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Upon generation of monoclonal antibodies to the T cell antigen receptor/CD3 (TCR/CD3) complex, we isolated mAb MT3, whose reactivity correlates inversely with the production of IFN‐γ by human peripheral blood T lymphocytes. Using eukaryotic expression cloning, we identified the MT3 antigen as myelin‐and‐lymphocyte (MAL) protein. Flow cytometry analysis demonstrates high surface expression of MAL on all naïve CD4+ T cells whereas MAL expression is diminished on central memory‐ and almost lost on effector memory T cells. MAL– T cells proliferate strongly in response to stimulation with CD3/CD28 antibodies, corroborating that MAL+ T cells are naïve and MAL– T cells memory subtypes. Further, resting MAL– T cells harbor a larger pool of Ser59‐ and Tyr394‐ double phosphorylated lymphocyte‐specific kinase (Lck), which is rapidly increased upon in vitro restimulation. Previously, lack of MAL was reported to prevent transport of Lck, the key protein tyrosine kinase of TCR/CD3 signaling to the cell membrane, and to result in strongly impaired human T cell activation. Here, we show that knocking out MAL did not significantly affect Lck membrane localization and immune synapse recruitment, or transcriptional T cell activation. Collectively, our results indicate that loss of MAL is associated with activation‐induced differentiation of human T cells but not with impaired membrane localization of Lck or TCR signaling capacity., Myelin‐and‐lymphocyte protein isoform‐A (MAL‐A) is highly expressed on human naive CD4+ T cells. Activation‐induced proliferation and differentiation is associated with a gradual loss of MAL expression, resulting in highly signalling‐competent effector‐memory cells with enhanced IFN‐γ‐secretion. Knockout of MAL does not affect recruitment of lymphocyte‐specific kinase Lck and TCR downstream signalling.

For decades, the benefits of utilizing hollow fiber bioreactors for continuous cell culture to produce monoclonal antibodies have been widely recognized. However, the suitability of this technology for laboratories or centers with limited resources and expertise seeking to expand their production capacity is uncertain, mainly due to unknown cost-effectiveness. In this study, a hollow fiber bioreactor with a 4.7-mL culture volume was used to culture a hybridoma clone producing immunoglobulin G antibody specific to hemoglobin F (HbF). The antibody reached a maximum concentration of 1.22 g/L and totaled 21 mg over a 44-day culture period. This preliminary production data was used to estimate the cost of consumables required for using the hollow fiber bioreactor to produce 130 mg of monoclonal antibodies, which was found to be THB35.8k (£880). The cost was slightly more expensive than batch cultivation in typical culture dishes, which ranged from THB27.8 to 30.2k (£680 to 740). Despite the advantages in terms of reduced hands-on time, shorter production duration, and highly concentrated products, the primary challenges associated with using hollow fiber bioreactors were the cost and availability of the cartridges.

CD147/Basigin/EMMPRIN is overexpressed in several cancerous tissues and it has been shown to induce matrix metalloproteinases (MMPs) whose expression is associated with cancer metastasis. Thus, targeting CD147 with monoclonal antibodies (mAbs) potentially has therapeutic applications in cancer immunotherapy. Here, we report the use of anti-CD147 mAbs targeting domain 1 of CD147, namely M6-1D4 (IgM), M6-1F3 (IgM), M6-2F9 (IgM) and M6-1E9 (IgG2a), against several human cancer cell lines. Strikingly, IgM but not IgG mAbs against CD147, especially clone M6-1D4, induced acute cellular swelling, and this phenomenon appeared to be specifically found with hepatocellular carcinoma (HCC) cells. Furthermore, molecular investigation upon treating HepG2 cells with M6-1D4 showed unfolded protein response (UPR) activation, autophagosome accumulation, and cell cycle arrest, but without classic apoptosis related features. More interestingly, prolonged M6-1D4 treatment (24 h) resulted in irreversible oncosis leading to necroptosis. Furthermore, treatment with a mixed lineage kinase domain-like psuedokinase (MLKL) inhibitor and partial knockout of MLKL resulted in reduced sensitivity to necroptosis in M6-1D4-treated HepG2 cells. Surprisingly however, the observed necroptotic signaling axis appeared to be non-canonical as it was independent of receptor-interacting serine/threonine-protein kinase (RIPK) phosphorylation. In addition, no cytotoxic effect on human dermal fibroblast (HDF) was observed after incubation with M6-1D4. Taken together, this study provides clues to target CD147 in HCC using mAbs, as well as sheds new light on a novel strategy to kill cancerous cells by the induction of necroptosis.

