ABSTRACT
We have previously shown the expression of group X secretory phospholipase A(2) (sPLA(2)-X) in mouse splenic macrophages and its powerful potency for releasing fatty acids from various intact cell membranes. Here, we examined the potency of sPLA(2)-X in the production of lipid mediators in murine peritoneal macrophages. Mouse sPLA(2)-X was found to induce a marked release of fatty acids including arachidonic acid and linoleic acid, which contrasted with little, if any, release by the action of group IB and IIA sPLA(2)s. In resting macrophages, sPLA(2)-X elicited a modest production of prostaglandin E(2) and thromboxane A(2). After the induction of cyclooxygenase-2 (COX-2) by pretreatment with lipopolysaccharide, a dramatic increase in the production of these eicosanoids was observed in sPLA(2)-X-treated macrophages, which was completely blocked by the addition of either the specific sPLA(2) inhibitor indoxam or the COX inhibitor indomethacin. In accordance with its higher hydrolyzing activity toward phosphatidylcholine, mouse sPLA(2)-X induced a potent production of lysophosphatidylcholine. These findings strongly suggest that sPLA(2)-X plays a critical role in the production of various lipid mediators from macrophages. These events might be relevant to the progression of various pathological states, including chronic inflammation and atherosclerosis.
Subject(s)
Macrophages, Peritoneal/metabolism , Phospholipases A/pharmacology , Animals , Carbamates/pharmacology , Cells, Cultured , Dinoprostone/metabolism , Eicosanoids/metabolism , Enzyme Inhibitors/pharmacology , Fatty Acids/metabolism , Indolizines/pharmacology , Isoenzymes/metabolism , Lipid Metabolism , Lipopolysaccharides , Lysophosphatidylcholines/metabolism , Macrophage Activation , Mice , Phospholipases A/antagonists & inhibitorsABSTRACT
Group X secretory phospholipase A2 (sPLA2-X) has recently been shown to possess a powerful potency for releasing arachidonic acid from cell membrane phospholipids. Here, we report the purification of mouse pro- and mature forms of sPLA2-X, as well as its expression and biological functions. Purified pro-sPLA2-X was found to possess a propeptide of 11 amino acid residues attached at the NH2-terminals of the mature protein, and showed as little as 8% of the PLA2 activity of the mature form. Limited proteolysis of pro-sPLA2-X with trypsin resulted in the appearance of the mature form with a concomitant increase in PLA2 activity, suggesting a requirement of proteolytic removal of the propeptide for the optimal activity. The expression of sPLA2-X mRNA was detected in various tissues including the lung, thymus, and spleen, and immunohistochemical analysis revealed its expression in splenic macrophages. In the spleen cells, mature sPLA2-X elicited a prompt release of arachidonic acid with significant production of prostaglandin E2 more efficiently than group IB and IIA sPLA2s. In addition, sPLA2-X was identified as a high-affinity ligand for both native and recombinant form of mouse PLA2 receptor (PLA2R). However, there was no significant difference in the sPLA2-X-induced arachidonic acid release responses in the spleen cells between wild-type and PLA2R-deficient mice. These findings strongly suggest that sPLA2-X possesses two distinct biological functions in mice: it elicits a marked release of arachidonic acid from membrane phospholipids leading to the production of lipid mediators based on its enzymatic potency, and it acts as a natural ligand for the PLA2R that has been shown to play a critical role in the production of inflammatory cytokines during endotoxic shock.
Subject(s)
Arachidonic Acid/metabolism , Phospholipases A/metabolism , Receptors, Cell Surface/metabolism , Spleen/metabolism , Animals , Enzyme Precursors/genetics , Enzyme Precursors/metabolism , Female , Group II Phospholipases A2 , Group X Phospholipases A2 , Immunohistochemistry , In Vitro Techniques , Kinetics , Ligands , Male , Mice , Mice, Inbred C57BL , Molecular Sequence Data , Phospholipases A/genetics , Phospholipases A2 , RNA, Messenger/genetics , RNA, Messenger/metabolism , Receptors, Phospholipase A2 , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Tissue DistributionABSTRACT
In the presence of a stoichiometric amount of CrCl(3) and trimethylchlorosilane (TMSCl), nucleophilic addition of arylzinc compounds 1c-h to arylaldehydes 2a,b,g smoothly proceeded at room temperature to yield corresponding benzhydrols 4a-f in good yields. From arylzinc compounds 1a,b, 3-aryl-1(3H)-isobenzofuranones 3a-f were given by the CrCl(3)-mediated reaction with arylaldehydes 2a-f. Diaryl ketones 5a-e were obtained in good yields by the addition of excess amount of benzaldehyde as an oxidant to the resulting solution after the CrCl(3)-mediated reaction between arylzinc compounds 1c-g and arylaldehydes 2b,g was completed. In the nucleophilic additions of arylzinc compounds 1a,d,f to alkyladehydes 6b-f, the treatment of arylzinc compounds with CrCl(3) was required prior to the addition of the aldehydes in order to prevent the fast protodezincation of arylzinc compounds by the enolizable aldehydes. In these CrCl(3)-mediated nucleophilic additions of arylzinc compounds to aldehydes, arylchromium(III) species are probably reactive intermediates.
