample.Aim. The aim of this study was to evaluate monoclonal versus polyclonal antibody and immunoturbidimetric versus immunonephelometric detection technology. Does different detection tehnology – besides different used antibody – contribute to greater variability in results?Method. In this study we compared results of 40 samples measured with polyclonal antibody (The Binding Site Ltd., Birmingham, UK) and monoclonal antibody (N Latex FLC, Siemens Healthcare Diagnostics, Marburg, Germany). In other 40 samples we compared results achieved with different antibodies and different analytical platforms (Siemens Nephelometer with Roche Cobas). Results were statistically analyzed using MedCalc software.Results. Results are shown in Table 1. Comparing all results, it is evident that there is at least proportional error when comparing different antibodies and different analytical systems. Although it is known that immunoturbidimetry is less sensitive than immunonephelometric method, greater discrepancies in results were not found. When we categorized patients as positive and negative according to manufacturer’s reference interval for kf/lf ratio, agreement between groups with different antibody and same detection technology was 63% (weighted kappa 0.30). Agreement between groups with different antibodies and different detection technology was 86% (weighted kappa 0.22). Although we have not measured the same samples when testing antibody and analytical platform, the selected analytical platform has, according to our results, no additional impact on the variability of results.Abstract 5680. Table 1Comparison of FLC results using different antibody and different analytical platformsMethodFLC kappa polyclonal Ab Nephelometer SiemensFLC kappa monoclonal Ab Nephelometer SiemensFLC lambda polyclonal Ab Nephelometer SiemensFLC lambda monoclonal Ab Nephelometer SiemensResults (min-max)6.59-5210.006.34-1600.001.67-3010.001.00-1600.00Passing-Bablok fitintercept (95% Cl)8.2442.9255 to 14.92491.0945-1.5910 to 5.5631slope (95% Cl)0.5950.4564 to 0.78521.87981.5336 to 2.1045Correlation rs (p<0.0001)0.9110.887MethodFLC kappa polyclonal Ab Cobas RocheFLC kappa monoclonal Ab Nephelometer SiemensFLC lambda polyclonal Ab Cobas RocheFLC lambda monoclonal Ab Nephelometer SiemensResults (min-max)2.40-453.201.10-342.002.30-452.1010.30-301.20Passing-Bablok fitintercept (95% Cl)2.622-3.8207 to 9.30230.1585-3.9731 to 4.5892slope (95% Cl)1.51.2972 to 1.99330.74160.6174 to 0.8708Correlation rs (p<0.0001)0.8730.929Conclusion. Physicians and especially clinical biochemists must be aware of the technical shortcomings of this test, such as the variability between different series (lots) reagents, non-linearity, unreliable detection of excess antigen and overestimation of FLC concentrations due to nonspecific interference or polymerization.Although initial results are not discouraging, it will be necessary to collect much more evidence, especially bearing in mind that use of monoclonal antibodies along with advantages has certain disadvantages. In the future, it will probably be necessary to incorporate into the guidelines a recommendation to report the method used, like for other tumor markers. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Antibody-secreting cell specificity in labial salivary glands reflects the clinical presentation and serology in patients with Sjögren's syndrome.

The serologic hallmark of primary Sjögren's syndrome (SS) is the presence of IgG antibodies specific for Ro (SSA) and La (SSB). The molecular characteristics of gland-derived B cells at the site of primary SS inflammation have been described previously; however, parallels between glandular antibody-secreting cells (ASCs) and serologic antibody specificities have not been evaluated. We used recombinant monoclonal antibody (mAb) technology to study the specificities of salivary gland (SG)-derived ASCs, evaluate their molecular characteristics, and identify IgG antibody specificity. Human antibodies were generated from glandular IgG ASCs. Heavy chain and light chain use and immunoglobulin subclass were analyzed by sequencing. Enzyme-linked immunosorbent assay, indirect immunofluorescence, enzyme immunoassay, and (35) S-labeled protein immunoprecipitation analysis were used to determine antibody specificity. Evaluation of single ASCs in SG biopsy specimens from a patient with primary SS and a patient with SS and overlapping systemic lupus erythematosus revealed significant concordance between serum autoantibody and glandular ASC specificities. Gland-derived ASC heavy chains and light chains were extensively somatically hypermutated, which is indicative of antigen-driven responses. Specifically, we produced the first fully human mAb derived from SGs. In patients with SS, the SGs are a site for the production of antibodies that extend beyond the canonical Ro and/or La SS specificities. Glandular antibody production strongly reflected the serologic humoral response in the 2 patients whom we studied.

