In addition to T cell-dependent (TD) Ab responses, T cells can also regulate T cell-independent (TI) B cell responses in the absence of a specific major histocompatibility complex (MHC) class II and antigenic peptide-based interaction between T and B cells. The elucidation of T cells capable of supporting TI Ab responses is important for understanding the cellular mechanism of different types of TI Ab responses. Natural killer T (NKT) cells represent 1 type of helper T cells involved in TI Ab responses and more candidate helper T cells responsible for TI Ab responses may also include γδ T cells and recently reported B-1 helper CD4⁺ T cells. Marginal zone (MZ) B and B-1 cells, 2 major innate-like B cell subsets considered to function independently of T cells, interact with innate-like T cells. Whereas MZ B and NKT cells interact mutually for a rapid response to blood-borne infection, peritoneal memory phenotype CD49d(high)CD4⁺ T cells support natural Ab secretion by B-1 cells. Here the role of innate-like T cells in the so-called TI Ab response is discussed. To accommodate the involvement of T cells in the TI Ab responses, we suggest an expanded classification of TD Ab responses that incorporate cognate and non-cognate B cell help by innate-like T cells.
Antibody Formation* ; Antigen-Antibody Reactions ; B-Lymphocyte Subsets ; B-Lymphocytes ; Classification ; Major Histocompatibility Complex ; Memory ; Natural Killer T-Cells ; Phenotype ; T-Lymphocytes* ; T-Lymphocytes, Helper-Inducer

Hematoxylin and eosin staining is simple and one of the most important techniques in pathological diagnosis. However, it cannot provide complete information about the disease of a patient. Immunohistochemical staining (IHC) is an important method for demonstrating the distribution of a certain molecule or antigen in tissues using specific antigen-antibody reactions. It is used in routine diagnostic work and research to explore biomarkers. In this review, I aim to provide an adequate interpretation of the results of IHC and pathological diagnosis for clinicians.
Antigen-Antibody Reactions ; Biomarkers ; Diagnosis* ; Eosine Yellowish-(YS) ; Hematoxylin ; Humans ; Methods

Immunohistochemistry (IHC) is an important auxiliary method for pathologists in routine diagnostic work as well as in basic and clinical research including exploration of biomarkers, as IHC allows confirmation of target molecule expressions in the context of microenvironment. Although there has been a considerable progress in automation and standardization of IHC, there are still many things to be considered in proper optimization and appropriate interpretation. In this review, we aim to provide possible pitfalls and useful tips for practicing pathologists and residents in pathology training. First, general procedure of IHC is summarized, followed by pitfalls and tips in each step and a summary of troubleshooting. Second, ways to an accurate interpretation of IHC are discussed, with introduction to general quantification and analysis methods. This review is not intended to provide complete information on IHC, but to be used as a basic reference for practice and publication.
Antigen-Antibody Reactions ; Automation ; Biomarkers ; Immunohistochemistry* ; Methods ; Pathology ; Publications

In order to obtain the lead compound for treatment of rheumatoid arthritis (RA), in this study, therapeutic efficacy of three bispecific antibodies (BsAB-1, BsAB-2 and BsAB-3) against both hIL-1beta and hIL-17 were compared on CIA model mice. First, by ELISA method we compared the binding capacity of the three bispecific antibodies to the two antigens. The results showed that all three antibodies could simultaneously bind both antigens, among these antibodies, BsAB-1 was superior over BsAB-2 and BsAB-3. CIA model was established with chicken type II collagen (CII) and developed RA-like symptoms such as ankle swelling, skin tight, hind foot skin hyperemia. The CIA mice were treated with three antibodies once every two days for total of 29 days. Compared with the CIA model mice, the RA-like symptoms of the antibody treated-mice significantly relieved, while the BsAB-1 treated-mice were almost recovered. CII antibody level in the serum and cytokines (IL-2, IL-1beta, IL-17A and TNF-alpha) expression in the spleen were examined. Compared with the CIA model mice, all three antibodies could significantly reduce CII antibody and cytokine expression levels. BsAB-1 antibody was more potent than BsAB-2 and BsAB-3. In summary, BsAB-1 is superior over BsAB-2 and BsAB-3 in amelioration of RA symptoms and regulation of CII antibody production and pro-inflammatory cytokine expression, therefore, BsAB-1 can be chosen as a lead compound for further development of drug candidate for treatment of RA.
Animals ; Antibodies ; metabolism ; Antibodies, Bispecific ; immunology ; therapeutic use ; Antigen-Antibody Reactions ; Arthritis, Experimental ; chemically induced ; metabolism ; therapy ; Arthritis, Rheumatoid ; chemically induced ; metabolism ; therapy ; Collagen Type II ; immunology ; Interleukin-17 ; metabolism ; Interleukin-1beta ; metabolism ; Interleukin-2 ; metabolism ; Male ; Mice ; Spleen ; metabolism ; Tumor Necrosis Factor-alpha ; metabolism