CD147, a member of the immunoglobulin (Ig) superfamily, is widely expressed in several cell types. CD147 molecules have multiple cellular functions, such as migration, adhesion, invasion, energy metabolism and T cell activation. In particular, recent studies have demonstrated the potential application of CD147 as an effective therapeutic target for cancer, as well as autoimmune and inflammatory diseases. In this study, we elucidated the functional epitopes on CD147 extracellular domains in T cell regulation using specific monoclonal antibodies (mAbs). Upon T cell activation, the anti-CD147 domain 1 mAbs M6-1E9 and M6-1D4 and the anti-CD147 domain 2 mAb MEM-M6/6 significantly reduced surface expression of CD69 and CD25 and T cell proliferation. To investigate whether functional epitopes of CD147 are differentially expressed on distinct leukocyte subsets, PBMCs, monocyte-depleted PBMCs and purified T cells were activated in the presence of anti-CD147 mAbs. The mAb M6-1E9 inhibited T cell functionsviaactivation of CD147 on monocytes with obligatory cell-cell contact. Engagement of the CD147 epitope by the M6-1E9 mAb downregulated CD80 and CD86 expression on monocytes and IL-2, TNF-α, IFN-γ and IL-17 production in T cells. In contrast, the mAb M6-1D4 inhibited T cell functionviaactivation of CD147 on T cells by downregulating IL-2, TNF-α and IFN-γ. Herein, we demonstrated that certain epitopes of CD147, expressed on both monocytes and T cells, are involved in the regulation of T cell activation.

Natural killer (NK) cells are innate lymphoid cells having potent cytolytic function that provide host defense against microbial infections and tumors. Using our generated monoclonal antibody (mAb), named FE-1H10, new NK cell sub-populations in peripheral blood were identified. The molecules recognized by mAb FE-1H10 were expressed on a sub-population of CD3−CD56dim NK cells. The epitope recognized by mAb FE-1H10 was demonstrated to be N-glycan and proven to be different from CD57. Upon K562 stimulation, the CD56dimFE-1H10+ NK cell sub-population exhibited significantly lower cytolytic function with low ability to degranulate and release cytolytic granules compared to the CD56dimFE-1H10− NK cell sub-population. Moreover, the CD56dimFE-1H10+ NK cells produced less IFN-γ and TNF-α than the CD56dimFE-1H10− NK cells. We demonstrated here that mAb FE-1H10 could identify two sub-populations of circulating CD56dim NK cells with different functions. Our discovery of new sub-populations of NK cells improves our understanding of NK cell biology and may lead to the development of new approaches for NK cell therapy.

Objective CD99, a leukocyte surface molecule, reportedly plays an important role in several cellular processes. However, the role of CD99 in T cell regulation remains unclear, as the CD99 ligand associated with T-cell regulation has not yet been identified. Our previous study showed that recombinant CD99 bound to CD99 ligands was expressed on monocytes, NK cells and dendritic cells. This interaction regulates the expression of IL-6 and TNF-α in CD3 + T cells following T cell activation. In the present study, we confirmed the presence of CD99 ligands in immune cells. Results A recombinant CD99-human IgG fusion protein, CD99HIgG, was produced and used to search for CD99 ligand expression in various hematopoietic cell lines. Among several cell lines, THP-1 monocytic cell line showed strong positive reaction for CD99HIgG, and CD99 and CD99 ligand complexes were pulled-down using a DTSSP cross-linker. The study demonstrated the presence of the membrane bound CD99 ligand, and CD99 ligand candidates were identified via LC–MS/MS. These results may be useful to further identify the CD99 ligands, and to fully comprehend the role of CD99 in immunoregulation.

