Immunoelectrophoresis Research Articles

Feeding Medium-Chain Triglycerides to Rats Decreases Degradation of Sucrase-Isomaltase Complex in the Jejunum

We demonstrated previously that feeding a diet containing medium-chain triglycerides (MCT) to rats causes an increase in the sucrase activity in jejunum relative to diets containing long-chain triglycerides (LCT). To explore the mechanism whereby MCT affect jejunal sucrase activity, 7-wk-old rats were fed a high LCT diet for 7 d, and then they were force-fed either the high LCT diet, a high MCT diet or a high carbohydrate (alpha-methylglucoside) diet for the subsequent 12 h. Feeding the high carbohydrate diet produced significantly greater sucrase and isomaltase activities in both upper and lower jejunum than feeding the LCT diet. Feeding the high MCT diet led to significantly greater sucrase activity in the lower jejunum, but isomaltase activity was not elevated. Rocket immunoelectrophoresis revealed that in the lower jejunum of the rats fed the high MCT diet the proportion of sucrase-isomaltase complex to its degradation product (i.e., isomaltase monomer) was elevated, suggesting that dietary MCT affects sucrase activity by retardation of degradation of sucrase-isomaltase. Because the amounts of total bile acids in the upper jejunum were 50% lower in rats fed the high MCT diet relative to animals fed the high LCT diet, it is likely that MCT feeding decreases secretion of bile into the lumen, which in turn decreases degradation of the sucrase subunit of the sucrase-isomaltase complex.

Bird fancier's lung serodiagnosis by automated r-IgLL1 ELISA

ContextBird fancier's lung (BFL) is the most prevalent form of hypersensitivity pneumonitis (HP) worldwide. The current techniques used for the serological diagnosis of BFL all use crude extracts from feathers, droppings, and blooms as test antigens, which is associated with a lack of standardization and variability of the results. An antigenic protein, immunoglobulin lambda-like polypeptide-1 (IgLL1), isolated from pigeon droppings, was recently identified to be associated with BFL. We used genetic engineering to produce IgLL1 as a recombinant antigen. AimWe aimed to prospectively validate the use of an automated ELISA based on recombinant IgLL1 protein (r-IgLL1) as the test antigen for the serological diagnosis of BFL. MethodsImmunoprecipitation (IP) techniques (immunodiffusion (ID), immunoelectrophoresis (IEP)) and ELISA using r-IgLL1 were performed concomitantly over 10 months on 634 sera from patients with a BFL serodiagnosis request. Questionnaires were sent to obtain details on the avian exposure, clinical data, and final diagnosis. Concordance, sensitivity (Se), and specificity (Sp) of the two techniques were compared. ResultsIn total, 72 completed questionnaires were returned with 18 cases of BFL diagnosed and 54 of non-BFL. The concordance between the ELISA and ID+IEP precipitation techniques was 71%. The combination of immunoprecipitation techniques showed a Se of 78% and a Sp of 67%. The ELISA using r-IgLL1 showed a Se of 89% and a Sp of 91%. The automated r-IgLL1 ELISA test is sufficiently efficient to be used alone for the diagnosis of patients exposed solely to Columbidae.In cases of other avian exposure, the Se and Sp of the r-IgLL1 ELISA used for screening combined with the immunodiffusion test for confirmation were 89% and 93%, respectively. ConclusionsThe automated ELISA using r-IgLL1 is a promising tool for BFL serodiagnosis. Replacing immunodiffusion by the automated ELISA using r-IgLL1 as a screening technique will be the basis of our future strategy for BFL serodiagnosis.