The N-terminal fragment of prohormone brain natriuretic peptide (NT-proBNP) is a commonly used biomarker for the diagnosis of congestive heart failure, although its biological function is not well known. NT-proBNP exhibits heavy O-linked glycosylation, and it is quite difficult to develop an antibody that exhibits glycosylation-independent binding. We developed an antibody that binds to the recombinant NT-proBNP protein and its deglycosylated form with similar affinities in an enzyme immunoassay. The epitope was defined as Gly63-Lys68 based on mimetic peptide screening, site-directed mutagenesis and a competition assay with a peptide mimotope. The nearest O-glycosylation residues are Thr58 and Thr71; therefore, four amino acid residues intervene between the epitope and those residues in both directions. In conclusion, we report that an antibody reactive to Gly63-Lys68 of NT-proBNP exhibits O-glycosylation-independent binding.
Amino Acid Sequence ; Animals ; Antibodies/*immunology ; Antigen-Antibody Reactions ; Epitope Mapping ; Epitopes/chemistry/genetics/*immunology ; Glycosylation ; HEK293 Cells ; Heart Failure/immunology ; Humans ; Molecular Sequence Data ; Mutagenesis, Site-Directed ; Natriuretic Peptide, Brain/chemistry/genetics/*immunology ; Peptide Fragments/chemistry/genetics/*immunology ; Rabbits ; Recombinant Fusion Proteins/chemistry/genetics/immunology

The amino group PEGylation of rhIFNomega with monomethoxy polyethylene glycol succinimidyl succinate (mPEG-SS, 20 000) was investigated, and the modified mixture was separated and purified by ion exchange chromatography and gel filtration chromatography. Under the optimized purification conditions, the average content ofmono PEG-rhIFNomega in the collect liquid reached 182 microg x mL(-1). The average purified yield of mono PEG-rhIFNomega exceed to 22%, and the purity of mono PEG-rhIFNomega was greater than 98% by SDS-PAGE and RP-HPLC. Relative molecular mass of mono PEG-rhIFNomega was 43 790 detected by MALDI-TOF MS. The apparent molecular mass measured by SDS-PAGE was about 60 810. The purified PEG-rhIFNomega has the characteristics of typical PEGylated protein. Activity reservation rate of mono PEG-rhIFNomega was 15.0%, while the antigenicity decreased by at least 64 folds. In addition, the acid stability, thermal stability and stability in serum and trypsin solution of mono PEG-rhIFNomega were markedly better than those of the rhIFNomega. The pharmacological properties of mono PEG-rhIFNomega were significantly improved. The prepared PEG-rhIFNomega might be developed to a novel safe and long-acting interferon.
Animals ; Antigen-Antibody Reactions ; Chromatography, Gel ; Chromatography, High Pressure Liquid ; Chromatography, Ion Exchange ; Drug Stability ; Electrophoresis, Polyacrylamide Gel ; Interferon Type I ; chemistry ; immunology ; Molecular Weight ; Polyethylene Glycols ; chemistry ; Rabbits ; Recombinant Proteins ; chemistry ; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization

The lower expression of CD20 antigen molecules on the B cell membrane is the primary characteristic of B-chronic lymphocytic leukemia (B-CLL). In this paper, we combined laser scanning confocal microscopy (LSCM) and quantum dots labeling to detect the expression and distribution of CD20 molecules on CD20+B lymphocyte surface. Simultaneously, we investigated the morphology and ultrastructure of the B lymphocytes that belonged to the normal persons and B-CLL patients through utilizing the atomic force microscope (AFM). In addition, we measured the force spectroscopy of CD20 antigen-antibody binding using the AFM tips modified with CD20 antibody. The fluorescent images indicated that the density of CD20 of normal CD20+B lymphocytes was much higher than that of B-CLL CD20+B cells. The AFM data show that ultrastructure of B-CLL CD20+B lymphocytes became more complicated. Moreover, the single molecular force spectroscopy data show that the special force of CD20 antigen-antibody was four times bigger than the nonspecific force between the naked AFM tip and cell surface. The force map showed that CD20 molecules distributed homogeneously on the normal CD20+B lymphocytes, whereas, the CD20 molecules distributed heterogenous on B-CLL CD20+B lymphocytes. Our data provide visualized evidence for the phenomenon of low-response to rituximab therapy on clinical. Meanwhile, AFM is possible to be a powerful tool for development and screening of drugs for pharmacology use.
Antigen-Antibody Reactions ; immunology ; Antigens, CD20 ; immunology ; B-Lymphocytes ; immunology ; ultrastructure ; Binding Sites, Antibody ; Cell Membrane ; immunology ; Humans ; Leukemia, Lymphocytic, Chronic, B-Cell ; immunology ; Microscopy, Atomic Force ; Microscopy, Confocal ; Quantum Dots

