间日疟原虫乳酸脱氢酶的表达、纯化及免疫活性的鉴定

目的在大肠杆菌中表达间日疟原虫乳酸脱氢酶（PvLDH）与谷胱甘肽S-转移酶（GST）融合蛋白，对重组蛋白进行纯化并测定其免疫活性。方法将构建的LDH／pGEX-4T-1重组菌BL21接种于LB培养基中，异丙基硫代半乳糖苷（IPTG）诱导重组融合蛋白的表达，重组蛋白纯化后免疫小鼠制备特异性血清，ELISA检测血清效价，Western-blotting鉴定其免疫活性。结果重组质粒在大肠杆菌中表达PvLDH与谷胱甘肽S-转移酶（GST）的融合蛋白，表达产物主要以包涵体形式存在，经复性、纯化后免疫小鼠，能诱导小鼠产生特异性体液免疫应答．ELISA法测效价为1：51200．Western-blotting结果显示该血清能特异性与间日疟和恶性疟患者全血发生反应．而不能与正常人起交叉反应。结论PvLDH在大肠杆菌中获得高效表达且表达产物具有良好的抗原性和免疫原性。     

Antibodies to lactate dehydrogenase of Plasmodium knowlesi are specific to Plasmodium species

Polyclonal immune monkey serum raised against schizonts of Plasmodium knowlesi (H-strain) showed the presence of antibodies to lactate dehydrogenase (LDH) of P. knowlesi by immunodot enzyme staining method. The anti-LDH antibodies are most probably directed towards an epitope distinct from the catalytic site as shown by the specific enzyme staining of LDH after binding with antibody on nitrocellulose paper. These antibodies showed reactivity with LDH from different strains (H, P and W1 strains of P. knowlesi) and species (P. cynomolgi B, P. berghei, P. yoelii, P. falciparum and P. vivax) of malarial parasites but did not cross-react with three isoenzymic forms of mammalian LDH (A4, B4 and C4) as well as with LDH from some protozoan and helminth parasites. These findings suggest that the anti-LDH antibodies have defined specificity to Plasmodium spp.     

EB病毒抗原单克隆抗体的特异性研究

5种EB病毒细胞系通过三步放大间接免疫过氧化物酶方法,检测抗EB病毒抗原的单克隆抗体(McAb)—病毒壳抗原(VCA)、膜抗原(MA),早期抗原(EA)、早期弥漫性抗原(EA—D)、早期限制性抗原(EA—R)、核抗原(EBNA)、核抗原第2型(EBNA—2)及潜在性膜蛋白(LMP)。结果表明McAb、EBNA、EBNA—2和EA有较高特异性,VCA和EA—R有一定特异性,MA、LMP和EA—D未得到预期的特异性反应。     

Antibodies to Plasmodium falciparum rifin proteins are associated with rapid parasite clearance and asymptomatic infections

Plasmodium falciparum rifin proteins, belonging to the largest known family of variable infected-erythrocyte surface-expressed proteins encoded by rif genes, were recently shown to be capable of inducing a strong immune response in P. falciparum-infected adults living in an area in Gabon where malaria is endemic. In the present study, the levels of antirifin antibodies were analyzed in serum obtained from 60 children from the same area who were admitted to hospital and diagnosed with severe malaria. High antirifin antibody concentrations in these individuals correlated significantly with their capacity to rapidly clear their parasites from the circulation after the start of chemotherapy. A doubling of antirifin antibody concentrations reduced the clearance time by 5 h (95% confidence interval, 4.1 to 6.9 h). In the same group of children, who were followed up for 2 years, antirifin antibody levels did not correlate with a reduced rate of reinfection or with a delay in the time to the first reinfection. However, the initial antirifin antibody levels were sustained over the study period. The likelihood that these antibodies could confer a certain degree of protection against malaria is supported by our findings of statistically higher levels of antirifin antibodies to all four rifin proteins in a group of 42 asymptomatic parasitemic children.     