Complement C3d is not associated with axial spondyloarthritis and magnetic resonance imaging changes at the sacroiliac joint

Objective: To investigate the associations between complement C3d and inflammatory and structural changes by magnetic resonance imaging (MRI) at the sacroiliac joints (SIJ) suggestive of axial spondyloarthritis, according to the Assessment of SpondyloArthritis international Society (ASAS) criteria, in patients with low back pain.Method: This was a cross-sectional study of patients referred to the Spine Centre of Southern Denmark owing to unspecified low back pain (Spines of Southern Denmark cohort). The patients were divided into three groups: group 1: patients fulfilling the ASAS criteria for axial spondyloarthritis (axSpA, n = 96); group 2: patients with either a positive MRI of the SIJ and no spondyloarthritis features, or a negative MRI of the SIJ but positive human leucocyte antigen-B27 and one spondyloarthritis feature (non-axSpA, n = 38); group 3: patients with unspecified low back pain for > 3 months (control group, n = 82). Complement C3d was measured with double-decker rocket immunoelectrophoresis and evaluated in relation to the group division and baseline findings by SIJ MRI.Results: In total, 184 C3d analyses were performed. The mean ± sd level of C3d was 33.8 ± 8.1 AU/mL. There were no differences in C3d levels between the three patient groups, mean values being: axSpA = 34.3 ± 7.9 AU/mL, non-axSpA = 33.5 ± 6.9 AU/mL, and controls = 33.4 ± 9.2 AU/mL. The level of C3d was not related to MRI findings.Conclusions: In these patients, complement C3d was not associated with active or structural SIJ changes on MRI suggestive of axial spondyloarthritis.

Establishment of 2nd National Reference Standard for Purified Vi Polysaccharide Typhoid vaccine

This study aimed to establish a national reference standard for the purified Vi typhoid vaccine. The first national standard for the typhoid vaccine established in 2009 is currently outdated; hence, Boryung Biopharma Co. produced 4,070 vials of a candidate vaccine as per the second standard outlined in 2018. The large-scale manufacture of the standard candidate was guaranteed through quality tests and accelerated and long-term stability tests. Reference standards ensure the accuracy and reliability of the results of quality control tests for vaccines. Thus, a collaborative study was conducted to estimate the potency of the candidate material as per the second national standard using the test methods of HPAEC-PAD (High Performance Anion-Exchange Chromatography with Pulsed Amperometric Detection) and rocket immunoelectrophoresis. The HPAEC-PAD result was 51.5 μg/vial (95% confidence interval [CI]: 50.70-52.29 μg/vial). On the other hand, the rocket assay result was 63.4 μg/vial (95% CI: 61.08-65.89 μg/vial). In addition, the precision and proficiency of the institutes was confirmed by the CV and robust Z-score, respectively. The candidate will be registered for use as the second national standard for the purified Vi typhoid vaccine after evaluating its long-term stability. After registration, it will contribute to securing the reliability and consistency of the quality control test results for typhoid vaccines by being used in the national release system and quality control of the manufacturer.

Новые белковые маркеры в прогнозе внутриутробной инфекции при преждевременных родах

Objective. To identify prognostic markers of intrauterine infection (IUI) of the fetus and newborn in maternal serum, amniotic fluid, and umbilical cord serum in case of preterm birth. Patients and methods. We examined 93 pregnant women who had preterm birth (PB) from 24 to 33 weeks of gestation. Thirtyfive women delivered babies without any signs of IUI, while 30 women had newborns with mild IUI (conjunctivitis, lymphadenitis, pyoderma) and 28 women had newborns with severe IUI (early neonatal sepsis, advanced herpesvirus infection, chlamydiosis, candidiasis, pneumonia, and meningitis). We measured the levels of alpha 2-macroglobulin (α2-MG) in maternal serum (MS), umbilical cord serum (UCS) using rocket immunoelectrophoresis and in amniotic fluid (AF) using enzyme-linked immunosorbent assay (ELISA). The level of lactoferrin (LF) was assessed using ELISA. MS and UCS levels of albumin (ALB) were measured using biochemical methods, while AF level ALB was evaluated using rocket immunoelectrophoresis. Statistical analysis was performed using logistic regression. Results. We found no association between the concentration of LF in all biological fluids and the condition of newborns. Levels of ALB in MS and UCS also demonstrated no correlation with the condition of newborns. AF ALB in women who delivered babies with IUI (any grade of severity) was significantly higher than that in women who delivered babies without IUI. Women who delivered babies with severe IUI demonstrated the lowest concentration of α2-MG in their serum, whereas women who delivered babies with mild IUI had the highest α2-MG concentration. The AF α2-MG level was 10 to 20 times higher in women who had babies with IUI (any grade of severity) compared to those who had babies without IUI. Conclusion. Low α2-MG level in MS (<2.2 g/L) along with elevated α2-MG level in AF (≥6.0 mg/L) in 86–89% of PB cases indicated generalized fetal IUI and required urgent delivery without prolongation of pregnancy. Key words: albumin, alpha-2-macroglobulin, intrauterine infection, lactoferrin. premature birth