A Short History of the B-Cell-Associated Surface Molecule CD40.

This perspective traces developments using monoclonal antibody technology that led to the discovery of CD40, a receptor that on B cells mediates “T cell help” and on dendritic cells helps to program CD8 T cell responses. I discuss some things that we got right during the path of discovery and some things we missed. Immunotherapies that block or stimulate the CD40 pathway hold great promise for treatment of autoimmune diseases and cancers.

Deciphering the therapeutic stem cell strategies of large and midsize pharmaceutical firms.

The slow adoption of cytotherapeutics remains a vexing hurdle given clinical progress achieved to date with a variety of stem cell lineages. Big and midsize pharmaceutical companies as an asset class still delay large-scale investments in this arena until technological and market risks will have been further reduced. Nonetheless, a handful of stem cell strategic alliance and licensing transactions have already been implemented, indicating that progress is actively monitored, although most of these involve midsize firms. The greatest difficulty is, perhaps, that the regenerative medicine industry is currently only approaching the point of inflexion of the technology development S-curve, as many more clinical trials read out. A path to accelerating technology adoption is to focus on innovation outliers among healthcare actors. These can be identified by analyzing systemic factors (e.g., national science policies and industry fragmentation) and intrinsic factors (corporate culture, e.g., nimble decision-making structures; corporate finance, e.g., opportunity costs and ownership structure; and operations, e.g., portfolio management strategies, threats on existing businesses and patent expirations). Another path is to accelerate the full clinical translation and commercialization of an allogeneic cytotherapeutic product in any indication to demonstrate the disease-modifying potential of the new products for treatment and prophylaxis, ideally for a large unmet medical need such as dry age-related macular degeneration, or for an orphan disease such as biologics-refractory acute graft-versus-host disease. In times of decreased industry average research productivities, regenerative medicine products provide important prospects for creating new franchises with a market potential that could very well mirror that achieved with the technology of monoclonal antibodies.

Robust, quantitative analysis of proteins using peptide immunoreagents, in vitro translation, and an ultrasensitive acoustic resonant sensor.

A major benefit of proteomic and genomic data is the potential for developing thousands of novel diagnostic and analytical tests of cells, tissues, and clinical samples. Monoclonal antibody technologies, phage display and mRNA display, are methods that could be used to generate affinity ligands against each member of the proteome. Increasingly, the challenge is not ligand generation, rather the analysis and affinity rank-ordering of the many ligands generated by these methods. Here, we developed a quantitative method to analyze protein interactions using in vitro translated ligands. In this assay, in vitro translated ligands generate a signal by simultaneously binding to a target immobilized on a magnetic bead and to a sensor surface in a commercial acoustic sensing device. We then normalize the binding of each ligand with its relative translation efficiency in order to rank-order the different ligands. We demonstrate the method with peptides directed against the cancer marker Bcl-xL. Our method has 4- to 10-fold higher sensitivity, using 100-fold less protein and 5-fold less antibody per sample, as compared directly with ELISA. Additionally, all analysis can be conducted in complex mixtures at physiological ionic strength. Lastly, we demonstrate the ability to use peptides as ultrahigh affinity reagents that function in complex matrices, as would be needed in diagnostic applications.