Regulatory Role of Supramolecular Alcohol Dehydrogenase and Lactate Dehydrogenase Complex in Cell Mitochondria

The existence of a supramolecular alcohol dehydrogenase–lactate dehydrogenase complex was demonstrated. Enzyme activities were evaluated in a preparation of mitochondrial membranes from mouse liver. The role of the enzyme complex (alcohol dehydrogenase–lactate dehydrogenase) in the regulation of pyruvate and acetaldehyde metabolism in the cell was studied.     

Antibodies to variable Plasmodium falciparum-infected erythrocyte surface antigens are associated with protection from novel malaria infections

In areas of unstable transmission malaria affects all age groups, but the malaria incidence is lower in adults compared to children and teenagers. Under such conditions subclinical Plasmodium falciparum infections are common and some infections are controlled, because blood parasitaemia is maintained at low densities. Here, we test the hypothesis that the presence or absence of antibodies against variant antigens on the surface of P. falciparum-infected erythrocytes protect individuals against some infectious challenges and render them susceptible to others. Plasma collected in Daraweesh, eastern Sudan, before and after the malaria season from individuals who had (susceptible) or did not have malaria (protected) during the season, were tested for reactivity against variant antigens on the surface of nine parasite isolates by flow cytometry. Both protected and susceptible individuals acquired antibodies to variant antigens during the malaria season. The presence of antibody to a Ghanaian isolate before the season was statistically significantly associated with protection against malaria. When considering all nine isolates, the patterns of antibody acquisition differed between susceptible and protected individuals. Together, the results indicate that pre-existing anti-PfEMP1 antibodies can reduce the risk of contracting clinical malaria when challenged by novel parasite clones expressing homologous, but not heterologous variable surface antigens. The results also confirm that antibodies to variant antigens are induced by both clinical and subclinical infections, and that antibodies against several var sero-types are induced during an infection.     

Specific Antibodies are Responsible for the Low Dose Suppression of the Immune Response to Thymus-Independent Antigens

Animals primed to the thymus-independent antigen native dextran B512 could not respond to the FITC hapten after immunization with native FITC-dextran. The degree of suppression of the anti-FITC response paralleled the increase of the anti-α1-6 PFC after priming with different doses of dextran. Suppression could be passively transferred with serum from dextran primed animals. Young animals that are very low- or non-responders to the α1-6 epitope of dextran B512, failed to suppress the anti FITC PFC response after priming with native dextran. We conclude that antibodies against the dextran carrier were responsible for the suppressed response to the FITC epitope in FITC-dextran immunized animals that had been primed with low doses of the dextran carrier.     

Antibodies to Citrullinated Vimentin are a Specific and Sensitive Marker for the Diagnosis of Rheumatoid Arthritis

Background

The last 5 years have seen the emergence and establishment of antibodies to citrullinated antigens as the diagnostic marker for rheumatoid arthritis (RA). Initially, these were detected using a synthetic peptide, which has undergone a number of modifications to give a diagnostic test with a sensitivity of 65–80% and a specificity of >95%. Antibodies to citrullinated vimentin were first described in 1994 as a highly specific marker for RA (anti-Sa). However, no easily performed assay for these antibodies has been available.

Methods

We have examined the use of a ELISA-based assay with a mutated citrullinated vimentin (MCV) antigen (Orgentec, Mainz, Germany) to assess the diagnostic and prognostic utility of this antibody in RA.

Results

Antibodies to MCV were detected in the sera of 74% RA patients (specificity 96%), 2% systemic lupus erythematosus, 14% Sjögren’s syndrome, and 2% scleroderma. Anti-MCV was not detected in sera from healthy blood donors. There was no difference in the frequency of antibodies detected in RA patients with early (<2 years) or chronic (>2 years) disease. There was no significant variation in anti-MCV antibody concentrations in early RA patients over a 52-week period. No significant change was observed with time between the two treatment groups of methotrexate alone or methotrexate plus infliximab.

Conclusions

Antibodies to MCV are a specific and sensitive marker for the diagnosis of RA.