ABSTRACT
Although the cyclooxygenase-2 (COX-2) pathway of the arachidonic acid cascade has been suggested to play an important role in colon carcinogenesis, there is little information concerning the identity of phospholipase A(2) (PLA(2)) involved in the arachidonic acid release in colon tumors. Here, we compared the potencies of three types of secretory PLA(2)s (group IB, IIA and X sPLA(2)s) for the arachidonic acid release from cultured human colon adenocarcinoma cells, and found that group X sPLA(2) has the most powerful potency in the release of arachidonic acid leading to COX-2-dependent prostaglandin E(2) (PGE(2)) formation. Furthermore, immunohistological analysis revealed the elevated expression of group X sPLA(2) in human colon adenocarcinoma neoplastic cells in concert with augmented expression of COX-2. These findings suggest a critical role of group X sPLA(2) in the PGE(2) biosynthesis during colon tumorigenesis.
Subject(s)
Adenocarcinoma/enzymology , Colonic Neoplasms/enzymology , Dinoprostone/metabolism , Phospholipases A/metabolism , Prostaglandin-Endoperoxide Synthases/metabolism , Adenocarcinoma/pathology , Adenocarcinoma/physiopathology , Colon/enzymology , Colon/pathology , Colonic Neoplasms/pathology , Colonic Neoplasms/physiopathology , Colorectal Neoplasms/enzymology , Colorectal Neoplasms/pathology , Cyclooxygenase 1 , Cyclooxygenase 2 , Group II Phospholipases A2 , Humans , Isoenzymes/genetics , Isoenzymes/metabolism , Membrane Proteins , Prostaglandin-Endoperoxide Synthases/genetics , Recombinant Proteins/metabolism , Tumor Cells, CulturedABSTRACT
Group X secretory phospholipase A(2) (sPLA(2)-X) possesses several structural features characteristic of both group IB and IIA sPLA(2)s (sPLA(2)-IB and -IIA) and is postulated to be involved in inflammatory responses owing to its restricted expression in the spleen and thymus. Here, we report the purification of human recombinant COOH-terminal His-tagged sPLA(2)-X, the preparation of its antibody, and the purification of native sPLA(2)-X. The affinity-purified sPLA(2)-X protein migrated as various molecular species of 13-18 kDa on SDS-polyacrylamide gels, and N-glycosidase F treatment caused shifts to the 13- and 14-kDa bands. NH(2)-terminal amino acid sequencing analysis revealed that the 13-kDa form is a putative mature sPLA(2)-X and the 14-kDa protein possesses a propeptide of 11 amino acid residues attached at the NH(2) termini of the mature protein. Separation with reverse-phase high performance liquid chromatography revealed that N-linked carbohydrates are not required for the enzymatic activity and pro-sPLA(2)-X has a relatively weak potency compared with the mature protein. The mature sPLA(2)-X induced the release of arachidonic acid from phosphatidylcholine more efficiently than other human sPLA(2) groups (IB, IIA, IID, and V) and elicited a prompt and marked release of arachidonic acid from human monocytic THP-1 cells compared with sPLA(2)-IB and -IIA with concomitant production of prostaglandin E(2). A prominent release of arachidonic acid was also observed in sPLA(2)-X-treated human U937 and HL60 cells. Immunohistochemical analysis of human lung preparations revealed its expression in alveolar epithelial cells. These results indicate that human sPLA(2)-X is a unique N-glycosylated sPLA(2) that releases arachidonic acid from human myeloid leukemia cells more efficiently than sPLA(2)-IB and -IIA.