Additional file 5: Table S1. List of proteins obtained from in-gel digestion of the BSA protein control followed by LC–MS/MS analysis and identified by MASCOT.

Additional file 3: Figure S2. Verification the binding activity of purified CD99HIgG.

Additional file 1. Methods for identification of CD99 ligands by LC-MS/MS.

Additional file 2: Figure S1. Validation of the purity and structure of purified CD99HIgG.

Additional file 4: Figure S3. The expression of CD99 ligands on hematopoietic cells.

Additional file 6: Table S2. List of proteins obtained from in-gel digestion of purified CD99HIgG protein control followed by LC–MS/MS analysis and identified by MASCOT.

α0-Thalassemia is an inherited hematological disorder caused by the deletion of α-globin genes. The Southeast Asian deletion (--SEA) is the most common type of α0-thalassemia observed in Southeast Asian countries. Regarding WHO health policy, an effective α0-thalassemia screening strategy is needed to control new severe α-thalassemia cases. In this study, a monoclonal antibody panel was used to develop immunochromatographic (IC) strip tests for detecting the Hb Bart's and ζ-globin chain. Among 195 samples, all α0-thalassemia traits (78 α0-thalassemia (--SEA) and 4 α0-thalassemia (--THAI)) had low MCV or MCH values. The sensitivity, specificity, PPV and NPV of the IC strip tests for ζ-globin and Hb Bart's for screening α0-thalassemia (--SEA) within the low MCV or MCH samples were 100%, 65.2%, 90.7%, 100% and 96.2%, 47.8%, 86.6%, 78.6%, respectively. All 4 α0-thalassemia (--THAI) traits were negative for ζ-globin chains but positive for Hb Bart's using the IC strip tests. These results led to a α0-thalassemia screening being proposed in which blood samples are first evaluated by MCV, MCH and Hb typing. Samples with high MCV and MCH values are excluded for the presence of the α0-thalassemia gene. Samples with low MCV or MCH values are assayed using the developed IC strip tests, where only samples testing positive are further assayed for α0-thalassemia by PCR. Patients with Hb H, EA Bart's or EF Bart's diseases do not need to use this IC strip assay. Thus, in this study, a simple and cost effective α0-thalassemia point of care test was developed.

Background Couples who carry α-thalassemia-1 deletion are at 25% risk of having a fetus with hemoglobin Bart’s hydrops fetalis. Southeast Asian deletion (−-(SEA)) is the most common type of α-thalassemia 1 among Southeast Asian populations. Thus, identification of the (−-(SEA)) α-thalassemia 1 carrier is necessary for controlling severe α-thalassemia in Southeast Asian countries. Results Using our generated anti ζ-globin chain monoclonal antibodies (mAbs) clones PL2 and PL3, a simple immunostick test for detecting ζ-globin chain presence in whole blood lysates was developed. The procedure of the developed immunostick test was as follows. The immunostick paddles were coated with 50 μg/mL of mAb PL2 as capture mAb, or other control antibodies. The coated immunostick was dipped into cocktail containing tested hemolysate at dilution of 1:500, 0.25 μg/mL biotin-labeled mAb PL3 and horseradish peroxidase-conjugated streptavidin at dilution of 1:1000. The immunostick was then dipped in precipitating substrate and the presence of ζ-globin chain in the tested sample was observed by the naked eye. Upon validation of the developed immunostick test with various types of thalassemia and normal subjects, 100% sensitivity and 82% specificity for detection of the (−-(SEA)) α-thalassemia-1 carriers were achieved. The mAb pre-coated immunostick can be stored at room temperature for at least 20 weeks. Conclusion In this study, a novel simple immunostick test for the screening of (−-(SEA)) α-thalassemia 1 carriers was presented. The developed immunostick test, within a single test, contains both positive and negative internal procedural controls.