     

Lactate Dehydrogenase Isoenzyme Pattern: Marker for Differentiation of Lymphocytes

The lactate dehydrogenase isoenzyme pattern has been determined in different murine thymocyte cell populations. Enrichment of thymocytes with more mature cells through total irradiation results in a smaller percentage of LDH-1, LDH-2, LDH-3 and a greater percentage of LDH-4 and LDH-5. Fractionation of normal thymocytes by velocity sedimentation at unit gravity yields a fraction of cells with a high sedimentation rate. LDH-1 and LDH-2 formed a smaller percentage of the total enzyme activity in these cells. These findings indicate that the LDH isoenzyme distribution is a marker for differentiation of thymocytes.     

Isoenzymes of Lactate Dehydrogenase in Amnionic Fluid in Early Pregnancy

Electrophoretic fractionation of lactate dehydrogenase in amnionic fluid was utilized to demonstrate the five isoenzymes usually found in a variety of human tissues. The anaerobic portion of the isoenzyme spectrum was prominent, and the percentage distribution may be related to the metabolic activities of tissues contributing to the formation of the fluid. In contrast to the placenta, an “extra” isoenzyme was not found in the fluid.     

Prevalence of Specific Antibodies to Chlamydia pneumoniae in Korea

To clarify the endemic status of Chlamydia pneumoniae in Korea, the incidence of antibodies in 564 serum samples from healthy individuals, patients with respiratory infection, and cord blood specimens was evaluated. We conclude that C. pneumoniae infection is highly endemic in Korea and that this infection is associated with acute respiratory diseases.     

Composition of a Suppressor Factor That Inhibits the Immune Response to Lactate Dehydrogenase B

Hybridomas obtained by fusion of lactute dehydrogenase B (LDH_B)-aciivatcd suppressor T (Ts) cells with the BW5147 thymoma produce a suppressor factor (TsF) that inhibits the proliferation of LDH_B-activated helper T (Th) cells. A similar factor (TsE) is contained in the extract of suppressor hybridomas. Both TsF and TsE are specifically retained by LDH_B-immunoadsorbent columns. Both consist of two components, an antigen-binding component (ABC) and possibly a major histocompatibility complex (MHC) component. The latter reacts with certain monoclonal antibodies specific for MHC determinants. The two components arc covalently associated in the IsF and noncovalcntly associated in TsE. Mixing of the two components reconstitutes the activity of the TsF or TsE. Disruption of the ABC's tertiary structure results in its inability to reconstitute suppressive activity on mixing with the MHC components. The ABC may contain an intrachain disulphide hond(s). Suppression is obtained when Th cells are incubated first with the ABC and then with the MHC component or vice versa, provided that the incubation period is at least 4 h. The MHC component is also produced by nonsuppressor hybridomas but not by mitogen-stimulated blasts or by the parental thymoma. The TsF is a glyeoprotein with a molecular weight ot about 120.000 to 160.000. The molecular weight of the ABC is about 76,000–86,000 and of the MHC component about 30,000–37,000.     

Sulphonamide Antibodies: From Specific Polyclonals to Generic Monoclonals

Polyclonal antibodies (PAbs) against eight different sulphonamides were raised in rabbits. The aromatic amino group, common to all sulphonamides, was used for linking the different sulphonamides to the carrier proteins (bovine serum albumin (BSA) and keyhole limpet haemocyanin (KLH)) and enzyme (horseradish peroxidase (HRP)), using different coupling procedures. The competitive direct ELISAs (cdELISAs) developed with these antisera and HRP-conjugates showed high sensitivity (0.2- 8.0 ng ml^-1 at 50% inhibition) and high specificity. The performances of these antibodies were compared with PAbs raised in mice against two sulphonamide derivatives (N¹ -[4-(carboxymethyl)-2-thiazolyl]sulphanilamide (TS) and N¹-[4-methyl-5-\[2-(4-carboxyethyl-1-hydroxyphenyl)]-azo-2-pyridyl]sulphanilamide (PS)) linked to proteins (BSA and KLH) in such a way that the common aromatic amino group was distal to the protein. In competitive indirect ELISAs (ciELISAs), these PAbs recognized several structurally different sulphonamides. The PAbs from mice immunized with TS-BSA reacted with sulphonamides containing thiazolyl, thiadiazolyl, pyridazinyl and isoxazolyl groups. The PAbs from mice immunized with PS-KLH reacted with sulphonamides containing pyrimidinyl, pyridazinyl, quinoxalinyl and pyridinyl groups. The spleen cells of the mice were fused with myeloma cells to obtain monoclonal antibodies (MAbs) producing hybridomas. So far, with only one of the mice (immunized with TS-BSA), this resulted in four different MAbs which recognized several sulphonamides. By use of the best MAbs (27G3A9B10 and 4E10B12B6E12) and an optimized ciELISA protocol, eight structurally different sulphonamides showed 50% inhibition at concentrations less than 100 ng ml^-1 or 5 ng/well. However, other relevant sulphonamides (such as sulphadimidine, sulphatroxazole and sulphachloropyrazine) were detected at a high level only.     