Subject(s)
Arachidonic Acid/metabolism , HL-60 Cells/metabolism , Phospholipases A/isolation & purification , Animals , Antibodies/immunology , Antibodies/isolation & purification , Antibodies/pharmacology , Arachidonic Acids/pharmacology , CHO Cells , COS Cells , Cell Line , Cricetinae , Dinoprostone/metabolism , Enzyme Inhibitors/pharmacology , Fatty Acids/metabolism , Group II Phospholipases A2 , HL-60 Cells/cytology , HL-60 Cells/drug effects , Humans , Immunohistochemistry , Leukemia, Myeloid/metabolism , Leukemia, Myeloid/pathology , Lung/enzymology , Peptide Fragments/chemistry , Peptide Fragments/immunology , Peptide Fragments/pharmacology , Phospholipases A/genetics , Phospholipases A/metabolism , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/isolation & purification , Recombinant Fusion Proteins/metabolism , Substrate Specificity , Tritium , Tumor Cells, CulturedABSTRACT
Human T-cell leukemia virus type 1 (HTLV-1) remains latent throughout the life of the carrier, with cells containing the provirus and viral gene expression efficiently down-regulated. On a molecular level, exactly how viruses are down-regulated in vivo remains unresolved. We described here the possibility that down-regulation results from the presence of inhibitory elements within the gag-env region of the provirus in fresh peripheral blood mononuclear cells from carriers. In vitro experiments then revealed that potent cis-acting inhibitory elements (CIEs) are indeed contained in two discrete fragments from the pol region and weaker ones in the env region. The effect of CIEs is relieved by the HTLV-1 posttranscriptional regulator Rex through binding to the Rex-responsive element (RxRE), suggesting that Rex might interfere with pre-mRNA degradation and/or activate the export of mRNA molecules harboring both of the inhibitory elements and RxRE on the same RNA molecule. Thus, we propose the hypothesis that such functions of CIEs may be involved in HTLV-1 persistence.
Subject(s)
DNA, Viral/genetics , Gene Expression Regulation, Viral , Genes, env , Genes, pol , Human T-lymphotropic virus 1/genetics , Regulatory Sequences, Nucleic Acid , Virus Latency , Cell Line , Chromosome Mapping , Defective Viruses/genetics , Gene Products, rex/physiology , Humans , Proviruses/genetics , RNA, Messenger/genetics , RNA, Viral/genetics , Sequence DeletionABSTRACT
The human T-cell leukemia virus type I (HTLV-I) pX gene encodes three nonstructural proteins, p40tax, p27rex and p21X. So far, natural antibodies to p27rex and/or p21X have not been found in sera from HTLV-I-infected individuals, although antibodies to p40tax have been found. Recently, the viral transcripts specific for these proteins were detected in fresh peripheral blood mononuclear cells from HTLV-I-infected individuals by the polymerase chain reaction coupled to reverse transcription, showing the in vivo expression of these proteins. We detected antibodies to p21X and p27rex by an enzyme-linked immunosorbent assay (ELISA) system using a recombinantly produced p21X protein as a common antigen, because p21X is identical to the C-terminal portion of p27rex. The sensitivity of the ELISA was determined to be approximately 100 times greater than that of Western blotting. From the analyzed sera of 31 ATL patients, 30 asymptomatic carriers, 18 HAM patients and 100 healthy donors, three specimens from one ATL patient and two carriers were found to be positive for anti-p21X/p27rex antibodies. The specificity of the ELISA reaction was confirmed by the competitive ELISA test with the highly purified recombinant p21X protein. As of result, we first determined the presence of anti-p21X/p27rex antibodies in a small percentage (3.8%) of the sera from HTLV-I-infected individuals. Even sera from the ATL patients, whose fresh PBMCs contained the transcripts for these proteins, were not found to contain these antibodies, suggesting that the immune response to these proteins is low in HTLV-I-infected humans.