CD99 has been reported to be involved in T cell regulation. CD99 ligand involvement in the regulation of T cell activation has been postulated. In this study, recombinant CD99 proteins were produced and used as a tool for determining the role of CD99 and its ligand interaction. Recombinant CD99 proteins induced the upregulation of IL-6 and TNF-α expression, but not IFN-γ, in anti-CD3 monoclonal antibody activated T cells. The cytokine alteration was not observed in unstimulated T cells indicating the cytokine upregulation required the signal from T cell activation. The upregulation of IL-6 and TNF-α was, in addition, observed in CD3- mononuclear cell population including monocytes and NK cells. The recombinant CD99 proteins, however, did not affect either CD25, CD69 or MHC class II expression or T cell proliferation, upon T cell activation. The CD99 ligands were demonstrated to be expressed on monocytes, NK cells and dendritic cells, but not on B and T cells. Our results indicated the presence of CD99 ligands on leukocyte surface. Interaction between CD99 and its ligands involves the regulation of cytokine production.

Background Soluble CD147 (sCD147) is the shed form of membrane-bound CD147, which is involved in the regulation of cellular functions. The presence of sCD147 in body fluids is associated with several diseases. Objective In this study, we aimed to establish antibody (Ab) biosensors for the simultaneous differential detection of the general and truncated forms of sCD147. Method By combining biolayer interferometry technology (BLItz) and different anti-CD147 monoclonal antibodies (mAbs) specific to different extracellular domains of the CD147 molecule, Ab-based biosensors were established to rapidly measure and characterize sCD147 isoforms. Results Two types of Ab-biosensors, desginated the single Ab-biosensor and double Ab-biosensor, were established for the measurment and characterization of sCD147 isoforms. For the single Ab-biosensor system, monoclonal antibodies specific for CD147 domain 1 (D1) or domain 2 (D2) were immobilized on the sensor tips and used for the quantification of sCD147 using a BLItz optical interferometric biosensor. For the double Ab-biosensor system, following the single Ab-biosensor step, secondary anti-CD147 mAbs specific for each domain of the CD147 molecule were added and monitored by a BLItz biosensor. By combining the results obtained from the single Ab- and double Ab-biosensors, sCD147 isoforms including the general form (D1 linked to D2) and the truncated forms (sCD147 containing D1 or D2) could be determined. Conclusions This method may be a beneficial tool for the determination of sCD147 isoforms for disease diagnosis and prognosis as well as for the definition of the cellular mechanisms of the immune system.

Background Human trefoil factor (TFF) peptides consist of three members: TFF1, TFF2 and TFF3. TFF3 is the most abundant TFF peptide in saliva. TFF3 homodimer was suggested to be involved in apoptosis inhibition and malignancy. Determination of TFF3 homodimer expression profiles in saliva may lead to new information about oral biology and diseases. The objective of this study was to generate monoclonal antibodies (mAbs) against TFF3 and apply the produced mAbs for the establishment of ELISA for quantification of dimeric TFF3 in saliva. Results With our modified hybridoma technique, three hybridoma clones producing anti-TFF3 mAbs having IgG isotype were generated. The mAbs were specific for TFF3 with no cross-reactivity to other TFFs. Using the generated mAbs, a modified-sandwich ELISA with high sensitivity for the quantification of dimeric TFF3 in saliva was developed. Using this ELISA, the amount of dimeric TFF3 in saliva could be measured. Conclusions A modified-sandwich ELISA for the quantification of TFF3 dimeric form was established. The established ELISA will be a valuable tool for facilitating the investigation of the physiological roles and the diagnostic values of TFF3 in oral diseases. The concept of this modified-sandwich ELISA may be applied for the determination of other homodimeric peptides of interest.

