Nobuhiko Kubo

Development of Antibody Against Epitope of Lipoprotein(a) Modified by Oxidation Evaluation of New Enzyme-Linked Immunosorbent Assay for Oxidized Lipoprotein(a)

BackgroundRecently, the biological effects of oxidized lipoprotein(a) [Lp(a)] have been reported to be more potent than Lp(a), the arteriosclerosis-relevant lipoprotein. Thus, investigations with oxidized Lp(a) are expected to provide viewpoints different from the conventional ones based on Lp(a). Methods and ResultsAn anti-Lp(a) monoclonal antibody (161E2) was produced against synthetic peptide antigen (Arg-Asn-Pro-Asp-Val-Ala-Pro). This epitope was characterized as having various properties because its external exposure was induced as a result of oxidative modification. Using 161E2 antibody, we developed a new enzyme-linked immunosorbent assay to measure Lp(a) modified by oxidative stress. The present data demonstrated that oxidized Lp(a) that contains the epitope of 161E2 antibody was present in the serum of humans. Therefore, we used this new enzyme-linked immunosorbent assay to evaluate the role of oxidized Lp(a) in patients with hypertension, which induces oxidative stress . Interestingly, hypertensive patients with complications showed a significantly higher level of oxidized Lp(a) in serum than did normotensive subjects (P <0.01), whereas there was no significant difference in native Lp(a) between normotensive and hypertensive subjects. Importantly, positive immunostaining with 161E2 monoclonal antibody was found in the human arteriosclerotic tissue. ConclusionsWe developed a new antibody against an epitope in Lp(a) as a result of oxidation treatment but not in native Lp(a). The present data demonstrated in vivo the presence of oxidized Lp(a) in the atherosclerotic tissue and its elevation in hypertensive patients. The presence of oxidized Lp(a) may be important in understanding the role of Lp(a) in cardiovascular disease.

A new Lp(a) assay that is unaffected by apo(a) size polymorphism

We developed sandwich ELISA methods in which anti-apo(a) kringle 4 type 5 through protease (K4 x 5-Pro) domain monoclonal antibody (clone: 203E2) is employed in each instance as the capture antibody and one of the three species of monoclonal antibody [Mab] (clones: 108B8, 202A9, 2B3) is used as the labeled antibody. Using serum containing apo(a) with 34 repeats of kringle 4 as the calibrator, a commercial kit using anti-Lp(a) polyclonal antibody (Pab) or anti-apo(a) Mab overestimated the Lp(a) concentration in samples containing apo(a) with more than 34 repeats of kringle 4 and underestimated the Lp(a) concentration in samples containing apo(a) with fewer than 34 repeats of kringle 4. Moreover, it was demonstrated that the ratios of commercial kit values to anti-apo(a) K4 x 5-Pro Mab-based method values increased as the size of apo(a) increased. The ratios of apo(a) K5 x Pro Mab-based method values to anti-apo(a) K4 x 5-Pro Mab-based method values, however, remained almost constant regardless of the size polymorphism. Thus, we suggest that apo(a) size heterogeneity can significantly affect Lp(a) measurement in the Lp(a) assay using anti-Lp(a) Pab. The novel Lp(a) assay method, using only anti-apo(a) K4 x 5-Pro Mab, is not subject to this phenomenon.

Occurrence of heavy chain of 7S IgM half-molecule whose NH2-terminal sequence is identical with that of κ light chain sequence in patients with Waldenstrom macroglobulinemia

We report a rare case of a half molecule 7S IgM (HM 7SIgM) consisting of a unique mu heavy chain and kappa light chain found in blood and urine samples from a patient with primary Waldenstrom macroglobulinemia. A 64kDa abnormal immunoglobulin was detected in serum and urine by immunoblot method, and purified by a two-dimensional SDS-PAGE after separation from IgG and albumin fractions on gel filtration. NH2-terminal amino acid sequence analysis of the heavy chain revealed that residues 1-20 were identical to those of the NH2-terminal region of kappa light chain derived from the same patient. This sequence was then followed by a sequence that could not be identified by a computer homology search on the protein database. Using polypeptide segments obtained from the unique mu chain by digestion with endopeptidase, we identified a sequence spanning from residue 127 in the variable region of the known mu chain to residue 19 in the known CH1 domain and a sequence spanning from residues 67-82 in the heavy chain variable region class II. From these results, we concluded that the 64 kDa protein was an abnormal half molecule 7S IgM consisting of a kappa light chain and a unique mu heavy chain of 35 kDa polypeptide in which the NH2-terminal 20 amino acids were replaced by 20 amino acids derived from the sequence of kappa light chain in the NH2-terminal region.

Functional T cell subpopulations responsible for hyposecretion of IL-2 in patients with systemic lupus erythematosus

SummaryThe ability of T cells to secrete IL-2 in patients with systemic lupus erythematosus (SLE) was investigated. In patients with SLE, impaired IL-2 production by peripheral blood lymphocytes stimulated with mitogens is well known. In this paper, we report that purified T cells stimulated with mitogens, in the presence of Epstein-Barr virus transformed B cells (B-LCL) as an accessory cell, however, could secrete a large quantity of IL-2 as much as normal T cells. In order to study this potential capacity of T cells to secrete IL-2 in patients with SLE, IL-2 secreting T cells were examined. To obtain these cells, T cells were divided into cluster forming cells and noncluster forming cells after short culture of T cells with accessory cells in the presence of Con A. Then the ability of IL-2 production in two kinds of separated T cells was examined. We found that 1) after short culture with B-LCL, the cluster forming T cells could secrete IL-2 when cultured again, but noncluster forming T cells could not, even in the presence of B-LCL, 2) after short culture with macrophages, in normal donors and SLE patients, noncluster forming T cells were able to secrete a greater amount of IL-2 than cluster forming and undivided T cells when cultured with B-LCL. These results suggested that IL-2 secreting T cells activated with Con A in the presence of macrophages were shown to be not all of, but a part of them, and that in SLE T cells which secreted IL-2 in the presence of macrophages might be impaired but other IL-2 secreting T cells might remain intact.

Functional T-cell subset defined by cluster formation with EB virus transformed B-cells

SummaryThis study compares functional properties of T-cells capable of forming clusters with EB virus transformed B lymphoblastoid cells (B-LCL) as accessory cells (A -cells). T-cell functional properties examined include T-cell activation, interleukin 2 (IL-2) production and cell proliferation. In addition, functional properties were compared with the presence or absence of surface markers. T-cells were divided into those that formed clusters with the B-LCL (clustered T-cells) and those that failed to form clusters (nonclustered T-cells). Each subpopulation of T-cells was also incubated with B-LCL and Concanavalin A (Con A) to test for changes in proliferative capabilities. Functional studies indicated that IL-2 activity was higher in the culture supernatant fluids from clustered T-cells than nonclustered T-cells. Spontaneous proliferation of clustered T-cells was equivalent to proliferation of clustered T-cells stimulated with Con A or human IL-2. However, weak spontaneous proliferation by non clustered T cells was enhanced after culturing with Con A or human IL-2. The nonclustered T-cells also produced less IL-2 in cultures containing both B-LCL or Con A. Quantification of T-cell surface markers showed that the expression of Tac antigen was greater on the clustered T-cells than on the nonclustered T-cells. These data suggest that functionally different T-cell subsets can be identified and isolated by their capacity to form clusters with B-LCL A-cells.