Subject(s)
Enzyme-Linked Immunosorbent Assay/methods , Gene Products, rex/immunology , HTLV-I Antibodies/blood , HTLV-I Infections/immunology , Human T-lymphotropic virus 1/immunology , Retroviridae Proteins, Oncogenic/immunology , Animals , Base Sequence , Cell Line , DNA, Viral , Gene Expression , Guinea Pigs , HTLV-I Infections/blood , HTLV-I Infections/virology , Humans , Molecular Sequence Data , Rabbits , Recombinant Fusion Proteins/biosynthesis , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/isolation & purification , Retroviridae Proteins, Oncogenic/genetics , Retroviridae Proteins, Oncogenic/isolation & purification , VaccinationABSTRACT
Primary RNA transcripts of the human T-cell leukemia virus type 1 (HTLV-1) are processed into mature mRNA by a complex series of splicing events. In this paper, we report the finding of a novel doubly spliced pX mRNA in two out of eight HTLV-1-infected cell lines and in one out of 13 peripheral blood mononuclear cells from HTLV-1-infected individuals. The second splicing for this novel pX mRNA is different from that for the known doubly spliced pX mRNA. A novel acceptor site in this splicing was generated by a single point mutation (G to A) at nucleotide 7,337 of the pX gene. This mRNA contained a complete open reading frame that encodes an amino-terminal truncated p27rex protein with 189 amino acids. A new 25-kD protein was detected in the cell lines expressing the novel pX mRNA by an antibody against the carboxy-terminal peptide of p27rex and was termed p25rex. Although the function of p25rex is not clear, we clarified that p25rex is a cytoplasmic phosphoprotein and its function is different from the transcriptional regulator function of p27rex. The possibility that the mutated virus is replicable only in cells coinfected with the wild type HTLV-1 may explain why the incidence of the mutants observed here is low.
Subject(s)
Gene Products, rex/genetics , Human T-lymphotropic virus 1/genetics , Point Mutation , RNA Splicing , RNA, Messenger/genetics , RNA, Viral/genetics , Amino Acid Sequence , Base Sequence , Cell Line , DNA, Viral , Gene Products, tax/genetics , HTLV-I Infections/microbiology , Humans , Leukocytes, Mononuclear/microbiology , Molecular Sequence DataABSTRACT
In addition to the three typical transcripts such as genomic/gag-pol mRNA, env mRNA and tax/rex mRNA, we previously found the singly spliced pX mRNA, termed p21X mRNA, responsible for producing the p21X protein in human T-cell leukemia virus type 1 (HTLV-1)-infected cells. Our finding of the p21X mRNA being constitutively expressed in the fresh peripheral blood mononuclear cells (PBMCs) from patients with ATL has suggested that the expression mechanism is quite different from that of the others. In this paper, the expression mechanism of p21X mRNA was investigated by analyzing the organization of the proviral genomes present in the representative HTLV-1-infected cell lines which are positive or negative for the expression of p21X mRNA. Southern and PCR analyses show that most of the analyzed cell lines contain both one complete and one defective genome each. However, one cell line without the p21X mRNA expression, C91/PL, contains only the complete genome, suggesting that the complete HTLV-1 has no ability to express p21X mRNA in spite of having the ability to produce the infectious virus. The defective genomes of the p21X mRNA positive cell lines, MT-2 and H582, have a large deletion of the entire pol and parts of the gag and env regions including the common domain of the second exon of the doubly spliced tax/rex mRNA, while another defective genome of the p21X mRNA negative cell line, MT-1, has a deletion within the gag-pol gene. We show that these defective genomes have the ability to express their distinct, defective genomic mRNA, suggesting they are active. The defective genomic mRNAs in MT-2 and H582 cells retain the first splice donor and the second splice acceptor sites, suggesting the possibility of synthesizing p21X mRNA by splicing singly with these sites. These findings assume that defective HTLV-1 genomes deleting the second exon region acquire the ability to express p21X mRNA but no ability to express tax/rex mRNA. Such a deletion may explain the difference between the expression mechanisms in the p21X mRNA transcript and those in the other viral transcripts.