Synthesis of furaltadone metabolite, 3-amino-5-morpholinomethyl-2-oxazolidone (AMOZ) and novel haptens for the development of a sensitive enzyme-linked immunosorbent assay (ELISA)

A sensitive and specific monoclonal enzyme-linked immunosorbent assay (ELISA) for the determination of tissue-bound metabolite 3-amino-5-morpholinomethyl-2-oxazolidone (AMOZ) was developed. Three different haptens of AMOZ were synthesized. The haptens (I, III) were derived from 4-carboxybenzaldehyde and ethyl 4-bromobutyrate, respectively. Hapten II was based on hapten I but a CN bond in the parent structure was reduced. Corresponding immunogens and coating antigens were prepared to improve the assay sensitivity. The hybridomas 4 × 108 secreting antibodies against CPAMOZ were obtained from immunogens I-BSA by monoclonal antibody (mAb) technology. The antibody indicated that: (1) the small molecular AMOZ cannot induce the specific response against AMOZ; (2) the weakened conjugation effect improved the recognition of AMOZ. Assessment of twelve coating antigen/antibody combinations consequently resulted in the development of an indirect competitive enzyme-linked immunosorbent assay (icELISA). The results showed that the sensitivity was highly improved about fifteen-fold for the novel heterologous coating antigen at low hapten density. The IC50 value of the ELISA for CPAMOZ was 0.13 ng mL−1. The cross reactivity values of the assay with NPAMOZ, AMOZ and FTD were 118%, 2.3% and 16.3%. Recoveries from AMOZ fortified animal tissues were in a range of 82.6–108.4% and the CV values were less than 12.5%. The immunoassay was further validated by a LC-MS/MS method and the two methods showed good correlation (r2 = 0.9908). The proposed icELISA could be used for an accurate monitoring of AMOZ in animal tissues.

Prof. Dr. med. Otto Götze 1935–2013

Prof. Dr. med. Otto Götze 1935–2013

Len Herzenberg (1931–2013)

Leonard Arthur Herzenberg was born the 5th November 1931 in New York, USA. He studied Biology and Chemistry at Brooklyn College. Len did his PhD in biochemistry and immunology at the CalTech (Pasadena, CA, USA). When he finished his PhD in 1955 he moved to France for a Postdoc in the Institute Pasteur, where he worked with Jacques Monod. In 1957, he joined Harry Eagle at the National Institutes of Health (NIH), adding pyruvate to the recipe of “Eagle's medium”, and working on somatic cell genetics of cell lines. In 1959, Joshua Lederberg recruited Len to the Department of Genetics of the Stanford University (CA, USA). At that time, when somatic cell genetics were introduced into immunology, Len worked on H-2 and IgH genetics, in particular on IgH allotypes. The lack of methods to isolate rare genetic variants from cell lines was a limitation for his studies. Looking for technologies to solve this bottleneck, Len discovered that a group of engineers headed by Mack Fulwyler at Los Alamos National Laboratories (New Mexico, USA) had developed a particle sorter by combining a Coulter volume sensor with an ink jet printer device. Len asked them to add a detector for fluorescence of the particles, but they rather gave him the construction plans. Len collected the 14 000 $ required to build a prototype at the Stanford instrumentation laboratory. This prototype was presented in 1969 1, improved and in 1972, the instrument was baptized “Fluorescence-activated Cell Sorter”, also known as FACS. Soon, it turned out to be a key instrument for the analysis of the immune system. For the first time ever, FACS allowed the quantitation of expression of a particular protein in individual cells, stained with fluorescent, specific antibodies, and the isolation of individual cells according to their protein expression. Soon several proteins could be detected simultaneously, using antibodies with different fluorescent dyes. It was a breakthrough for cytometry and immunology. And although todays “flow-in-air” FACS machines incorporate all kind of devices not available in the 70's, they still operate according to the original principle. Today, the speed of sorting has increased from 2 000 cells per second to 40 000 cells per second, and the number of fluorescent parameters from 1 to 20. There is hardly no modern scientific concept in immunology that has not benefited from cytometric immunofluorescence and cell sorting. I personally, as a young scientist, was completely dependent on FACS, to isolate somatic variants of immunoglobulin expression for a molecular analysis of somatic hypermutation and antibody class switching 2. And the MACS technology was essentially developed to complement FACS too 3. The FACS technology continues to challenge engineers and immunologists alike and this is Len's heritage 4. At the beginning, two points were essential for the success of the FACS. First, Len succeeded in convincing Becton-Dickinson (BD) to commercialize the FACS technology, though initially, neither he nor BD believed that there was really a huge market for such machines 5. Second, the advent of monoclonal antibodies in 1975 was catapulting immunofluorescence into a new era. Len immediately recognized that, and in 1976 spent a sabbatical with Cesar Milstein at the MRC in Cambridge, UK. He returned then to Stanford with the new monoclonal antibody technology. The triade of immunofluorescence, cytometry and cell sorting became a unique and precious tool that we are still using today. Len himself, together with Lee, his wife of 60 years, and a host of talented young scientists, used the FACS technology from the start, to dissect the immune system. They visualized allelic exclusion, B cells as plasma cell precursors, discovered B1 cells, T-cell imbalances in diseases like HIV and isolated genetic variants of hybridoma cell lines. In short, they contributed essentially to our current understanding of the cellularity of the immune system 5. For his seminal contributions to immunology, Len was awarded with the Novartis Immunology prize and the Kyoto prize. Len's activities were not restricted to science, though. He was severely concerned about and reacting to McCarthyism, eugenics and HIV stigmas, activities he considered to be the responsibility of a scientist 6. Len Herzenberg died on the 27th of October 2013. Immunology has lost a great personality and scientist, an inspiring mentor and a dear friend to many of us!

