Identification of different functions of CD8+ T cell subpopulations by a novel monoclonal antibody.

The explicit identification of CD8+ T cell subpopulation is important for deciphering the role of CD8+ T cells for protecting our body against invading pathogens and cancer. Our generated monoclonal antibody (mAb), named FE-1H10, recognized two novel subpopulations of peripheral blood CD8+ T cells, FE-1H10+ and FE-1H10- CD8+ T cells. The molecule recognized by mAb FE-1H10 (FE-1H10 molecules) had a higher distribution on effector memory CD8+ T cell subsets. The functions of FE-1H10- and FE-1H10+ CD8+ T cells were investigated. T cell proliferation assays revealed that FE-1H10- CD8+ T cells exhibited a higher proliferation rate than FE-1H10+ CD8+ T cells, whereas FE-1H10+ CD8+ T cells produced higher levels of IFN-γ and TNF-α than FE-1H10- CD8+ T cells. In T cell cytotoxicity assays, FE-1H10+ CD8+ T cells were able to kill target cells better than FE-1H10- CD8+ T cells. RNA-sequencing analysis confirmed that these subpopulations were distinct: FE-1H10+ CD8+ T cells have higher expression of genes involved in effector functions (IFNG, TNF, GZMB, PRF1, GNLY, FASL, CX3CR1) while FE-1H10- CD8+ T cells have greater expression of genes related to memory CD8+ T cell populations (CCR7, SELL, TCF7, CD40LG). The results suggested that mAb FE-1H10 identifies two novel distinctive CD8+ T cell subpopulations. The FE-1H10+ CD8+ T cells carried a superior functionality in response to tumour cells. The uncover of these novel CD8+ T cell subpopulations may be the basis knowledge of an optional immunotherapy for the selection of potential CD8+ T cells in cancer treatment.

The ligation of CD4 molecules, expressed on monocytes by an anti-CD4 monoclonal antibody, inhibits T cell activation and monocyte mobility.

BACKGROUND: CD4, a leukocyte surface glycoprotein, is mainly expressed on CD4+ T cells, but is also expressed on monocytes. The difference in the expression level and structure of CD4 on T cells and monocytes predicts the different functions of this molecule in both cell types. Although the function of CD4 on T cells is well characterized, little is known about that expressed on primary monocytes. OBJECTIVE: In this study, we investigated the immunoregulation function of CD4 on peripheral blood monocytes. METHODS: Methods: CD4 molecule on monocyte was ligated by anti-CD4 monoclonal antibody (mAb), MT4/3. The effect of mAb MT4/3 on T cell proliferation, cytokine production, the expression of monocyte costimulatory molecules, monocyte migration, and macrophage differentiation were investigated. Moreover, the molecular weight of CD4 on peripheral blood monocyte was carried out by Western immunoblotting. RESULTS: We demonstrated that mAb MT4/3 inhibited anti-CD3 induced T cell proliferation, cytokine production, and the expression of monocyte costimulatory molecules. The ligation of only CD4 on monocytes was sufficient to inhibit T cell activation. Moreover, mAb MT4/3 could inhibit monocyte migration in a transwell migration assay, but not affect the differentiation of monocytes to macrophages. Using purified primary monocytes, the molecular weight of CD4 expressed on monocytes was identified as 55 kDa. CONCLUSIONS: The CD4 molecule expressed on monocytes might play an important role in the regulation of immune responses in both innate and adaptive immunity. Understanding the novel role of CD4 on monocytes in immunoregulation is valuable in the development of new therapeutic approaches.

Humoral and cellular immune responses against SARS-CoV-2 variants of concern induced by heterologous CoronaVac/ChAdOx-1 versus homologous ChAdOx-1 vaccination in the elderly.

BACKGROUND: The concept of heterologous vaccination against SARS-CoV-2 infection has been adopted in Thailand with limited data on the induction of humoral and cellular immunity, particularly the CoronaVac/ChAdOx-1 (CoVac/ChAd) regimen in the elderly. OBJECTIVE: In this study, the immune responses of the elderly induced by heterologous CoVac/ChAd and homologous ChAdOx-1 (ChAd/ChAd) vaccinations were demonstrated. METHODS: A prospective observational study involving healthy participants aged ≥ 60 years who received heterologous CoVac/ChAd or homologous ChAd/ChAd vaccination was conducted. Surrogate neutralizing antibody (NAb) and T-cell responses against the SARS-CoV-2 wild type (WT) and variants of concern were determined at pre and post vaccinations. RESULTS: At 4 and 12 weeks after heterologous or homologous vaccination, the NAb levels against WT, Alpha, Beta, and Delta variants between each group were not significantly different, except for significant lower NAb against the Beta variant in heterologous group at 12 weeks after vaccination. The NAb against the Omicron at 4 weeks post-vaccination were below the cutoff level for antibody detection in both groups. However, higher spike-specific CD4 T cell producing IFN-γ and TNF-α in the heterologous than the homologous vaccination were observed. Insignificant difference of cellular immune responses to spike-peptides of Alpha, Beta, and Delta variants and their WT homologues was demonstrated. CONCLUSIONS: In the elderly, heterologous CoVac/ChAd vaccination could induce NAb response against the WT and non-Omicron variants not different from the homologous ChAd/ChAd vaccination. Both regimens could not give adequate NAb of the Omicron strain. The heterologous vaccination, however, induced higher spike-specific Th1 cell response.