Subject(s)
Genes, env , Genes, pol , Human T-lymphotropic virus 1/genetics , Proviruses/genetics , RNA Splicing , Retroviridae Proteins, Oncogenic/genetics , Base Sequence , Blotting, Southern , Blotting, Western , Cell Line , DNA, Viral , Genome, Viral , Human T-lymphotropic virus 1/physiology , Humans , Molecular Sequence Data , Polymerase Chain Reaction , RNA, Messenger/biosynthesis , RNA, Viral/biosynthesis , Retroviridae Proteins, Oncogenic/metabolism , Sequence Deletion , T-Lymphocytes/cytology , T-Lymphocytes/microbiology , Transcription, Genetic , Virus ReplicationABSTRACT
We amplified the human T-cell leukemia virus type 1 (HTLV-1) protease gene fragment by polymerase chain reaction (PCR) and cloned it into a pUC plasmid vector. DNA sequencing data of the protease gene fragment indicated that it contained an open reading frame capable of encoding the active HTLV-1 protease. To express a fusion protein of beta-galactosidase linked with the HTLV-1 protease in Escherichia coli, a plasmid DNA was constructed by inserting the HTLV-1 protease gene DNA into a procaryotic expression vector, pUEX2, consisting of a lacZ gene directed by a lambda phage Pr promoter and designated pUEX-pro. By Western blot analysis using anti-beta-galactosidase antibody, a bigger molecular size band than that of the control beta-galactosidase molecule was observed in E. coli cells transformed with pUEX-pro but not with control pUEX2, suggesting that the particular fusion protein was successfully expressed. This recombinant protease protein in the E. coli cell lysate was demonstrated to be able to cleave the decapeptide substrates composed of amino acid sequences containing proteolytic cleavage sites in the HTLV-1 gag precursor polyprotein. The gag precursor polyprotein expressed in the mammalian cells by the recombinant vaccinia virus system was also expectedly cleaved by this enzyme. Significant inhibition of this protease activity by pepstatin A, an aspartic proteinase-specific inhibitor, confirms that HTLV-1 protease is a member of the aspartic proteinase group as suggested previously. Since the crude lysate without purification is utilized sufficiently as a native HTLV-1 protease reagent, this protease preparation is easily applicable to the large scale screening of HTLV-1 protease inhibitors for the treatment of diseases caused by HTLV-1.
Subject(s)
Aspartic Acid Endopeptidases/biosynthesis , Endopeptidases/biosynthesis , Escherichia coli/metabolism , Human T-lymphotropic virus 1/enzymology , Amino Acid Sequence , Aspartic Acid Endopeptidases/chemistry , Aspartic Acid Endopeptidases/metabolism , Base Sequence , Blotting, Western , Chromatography, High Pressure Liquid , Cloning, Molecular , DNA, Viral/analysis , Endopeptidases/chemistry , Endopeptidases/metabolism , Gene Products, gag/metabolism , Molecular Sequence Data , Pepstatins/pharmacology , Plasmids , Polymerase Chain Reaction , Recombinant Fusion Proteins/biosynthesis , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/metabolism , Sequence Analysis, DNA , Sequence Homology, Amino Acid , Transformation, Genetic , beta-Galactosidase/metabolismABSTRACT
Although the p21X protein of human T cell leukaemia virus type 1 (HTLV-1) is generally thought to be expressed from a doubly spliced mRNA transcript (tax/rex mRNA) that encodes the p40tax, p27rex and p21X proteins, we have shown previously that a novel, alternatively spliced mRNA transcript (p21X mRNA) is responsible for p21X production in HTLV-1-infected cell lines. In the present study, we analysed expression of p21X mRNA and tax/rex mRNA in uncultured and cultured peripheral blood mononuclear cells (PBMCs) from eight patients with adult T cell leukaemia by using a quantitative polymerase chain reaction coupled to reverse transcription. The results demonstrated that the expression of p21X mRNA occurs constitutively in all uncultured and cultured PBMCs, whereas the expression of tax/rex mRNA is inducible in the cultured PBMCs, as described previously. In uncultured and cultured PBMCs from the one specimen in which p21X mRNA was highly expressed, the p21X protein was detectable by Western blotting. On the other hand, p27rex protein was detectable only after cultivation. These findings indicate that p21X mRNA is constitutively expressed in vivo and is responsible for production of p21X protein.
Subject(s)
Gene Expression Regulation, Neoplastic , Leukemia, T-Cell/metabolism , Leukocytes, Mononuclear/microbiology , RNA, Messenger/biosynthesis , Retroviridae Proteins, Oncogenic/biosynthesis , Adult , Aged , Amino Acid Sequence , Base Sequence , Cells, Cultured , Female , Gene Products, rex/metabolism , Gene Products, tax/metabolism , Humans , Leukemia, Prolymphocytic, T-Cell/genetics , Leukemia, Prolymphocytic, T-Cell/metabolism , Leukemia, T-Cell/genetics , Leukemia-Lymphoma, Adult T-Cell/genetics , Leukemia-Lymphoma, Adult T-Cell/metabolism , Male , Middle Aged , Molecular Sequence Data , Polymerase Chain ReactionABSTRACT
A series of radioiodinated spiperone (2'-ISP) derivatives bearing amide N-alkyl substituents (N-methyl-2'-ISP, N-ethyl-2'-ISP, and N-propyl-2'-ISP) were synthesized and evaluated as potential singlet photon emission computed tomographic radiopharmaceuticals for visualizing dopaminergic receptors. The lipophilicity of these ligands (i.e., the partition coefficient for octanol-phosphate buffer) increased as the chain length increased. Investigation of blood-brain barrier permeability in rats showed a parabolic relationship between the brain uptake index and the partition coefficient. In vitro competitive binding studies showed that the relative affinity for the dopamine D2 receptor was in the order of N-propyl-2'-ISP greater than 2'-ISP greater than N-methyl-2'-ISP approximately N-ethyl-2'-ISP. In vivo biodistribution studies showed that the initial brain uptake correlated fairly well with the brain uptake index and that the kinetics of the radioactivity specifically bound to the striatum were strongly influenced by the dopamine receptor binding affinity of the compounds. Thus, the in vivo behavior of these N-alkylated 2'-ISP derivatives involved a complex interplay between receptor affinity, lipophilicity, and blood-brain barrier permeability.