Enamel-renal-gingival syndrome (ERGS; OMIM #204690), a rare autosomal recessive disorder caused by mutations in FAM20A, is characterized by nephrocalcinosis, nephrolithiasis, amelogenesis imperfecta, hypoplastic type, gingival fibromatosis and other dental abnormalities, including hypodontia and unerupted teeth with large dental follicles. We report three patients and their families with findings suggestive of ERGS. Mutation analysis of FAM20A was performed in all patients and their family members. Patients with homozygous frameshift and compound heterozygous mutations in FAM20A had typical clinical findings along with periodontitis. The other had a novel homozygous missense mutation in exon 10, mild gingival fibromatosis and renal calcifications. The periodontitis in our patients may be a syndrome component, and similar findings in previous reports suggest more than coincidence. Fam20a is an allosteric activator that increases Fam20c kinase activity. It is hypothesized that lack of FAM20A activation of FAM20C in our patients with FAM20A mutations might have caused amelogenesis imperfecta, abnormal bone remodeling and periodontitis. Nephrocalcinosis appears not to be a consistent finding of the syndrome and the missense mutation may correlate with mild gingival fibromatosis. Here we report three patients with homozygous or compound heterozygous mutations in FAM20A and findings that extend the phenotypic spectrum of this disorder, showing that protein truncation is associated with greater clinical severity.

Overexpression of a disintegrin and metalloproteinase 9 (ADAM9) has been shown in various types of cancer. Some studies have reported inconclusive findings regarding chromosomal aberrations in the ADAM9-containing region and ADAM9 expression in oral cancer. Therefore, in this study, ADAM9 protein expression was determined and compared between oral squamous cell carcinoma (OSCC) and normal oral tissues, and between oral cancer cell lines and human oral keratinocytes (HOKs). In total, 34 OSCC and 10 healthy paraffin-embedded tissue sections were probed with an anti-ADAM9 antibody, and the immunohistochemical score was determined by multiplying the percentage of positively stained cells with the intensity score. Four different oral cancer and eight independent HOK cell lines were cultured, and the expression of membrane ADAM9 and active ADAM9 at 84 kDa in these cell lines was assayed by flow cytometry and western blot hybridization, respectively. The results showed that the median immunohistochemical score of ADAM9 expression in OSCC tissues was significantly greater than that in normal tissues (P

Background: Monoclonal antibodies (mAbs) have become essential tools in life science research and in medicine, because of their extreme specificity. Several mAbs against leukocyte surface molecules have been generated in our laboratory. From these, mAb COS3A was selected for biochemical and functional analysis. Objective: To identify and characterize the molecule recognized by mAb COS3A. Methods: Cellular distribution was analyzed by immunofluorescence staining and flow cytometry. Biochemical characterization of the molecular target of COS3A was approached by immunoprecipitation, Western blotting and amino acid sequencing using LC-MS. N-glycosidase F treatment of COS3A-precipitated protein and culture of U937 cells in the presence of tunicamycin before cell lysate preparation were used to study the glycosylation state of proteins. Phagocytosis was examined by flow cytometry. Results: MAb COS3A bound specifically to a molecule expressed on the surface of various human hematopoietic cells and cell lines but not on red blood cells. The antigen had a molecular weight of 30-70 kDa, which was reduced to 25 kDa by elimination of N-linked glycan. LC-MS data and immunoprecipitation indicated that mAb COS3A bound specifically to CD63 molecule. Remarkably, functional analysis demonstrated that mAb COS3A dramatically reduced granulocyte phagocytosis. Conclusions: The mAb COS3A recognized the CD63 molecule and strongly diminished phagocytosis of bacteria by granulocytes, suggesting that CD63 may play a crucial role in the initial step of phagocytosis. MAb COS3A is therefore suitable for both biochemical and functional studies of CD63, and may be used for further study of the mechanism of phagocytosis and also in therapeutic approaches. DOI 10.12932/AP0735

Several CD4 lymphocyte enumeration methods have been developed to be used as alternatives to conventional flow cytometry. However, no consensus has been reached on which technique should be widely adopted. We introduce here a novel nonflow cytometric method, called CD4 Select, for enumeration of CD4 lymphocytes. Instead of using a flow cytometer, the method requires only an automatic hematoanalyzer (complete blood count [CBC] machine). The correlations of the percentage of CD4 lymphocytes and absolute CD4 counts with the CD4 Select method and standard flow cytometry were 0.932 and 0.922, respectively. The CD4 Select method yielded percentage of CD4 lymphocytes greater than flow cytometry by a mean of 0.06%, and the limits of agreement at 95% confidence interval (CI) were 9.46% to -9.56% using Bland-Altman analysis. The CD4 select method is cheap, valid, and simple enough to be conducted locally at small facilities. The method yields not only absolute CD4 lymphocytes, but also percentage of CD4 lymphocytes. The use of a hematoanalyzer also gives this method relatively high sample throughput and makes it less labor-intensive than other nonflow cytometric methods.