Differentiation and activation of human CD4 T cells is associated with a gradual loss of myelin and lymphocyte protein.

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.

Optimization of culture conditions for stable expression of recombinant fc-fused human extracellular CD99 in HEK293T cells.

CD99 has been demonstrated to play a key role in several biological processes, including the regulation of T-cell activation, cell adhesion, and cell migration. We have also demonstrated that CD99 and its ligands regulate proinflammatory cytokines in NK cells, monocytes and activated T cells. These data suggest CD99 as a potential therapeutic target in cancer. However, the molecular mechanisms by which CD99 and CD99 counter receptors participate in such processes are unclear. High-quality CD99 recombinant proteins produced in large amounts are essential for biological studies and clinical research. In this study, we optimized the various culture conditions for increasing amounts of recombinant protein production with good biological activity. Intracellular immunofluorescence staining was performed to identify the highly expressing CD99HIgG cells. We further investigated the culture conditions for recombinant protein production. A double antibody sandwich enzyme-linked immunosorbent assay was employed to determine the level of secreted CD99HIgG proteins in the culture supernatant of various culture conditions. Later, affinity chromatography using protein G was used to purify CD99HIgG proteins from the culture supernatant of three proper culture conditions. According to our previous report, which utilized Western blotting, the purified CD99HIgG obtained from all tested culture conditions is composed of the CD99 extracellular part fused with the human IgG Fc part in dimer form. For biological activity, the obtained CD99HIgG proteins showed the ability to ligate with the CD99 counter receptor, resulting in the induction of cytokine production.

Neutralizing antibody and T cell responses against SARS-CoV-2 variants of concern following ChAdOx-1 or BNT162b2 boosting in the elderly previously immunized with CoronaVac vaccine.

BACKGROUND: The existence of SARS-CoV-2 variants of concern (VOCs) in association with evidence of breakthrough infections despite vaccination resulted in the need for vaccine boosting. In elderly individuals, information on the immunogenicity of booster vaccinations is limited. In countries where the CoronaVac inactivated vaccine is the primary vaccine, the appropriate boosting regimen is not clear. Immunologic studies of the effects of booster vaccination against VOCs, particularly Delta and Omicron, following CoronaVac in elderly individuals are helpful for policy makers. In this study, we determined the immune responses against VOCs following ChAdOx-1 or BNT162b2 boosting in elderly individuals previously immunized with CoronaVac. RESULTS: Before boosting, the median % inhibition of neutralizing antibodies (NAbs) against the wild-type (WT), Alpha, Beta, Delta and Omicron variants in the ChAdOx-1 and BNT162b2 groups was 52.8% vs. 53.4, 36.6% vs. 39.9, 5.2% vs. 13.7, 34.3% vs. 44.9, and 20.8% vs. 18.8%, respectively. After boosting with ChAdOx-1 or BNT162b2, the % inhibition of NAbs were increased to 97.3% vs. 97.4, 94.3% vs. 97.3%, 79.9 vs. 93.7, 95.5% vs. 97.5, and 26.9% vs. 31.9% for WT, Alpha, Beta, Delta and Omicron variants, respectively. Boosting with BNT162b2 induced significantly higher NAb levels than boosting with ChAdOx-1 against the Alpha, Beta and Delta variants but not the WT and Omicron variants. NAb levels against Omicron variant were not significantly different before and after boosting with ChAdOx-1 or BNT162b2. To evaluate T-cell responses, S peptides of the WT, Alpha, Beta and Delta variants were used to stimulate T cells. Upon stimulation, the expression of IL-17A in CD8 T cells was higher in the BNT162b2 group than in the ChAdOx-1 boosting group. However, IFN-γ production in CD4 and CD8 T cells did not significantly differ under all vaccination regimens. The expression of FasL in CD4 T cells, but not CD8 T cells, was higher in the BNT162b2-boosted group. CONCLUSION: Boosting with either ChAdOx-1 or BNT162b2 in CoronaVac-primed healthy elderly individuals induced high NAb production against all examined VOCs except Omicron. BNT162b2 stimulated higher NAb and some T-cell responses than ChAdOx-1. Vaccine boosting is, therefore, recommended for elderly individuals previously immunized with CoronaVac.