An Efficient Method for Producing Monoclonal Antibodies against Multi-pass Membrane Proteins

Antibodies have greatly contributed to the development of medical science and pharmacology, because of their high specificity. The cell fusion method has developed monoclonal antibodies (mAb) technology, such that massive amounts of mAb with a uniform structure can be produced. Although mAb have been produced against many proteins so far, the production of mAb against multi-pass transmembrane proteins, such as G-protein coupled receptor (GPCR) and various transporter proteins has been extremely difficult. The complicated structures, poorly extracellular regions, and high hydrophobicity of multiple-transmembrane proteins make it difficult to produce mAb against them. Production of mAb that recognize the extracellular region of living cells is thought to be important in determining the ability of a protein. Based on these findings, we tried to produce mAb against a multi-pass transmembrane transporter using green fluorescent protein (GFP)-fused full-length target proteins as immunogens. Furthermore, the immunizing method has proved to be important in generating functional mAb. We succeeded in producing functional mAb that react against the extracellular region of a 12-pass transmembrane transporter in a living cell. Based on this success, we began to produce mAb against seven-transmembrane GPCR. In this symposium, we report on the results of producing mAb against S1P receptors, a type of GPCR.

ICSH/ICCS practice guidelines special issue

ICSH/ICCS practice guidelines special issue

New method for analyzing pharmacodynamic material basis of traditional Chinese medicines by using specific knockout technology with monoclonal antibodies

Study on pharmacodynamic material basis of traditional Chinese medicines is one of the key issues for the modernization of traditional Chinese medicine. Having introduced the monoclonal antibody technology into the study on pharmacodynamic material basis of traditional Chinese medicines, the author prepared the immunoaffinity chromatography column by using monoclonal antibodies in active components of traditional Chinese medicines, so as to selectively knock out the component from herbs or traditional Chinese medicine compounds, while preserving all of the other components and keeping their amount and ratio unchanged. A comparative study on pharmacokinetics and pharmacodynamics was made to explicitly reveal the correlation between the component and the main purpose of traditional Chinese medicines and compounds. The analysis on pharmacodynamic material basis of traditional Chinese medicines by using specific knockout technology with monoclonal antibodies is a new method for study pharmacodynamic material basis in line with the characteristics of traditional Chinese medicines. Its results can not only help study material basis from a new perspective, but also help find the modern scientific significance in single herb or among compounds of traditional Chinese medicines.