Aims To exploit immunomagnetic separation combined with PCR with confronting two-pair primers (IMS-PCR-CTPP) as a rapid method for detection of Mycobacterium tuberculosis complex (MTC) and identification of Mycobacterium bovis from sputum specimens. Methods and Results Monoclonal antibody (mAb) against the mycobacterial antigen, 85B (Ag85B), was coupled with magnetic particles for specific immunomagnetic separation (IMS) of Mycobacterium spp. Immunofluorescence assay indicated the capability of mAb to bind to Ag85B in both the recombinant and the native form. The IMS combined with PCR-CTPP targeting the mycobacterial lep B gene was further implemented using 133 sputum samples with acid-fast bacilli grading from negative to 3+. The results showed the sensitivity and specificity of IMS-PCR-CTPP vs gold standard culture method were 89·9 and 88·6% respectively. Conclusions The IMS-PCR-CTPP method shortens the time for tuberculosis (TB) diagnosis from months to a day. This method is also suitable for investigation of MTC and epidemiological study of Myco. bovis in sputum specimens. Significance and Impact of the Study This study is the first report emphasizing the combination of IMS and PCR-CTPP for the detection of MTC and simultaneous identification of Myco. bovis from sputum. It could be used for TB diagnosis in resource-limited countries with high TB burden.

Autoantibodies against interferon-gamma (IFN-γ) can cause immunodeficiency and are associated with various opportunistic infections. In the present study, we investigated other cellular immune parameters for a better understanding of the immunodeficiency condition in the patients. The numbers of WBC, monocytes and NK cells were increased in patients with anti-IFN-γ autoantibodies (AAbs). Upon TCR activation, T cell proliferation and IL-2 receptor of the patients remained intact. Nonetheless, the Th1 cytokine (IFN-γ and TNF-α) production was up-regulated. The production of Th2 (IL-4) and Th17 (IL-17) cytokines was unchanged. We suggest that, in addition to the presence of anti-IFN-γ autoantibodies, alterations in the cellular immune functions may also contribute to this immunodeficiency.

A CD4 monoclonal antibody which reacts with CD4 + lymphocytes but does not react significantly with monocytes was generated and used to develop a simple method for CD4 count. In a comparison with standard flow cytometry, high correlation was obtained. The developed method can be an alternative to flow cytometry for resource-limited countries.

Background: Several molecules are known to be involved in T-cell activation via the TCR/CD3 complex and while the mechanisms of late T cell signaling have been well characterized, the very early events are still not fully understood. Objective: The aim of this study was to identify yet unknown molecules associated with the TCR/CD3 complex . Results: To identify new molecules associated with the TCR/CD3 complex, a monoclonal antibody termed MT3 was produced by immunoprecipitated beads immunization. C olocalization of the MT3 mAb recognizing molecules with the TCR/CD3 complexes was verified by confocal microscopic analysis. The surface antigen recognized by MT3 antibody was expressed on a subpopulation of CD3 + T cells, and on both CD4 + and CD8 + lymphocytes. The antigen was also expressed on naive CD4 + T cells and on a subset of memory CD4 + T cells. In contrast, in the CD8 population, the majority of MT3 + cells were found in the naive population. The MT3 mAb recognizing molecules were also expressed on red blood cells but only in particular subjects. Similar to peripheral blood leukocytes, MT3 mAb recognizing molecules are exclusively expressed on T cell lines. Conclusions: Based on the cellular distribution patterns and confocal microscopic analysis, the MT3 mAb recognizing molecule that we investigated is proposed to be a TCR/CD3 associated molecule and might be involved in the antigen recognition of T cells.