Transfusion-related acute lung injury (TRALI) is a serious adverse transfusion reaction that is presented as acute hypoxemia and non-cardiogenic pulmonary edema, which develops during or within 6 hr of transfusion. Major pathogenesis of TRALI is known to be related with anti-HLA class I, anti-HLA class II, or anti-HNA in donor's plasma. However, anti-HLA or anti-HNA in recipient against transfused donor's leukocyte antigens also cause TRALI in minor pathogenesis and which comprises about 10% of TRALI. Published reports of TRALI are relatively rare in Korea. In our cases, both patients presented with dyspnea and hypoxemia during transfusion of packed red blood cells and showed findings of bilateral pulmonary infiltrations at chest radiography. Findings of patients' anti-HLA antibodies and recipients' HLA concordance indicate that minor pathogenesis may be not as infrequent as we'd expected before. In addition, second case showed that anti-HLA class II antibodies could be responsible for immunopathogenic mechanisms, alone.
Acute Lung Injury/*diagnosis/*immunology/radiography ; Aged ; Anoxia/diagnosis ; Antigen-Antibody Reactions ; Blood Transfusion/*adverse effects ; Dyspnea/diagnosis ; Female ; HLA Antigens/*immunology ; Histocompatibility Antigens Class I/immunology ; Histocompatibility Antigens Class II/immunology ; Humans ; Isoantibodies/*blood ; Male ; Middle Aged

BACKGROUND: Granulocyte specific antibodies are associated with several clinical conditions including febrile transfusion reaction and transfusion-related acute lung injury as well as immune neutropenias. The identification of granulocyte specific antibodies is important for the diagnosis of these disorders. However, there have been rarely confirmed clinical reports in Korea since the testing techniques are complicated and difficult to maintain. In this study, development of in-house indicator cells and renewedly establishment of the mixed passive hemagglutination assay (MPHA) as a serologic test to detect and identify granulocyte specific antibodies were conducted. METHODS: The in-house indicator cells for MPHA were made by sensitizing human Rh(D) positive O RBCs with human IgG anti-Rh(D) (DiaMed AG, Switzerland) and then combining with AHG anti-IgG (Immucor Inc., USA). To determine the optimal conditions, various combinations of anti-Rh(D) IgG sensitization strengths of indicator cells, microwell coated antigens (intact granulocyte vs. extracted granulocyte) and reaction conditions were compared. RESULTS: The best test conditions for MPHA were as follows: optimal results were obtained with the anti-Rh(D) sensitization dilutions of 1/64-1/192 and the reaction condition of 4 hours incubation at room temperature in humid chamber. Extracted granulocytes coated at the plate showed better results than intact granulocytes. HLA antigens were completely removed from extracted granulocyte antigens after acidified chloroquine treatment. CONCLUSION: Granulocyte MPHA using in-house anti-Rh(D) sensitized indicator cells was developed for the first time in Korea. The newly established MPHA would be effectively used for the diagnosis and treatment of disorders associated with granulocyte specific antigen-antibody reactions in Korea.
Acute Lung Injury ; Antibodies ; Antibodies, Anti-Idiotypic ; Antigen-Antibody Reactions ; Blood Group Incompatibility ; Chloroquine ; Granulocytes ; Hemagglutination ; HLA Antigens ; Humans ; Immunoglobulin G ; Korea ; Neutropenia ; Serologic Tests

According to the serological screening methods of antigen-antibody reaction such as ELISA, it has been known that the complete detection of viral infections of HBV, HCV, and HIV-1 viruses in the blood and blood related-products is not much reliable. Therefore, nucleic acid amplification testing methods (NAT) adopted to detect the small quantitative viral nucleic acids could support the basis of using and supplying the blood and its related products safely. This research work is basically designed to describe the simultaneous blood screening system by multiplex or duplex tests for detection of HBV, HCV, and HIV-1 viruses in the blood at one time with low price and labor. It is aimed at easy detection by using the conventional agarose gel electrophoresis. Thus, we tried to detect and identify the viral components in the blood sample according to their different size of PCR products. We decided a set of consensus sequences to recognize each viral DNA fragments after running the multiplex PCR in one tube. This was done by nested RT-PCR using two different RNA viral genomic templates followed by multiplex PCR with addition of viral DNA and their primers after purifying the viral genomic nucleic acids. Those specific primers could be used without any interference to amplify each viral genome in the blood samples. The sensitivities with different viral loads were evaluated on the agarose gel electrophoresis. Three different viral agents in the blood samples could be tested by this multiplex (RT)-PCR with three different primers.
Antigen-Antibody Reactions ; Consensus Sequence ; DNA, Viral ; Electrophoresis, Agar Gel ; Enzyme-Linked Immunosorbent Assay ; Genome, Viral ; HIV-1 ; Mass Screening ; Multiplex Polymerase Chain Reaction ; Nucleic Acid Amplification Techniques ; Nucleic Acids ; Polymerase Chain Reaction ; Quality Control* ; RNA ; Running ; Viral Load ; Viral Structures