Characterization and functional analysis of novel circulating NK cell sub-populations.

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.

Immunostick Test for Detecting ζ-Globin Chains and Screening of the Southeast Asian α-Thalassemia 1 Deletion.

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.

Triggering of CD99 on monocytes by a specific monoclonal antibody regulates T cell activation.

CD99, a leukocyte surface glycoprotein, has been implicated in many cellular processes including cell adhesion, cell migration and T cell activation. Our previous study demonstrated the anti-CD99 monoclonal antibody (mAb) clone MT99/3 inhibited T cell activation; however, the mechanism is unclear. In this study, we demonstrated that CD99 expressed on monocytes played a role in the inhibition of T cell activation. Anti-CD99 mAb MT99/3 downregulated the expression of costimulatory molecule CD86, but upregulated IL-6, IL-10 and TNF-α production by monocytes. The inhibitory effect of mAb MT99/3 required cell to cell contact between monocytes and lymphocytes. The soluble mediators produced by monocytes alone were insufficient to induce hypo-function of T lymphocytes. In summary, we demonstrated that ligation of CD99 on monocytes by anti-CD99 mAb MT99/3 could mediate T cell hypo-responsiveness. These findings provide the first evidence of the role of CD99 on monocytes that contributes to T cell activation.

Sandwich antibody-based biosensor system for identification of Mycobacterium tuberculosis complex and nontuberculous mycobacteria.

Mycobacterial infection, leading to pulmonary disease, remains a world health problem. Clinical symptoms of pulmonary disease caused by Mycobacterium tuberculosis complex (MTBC) and nontuberculous mycobacteria (NTM) are very similar. A rapid method for the differentiation of MTBC and NTM infection is essential for appropriate therapy. In this study, we aim to establish an antibody-based biosensor system for the identification of MTBC and NTM infection. Monoclonal antibodies (mAbs) specific for Ag85B proteins of mycobacteria were generated and characterized. The generated anti-Ag85B mAb clones AM85B-5 and AM85B-8 reacted to Ag85B of Mycobacterium spp.; in contrast, clone AM85B-9 specifically reacted to Ag85B of MTBC. By employing the produced mAbs, single and sandwich antibody-based biosensors using bio-layer interferometry were established for determination of Ag85B proteins. The sandwich antibody-based biosensor system was demonstrated to be suitable for detection of Ag85B protein and identification of MTBC and NTM. Using anti-Ag85B mAbs AM85B-8 and AM85B-9 as immobilized antibodies on sensor chips and using mAb AM85B-5 as secondary antibody, the established sandwich antibody-based biosensor could discriminate MTBC and NTM. The developed biosensor system can be used for culture confirmation of mycobacteria and speciation to MTBC and NTM.

A modified sandwich ELISA for accurate measurement of HbF in α-thalassemia carriers containing Hb Bart's and Hb Portland 1.

Hemoglobin F (HbF) in blood lysate can be accurately measured by various methods, including immunoassay. In this study, we have produced polyclonal antibody (pAb) against HbF and established a modified sandwich-type ELISA for HbF quantification in blood lysates. The modified sandwich ELISA utilized anti-γ-globin monoclonal antibody clones Thal N/B as the capture antibody (Ab) coated on solid-phase, fluorescein isothiocyanate (FITC)-labeled pAb as the detecting Ab, and HPR-labeled anti-FITC Ab as the signal-generating Ab. By using an optimized blood lysate dilution, the HbF could be measured with no interference from hemoglobin Bart's (Hb Bart's) and hemoglobin Portland (Hb Portland 1) presented in α-thalassemia carriers. HbF levels measured by the modified sandwich ELISA were comparable to those quantified by the standard cation-exchange high performance liquid chromatography. We suggested that this modified sandwich ELISA was able to accurately measure HbF levels even in α-thalassemia carriers containing Hb Bart's and Hb Portland 1 and be an alternative method for HbF measurement.

Antibody biosensors for the measurement and characterization of soluble CD147 molecules.

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.

Production of Monoclonal Antibodies against Human Trefoil Factor 3 and Development of a Modified-Sandwich ELISA for Detection of Trefoil Factor 3 Homodimer in Saliva.

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.

Periodontal disease and FAM20A mutations.

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.

Exploitation of human CD99 expressing mouse myeloma cells as immunogen for production of mouse specific polyclonal antibodies.