Early diagnosis of cancer is a key factor for the success of treatment. For this reason, identification of highly sensitive and specific novel tumor markers is urgently needed. In the present study, the CM5 polyclonal antibody (CM5 pAb) raised against p53 of mouse origin was used to identify p53 structurally related protein(s) that may also play an important role in promoting or preventing lung cancer. Western blot analysis was performed on tumor tissues and corresponding normal tissues obtained from lung cancer patients. CM5 pAb reacted with a human protein with an apparent molecular weight of 90 kDa in the lung tumor tissue. The levels of this protein were greatly increased in 35 of the 37 (94.6%) lung tumor samples assessed, with only minimal expression in the normal adjacent tissues. The 90-kDa protein was immunoprecipitated by CM5 pAb and was subsequently identified by LC-MS/MS to be glucosidase II, a key protein involved in the quality control mechanism of glycoprotein folding. An investigation of the response to genotoxic stress and endoplasmic reticulum (ER) stress using A549 human lung adenocarcinoma cells demonstrated that glucosidase II exhibited a similar pattern of response as the p53 tumor suppressor. Protein levels of both p53 and glucosidase II were increased in response to UV irradiation but decreased in response to tunicamycin-induced ER stress. In conclusion, we demonstrated that a polyclonal antibody raised against mouse p53 could cross-react with human glucosidase II, which was found to be frequently overexpressed in human lung tumor tissues and exhibited a stress response similar to p53. The high frequency of glucosidase II overexpression, which to the best of our knowledge has not been previously described, indicates its crucial roles in lung tumorigenesis and is thus a valuable biomarker for facilitating the screening and/or diagnosis of lung cancer. However, further investigations concerning its relationship to p53 and its roles in ER and genotoxic stress are warranted.

In this study, we introduce an application of flow cytometry for the concurrent detection of phagocytotic cells and surface molecules involved in the phagocytic process. E. coli expressing green fluorescent protein (GFP) were applied as the phagocytosable particles. Blood samples were incubated with E. coli expressing GFP, followed by indirect immunofluorescence using four candidate monoclonal antibodies (mAbs). Granulocytes that had phagocytosed E. coli exhibited high levels of GFP intensity, in contrast to the nonphagocytosed cells. By comparing the level of expression of molecules expressed on phagocytosed granulocytes with that of nonphagocytosed cells by flow cytometry, it enabled the determination of the expression and alteration of the cell surface molecules upon phogocytosis. Of the four mAbs used in this study, upon phagocytosis, molecules recognized by mAbs WK13, COSA5A and COSA33NL were up-regulated. However, CD15 recognized by mAb VIMD5 was downregulated. The proposed method will benefit the study of phagocytic mechanisms in the future.

Monoclonal antibodies (MAb) specific to the protein of interest can be achieved following the classical hybridoma technique. However, obtaining a desired MAb is not always straightforward. The intrinsic quality of immunogen is one of the critical success factors. In this study, three sources of immunogens were compared for CD4 MAb production. CD4 proteins were isolated by immunoprecipitation and the CD4 immunoprecipitated (CD4-IP) beads were used as an immunogen. Recombinant CD4 protein-biotin carboxyl carrier protein (BCCP) fusion proteins (CD4-BCCP) were produced in Escherichia coli, isolated by streptavidin-coated beads, and the CD4-BCCP beads were used as an immunogen. CD4 expressing COS (CD4-COS) cells were generated, enriched by immunosorting, and used as an immunogen. After three immunizations, anti-CD4 antibodies could be observed in all immunized mice. The CD4 MAbs that were generated from CD4-IP bead and CD4-COS cell immunizations reacted with both CD4 expressed on transfected COS cells and lymphocytes. These MAbs could be used for immunoprecipitation of CD4 molecules from lymphocyte lysate and for enumerating CD4+ lymphocytes by flow cytometry. In contrast, the MAb generated from CD4-BCCP bead immunization reacted only with recombinant CD4-BCCP proteins but not with native CD4 expressed on CD4+ lymphocytes. Our results indicate that the proposed methods can facilitate the production of desired MAbs where the purified protein antigens are not available or difficult to prepare, but either the encoding cDNA or specific MAb is available.