A New Isoenzyme of Lactate Dehydrogenase Associated with Sickle Cell Hemoglobin

Electrophoretic fractionation of lactate dehydrogenase in the cytoplasm of red blood cells containing various types of hemoglobin has revealed a sixth isoenzyme occurring regularly in individuals in whom the major part of the hemoglobin is of the sickle cell type. The newly discovered fraction occurs between the normally present third and fourth components. This discovery marks the first time that an additional lactate dehydrogenase isoenzyme has been found in connection with a clinical abnormality. The isoenzyme may serve as a genetic and clinical marker.     

Antibodies are challenged

Studies on antibody were documented as early as in 1890. They are proteins found in blood or other body fluid of vertebrates, and are used by the immune system to identify and neutralize antigens (like foreign objects, pathogens like bacteria and virus etc). Antibodies are dominating the biomedical research field especially detection, imaging and inhibition of biological target molecules, and therapeutics so far. However, recently aptamer has been seen to compete with antibodies in all the above areas. Aptamers are single stranded oligonucleotides or peptides that fold into well defined three dimensional shapes, allowing them to bind their targets with high affinity and specificity. Aptamer technology is relatively new and discovered only in 1990. Because of synthetic origin and similar function as antibodies, they are often termed as chemical antibody. Within 25 years of discovery, the first generation of aptamer drug "Macugen" is already marketed and available for public use. The Global market for aptamer was $236 million in 2010 and is expected to be valued at nearly $1.8 billion by 2014, with a growing compound annual growth rate of 67.5%. Various drugs being on the pipeline for clinical trials this emerging field of medical biotechnology is raising significant interest. This article gives an overview how aptamers are similar yet distinctly different from antibodies in terms of synthesis, handling, and applicability.     

重组日本血吸虫乳酸脱氢酶（SjLDH）酶学特性的研究

目的获得重组SjLDH主要的酶动力学参数。方法分光光度计测定NADH在340nm处吸光度的变化，检测不同pH及温度下重组蛋白催化正、逆反应的效率以确定其最佳pH、最佳温度；分别固定底物NADH、丙酮酸、NAD＋及乳酸浓度，分别测定丙酮酸、NADH、乳酸及NAD^＋在不同浓度下的反应速度，计算机Lineweaver-Burk双倒数方程回归计算各底物的米氏常数（Km值）、最大反应速度（Vmax）。并比较各自的Vmax／Km值。结果重组蛋白酶活性为379U／mg。催化正、逆反应的最佳pH分别为pH6．0—7．0和pH9．0—10．0；催化正、逆反应的最佳温度分别为37—60℃及40—50℃，后者在70℃时仍有较高催化活性；在NADH辅酶作用下，重组SjLDH催化丙酮酸还原为乳酸的最大反应速度是催化NAD＋作用下乳酸氧化为丙酮酸的21倍。比较丙酮酸与乳酸的Vmax／Km值。前者是后者的23倍。而NADH的Vmax／Km值是NAD＋的7倍。结论重组蛋白在生理条件下主要催化丙酮酸还原为乳酸的反应。     