In this study, we describe the application of a molecular biology technique for the production of mouse polyclonal antibodies (pAbs) specific to human cell surface molecules. Production of the pAb specific to the human CD99 surface molecule was used as the study model. The retroviral expression system was employed to generate human CD99 expressing mouse myeloma cells. After cell sorting and single cell cloning, a myeloma clone which stably expressed high levels of human CD99 on its surface was established. The human CD99 expressing mouse myeloma cells were then used as the immunogen for immunization of BALB/c mice. As endogenous proteins of mouse myeloma cells possess self-non-immunogenicity for BALB/c mice, after immunization, only the expressed human CD99 molecules induce antibody response. After three immunizations, high titers of mouse anti-CD99 pAbs were successfully produced. The produced pAb specifically reacted to both recombinant human CD99 and native CD99 molecules expressed on human blood cells. The established technology is simple and valuable for the production of pAbs specific to human CD99 membrane proteins which can be used for characterization of the CD99 molecule.

A silicon nitride ISFET based immunosensor for Ag85B detection of tuberculosis.

A silicon nitride Ion Sensitive Field Effect Transistor (ISFET) based immunosensor was developed as a low-cost and label-free electrical detection for the detection of antigen 85 complex B (Ag85B). The sensing membrane of the ISFET was modified with 3-aminopropyltriethoxysilane (APTES) followed by glutaraldehyde (GA), yielding an aldehyde-terminated surface. This group is available for immobilization of a monoclonal antibody against a recombinant Ag85B protein (anti-Ag85B antibody). The optimal concentration for anti-Ag85B antibody immobilization onto the modified ISFET was 100 µg ml-1. This optimal condition provided the maximal binding capability and minimal non-specific background signal. The binding event between the recombinant Ag85B antigen and anti-Ag85B antibody on the ISFET surface is presented by monitoring the gate potential change at a constant drain current. The dose response for the recombinant Ag85B protein showed a linear response between 0.12 and 1 µg ml-1 without significant interference from other recombinant proteins. The analytical imprecision (CV%) and accuracy of this Ag85B protein biosensor were 9.73-10.99% and 95.29%, respectively. In addition, an irrelevant antibody and other recombinant proteins were employed as a negative control to demonstrate the non-specific interaction of the antigen and antibody. The success of this immunosensor system for Ag85B protein detection facilitates the construction of a promising device which can shorten the turnaround time for the diagnosis of tuberculosis compared to a standard culture method. Furthermore, this device could also be applied for real-time growth monitoring of Mycobacterium tuberculosis in a mycobacterial culture system.

Simultaneous flow cytometric measurement of antigen attachment to phagocytes and phagocytosis.

The current available assays cannot differentiate the stages of phagocytosis. We, therefore, established methods for concurrent detection of antigen attachment and engulfment by phagocyte using latex beads coated with lipopolysaccharide, rabbit IgG, and carboxyfluorescein diacetate succinimidyl ester. The generated beads were incubated with whole blood at 37°C for 1 hr and stained with PE-Cy5.5 anti-rabbit IgG antibody. By flow cytometry, attachment and phagocytic processes could be detected, simultaneously. The established method is a valuable tool for diagnosis of phagocytic disorder and study of molecules involved in phagocytosis.

Production and characterization of monoclonal antibodies against α-globin chain-containing human hemoglobins for detecting α-thalassemia disease.

Monoclonal antibodies against α-globin containing human Hbs, named AMS-Alpha1 and AMS-Alpha 2, were produced by the hybridoma technique using spleen cells enriched by the newly developed B lymphocyte enrichment protocol. These two monoclonal antibodies were of IgM class, reacting to only intact form of human Hbs A, A2, E, and F, which contain α-globin chain. By the indirect ELISA, the AMS-Alpha1 and AMS-Alpha 2 quantified less amount of α-globin chain containing hemoglobins in HbH disease than the SEA-α thalassemia 1 carriers and normal individuals. It was thus anticipated that these monoclonal antibodies can be used for detecting Hb Bart's hydrops fetalis in which no α-globin chain is produced.

Biochemical and functional analysis of COS3A, a novel CD63-specific monoclonal antibody.

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 analyze the properties and function of the mAb COS3A. METHOD: 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 erythrocytes. 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 the CD63 molecule. Remarkably, functional analysis demonstrated that mAb COS3A dramatically reduced granulocyte phagocytosis. CONCLUSIONS: The mAb COS3A recognized the CD63 molecule and strongly diminished granulocyte phagocytosis of E. coli, 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.

Identification of a T cell surface molecule using a monoclonal antibody produced by TCR/CD3 complex immunization.

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. Colocalization 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 na?ve 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 na?ve 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.