Subject(s)
Brain/diagnostic imaging , Receptors, Dopamine/drug effects , Spiperone/analogs & derivatives , Animals , Brain Chemistry , Chemical Phenomena , Chemistry, Physical , In Vitro Techniques , Iodine Radioisotopes , Male , Mice , Radionuclide Imaging , Rats , Receptors, Dopamine/metabolism , Spiperone/chemistry , Spiperone/pharmacokinetics , Tissue DistributionABSTRACT
The pX sequence of human T cell leukemia virus type 1 (HTLV-1) has been thought to be expressed as a doubly spliced mRNA that codes for p40tax, p27rex and p21X. However, we identified a novel alternatively spliced mRNA in the HTLV-1 infected cells by using reverse transcription followed by the polymerase chain reaction. This mRNA contains only the first and third exons of the doubly spliced mRNA and encodes only p21X. Our data that this mRNA is responsible for expressing p21X exists in most of HTLV-1 infected cells strongly suggests that p21X may play a crucial role for HTLV-1 replication.
Subject(s)
Human T-lymphotropic virus 1/genetics , RNA Splicing , RNA, Messenger/genetics , Retroviridae Proteins, Oncogenic/genetics , Transcription, Genetic , Base Sequence , Blotting, Southern , Cell Line , Cloning, Molecular , Gene Products, rex/genetics , Gene Products, tax/genetics , Humans , Molecular Sequence Data , Oligodeoxyribonucleotides , Oligonucleotide Probes , Polymerase Chain Reaction/methods , RNA, Viral/genetics , RNA, Viral/isolation & purificationABSTRACT
Human IgG of four subclasses, semi-purified from pooled human serum by a series of DEAE ion exchange and protein A affinity chromatographies, were used as immunogens and initial screening antigens to produce subclass-specific and -restricted monoclonal antibodies (McAbs). These McAbs were bound to CNBr-activated Sepharose 4B and utilized in immunoaffinity chromatography to prepare four polyclonal human IgG subclasses of satisfactory purities, which were then used as final screening antigens. Subclass-specific McAbs thus chosen were further evaluated for subclass- and especially allotype-specificity using a panel of monoclonal IgG myeloma proteins with representative Gm markers for each subclass in micro enzyme-linked immunosorbent assay (ELISA). A total of 10 clones of subclass-specific McAbs (one for anti-IgG1, three anti-IgG2, two anti-IgG3, four anti-IgG4) were established. Among them, IgG2-specific clones of HG2-30F and HG2-56F, IgG3-specific HG3-7C and HG3-32C, and IgG4-specific HG4-53G McAbs were superior to the corresponding specificity standard McAbs chosen by the Human Immunoglobulins Subcommittee of the WHO/International Union of Immunological Societies (IUIS) in 1985. As allotype-specific McAbs, HG1-1E for G1m(az) and HG3-3B for G3m(b) were obtained. In micro ELISA of this study as well as all protocols of the previous WHO/IUIS collaborative study, antigens (myeloma IgG subclasses) were immobilized or fixed to a solid phase, resulting in possible variations in their epitope expressions. We developed a new assay system, micro radioimmunoassay (RIA), in which reactivities of McAbs against free IgG subclasses in solution can be evaluated. HG2-30F, having extremely high reactivities to coated IgG2 in micro ELISA, remarkably reduced its reactivities to free IgG2 in solution in micro RIA. Two other clones also showed some different reactivities in micro RIA and micro ELISA. We believe that this micro RIA is valuable for evaluation of McAbs reactivities against native human IgG subclasses in solution.