Combining Parasite Lactate Dehydrogenase-Based and Histidine-Rich Protein 2-Based Rapid Tests To Improve Specificity for Diagnosis of Malaria Due to Plasmodium knowlesi and Other Plasmodium Species in Sabah,Malaysia

Plasmodium knowlesi causes severe and fatal malaria in Malaysia. Microscopic misdiagnosis is common and may delay appropriate treatment. P. knowlesi can cross-react with “species-specific” parasite lactate dehydrogenase (pLDH) monoclonal antibodies used in rapid diagnostic tests (RDTs) to detect P. falciparum and P. vivax. At one tertiary-care hospital and two district hospitals in Sabah, we prospectively evaluated two combination RDTs for malaria diagnosis by using both a pan-Plasmodium-pLDH (pan-pLDH)/P. falciparum-specific-pLDH (Pf-pLDH) RDT (OptiMAL-IT) and a non-P. falciparum VOM-pLDH/Pf-HRP2 RDT (CareStart). Differential cross-reactivity among these combinations was hypothesized to differentiate P. knowlesi from other Plasmodium monoinfections. Among 323 patients with PCR-confirmed P. knowlesi (n = 193), P. falciparum (n = 93), and P. vivax (n = 37) monoinfections, the VOM-pLDH individual component had the highest sensitivity for nonsevere (35%; 95% confidence interval [CI], 27 to 43%) and severe (92%; CI, 81 to 100%) P. knowlesi malaria. CareStart demonstrated a P. knowlesi sensitivity of 42% (CI, 34 to 49%) and specificity of 74% (CI, 65 to 82%), a P. vivax sensitivity of 83% (CI, 66 to 93%) and specificity of 71% (CI, 65 to 76%), and a P. falciparum sensitivity of 97% (CI, 90 to 99%) and specificity of 99% (CI, 97 to 100%). OptiMAL-IT demonstrated a P. knowlesi sensitivity of 32% (CI, 25 to 39%) and specificity of 21% (CI, 15 to 29%), a P. vivax sensitivity of 60% (CI, 42 to 75%) and specificity of 97% (CI, 94 to 99%), and a P. falciparum sensitivity of 82% (CI, 72 to 89%) and specificity of 39% (CI, 33 to 46%). The combination of CareStart plus OptiMAL-IT for P. knowlesi using predefined criteria gave a sensitivity of 25% (CI, 19 to 32%) and specificity of 97% (CI, 92 to 99%). Combining two RDT combinations was highly specific for P. knowlesi malaria diagnosis; however, sensitivity was poor. The specificity of pLDH RDTs was decreased for P. vivax and P. falciparum because of P. knowlesi cross-reactivity and cautions against their use alone in areas where P. knowlesi malaria is endemic. Sensitive P. knowlesi-specific RDTs and/or alternative molecular diagnostic tools are needed in areas where P. knowlesi malaria is endemic.     

Antibodies to malaria peptide mimics inhibit Plasmodium falciparum invasion of erythrocytes

Apical membrane antigen 1 (AMA1) is expressed on the surfaces of Plasmodium falciparum merozoites and is thought to play an important role in the invasion of erythrocytes by malaria parasites. To select for peptides that mimic conformational B-cell epitopes on AMA1, we screened a phage display library of >10(8) individual peptides for peptides bound by a monoclonal anti-AMA1 antibody, 4G2dc1, known to inhibit P. falciparum invasion of erythrocytes. The most reactive peptides, J1, J3, and J7, elicited antibody responses in rabbits that recognized the peptide immunogen and both recombinant and parasite AMA1. Human antibodies in plasma samples from individuals exposed to chronic malaria reacted with J1 and J7 peptides and were isolated using immobilized peptide immunoadsorbents. Both rabbit and human antibodies specific for J1 and J7 peptides were able to inhibit the invasion of erythrocytes by P. falciparum merozoites. This is the first example of phage-derived peptides that mimic an important epitope of a blood-stage malaria vaccine candidate, inducing and isolating functional protective antibodies. Our data support the use of J1 and J7 peptide mimics as in vitro correlates of protective immunity in future AMA1 vaccine trials.