Human cystic echinococcosis: Serological diagnosis by indirect hemagglutination test, enzyme-linked immunosorbent assay, immunoelectrophoresis, and immunoblotting in surgically confirmed patients versus cases diagnosed by imaging techniques

IntroductionHuman cystic echinococcosis (CE) diagnosis is based on imaging findings and serology. Serology is the first-line test followed by imaging tests. Materials and methodsA total of 268 serum samples from CE patients were included in this study. The serodiagnosis was made simultaneously by indirect hemagglutination assay (IHA), enzyme-linked immunosorbent assay (ELISA), immunoblotting test (IB), and immunoelectrophoresis (IEP). ResultsIn serum samples belonging to surgically confirmed CE patients, we observed a percentage of positivity of 83.7% [77.2%–89.0%] and 80.7% [73.9%–86.4%] for IHA and IgG-ELISA, respectively. IgG-IB was associated with a higher positivity rate than IEP with 81.3% [74.6%–86.9%] and 62.0% [54.2%–69.5%], respectively. IHA and ELISA results were analyzed using cut-off generated by receiver operating curves. The best diagnostic performances were achieved by IHA (cut-off ≥1/128) and ELISA (>1.16 index). Sensitivities reported in patients with suggestive imaging findings and positive Echinococcus IB were 86.2% [78.0%–92.2%], 72.5% [62.8%–80.9%], 49.0% [39.0%–59.1%] for IHA, IgG-ELISA, and IEP, respectively. All tests gave false negative results in the confirmed CE group. Overall, 18.6% of negative results were obtained by IgG-IB test. Cross-reactivities with non-hydatid serum samples were observed in all tests. Only one patient carrying Taenia saginata serum cross-reacted with 8/12 kDa band by IB. We observed specificity at 73% [63.2%–81.4%], 87% [78.8%–92.9%], 99% [94.6%–100.0%], and 99% [94.6%–100.0%] with IHA, IgG-ELISA, IEP, and IgG-IB, respectively. Serology was less sensitive (74%) in lung cysts. Sensitivity was better in liver cysts, especially by IgG-IB (96%).

Models of dependence of the quantity of the <i>Streptococcus pneumoniae biomass</i> and his capsular polysaccharide from the composition of the feeding environment

Aim.The development of a semi-synthetic nutrient medium that provides the maximum amount of capsular polysaccharide (CPS). Materials and methods. We used the strain 521 of S. pneumoniae serotype 23F. Cultivation was carried out in test tubes with 10 ml of polysynthetic nutrient medium of a specific composition. The amount of polysaccharide in the samples was determined using rocket immunoelectrophoresis. Building models and comparing the effects of various components was carried out according to the methodology specified in the tutorial. The calculation of the coefficients of the equation and the assessment of the adequacy of the equations themselves was carried out using RStudio version 1.0.153. Results. As a result of a series of experiments, the coefficients of the regression equations were calculated, their significance was evaluated, and models of dependence of biomass production and CPS were constructed depending on the composition of the nutrient medium. To solve the problem, an experiment was carried out according to the Box-Wilson method. The peptone and glucose concentrations were selected as optimized parameters. The step size ΔSi in the increasing gradient direction was calculated based on the coefficients of the regression equation. At the same time, the exact nature of the dependence was determined. The optimal calculated concentrations of peptone and glucose, at which the formation of CPS is maximum, are 32.6 and 12.1 g/l, respectively. In this case, the forecast yield of the polysaccharide is 239 mg/l. Conclusion. Using the method of fractional factorial experiment, models of the dependence of the biomass amount of S. pneumoniae and its capsular polysaccharide on the composition of the nutrient medium were obtained. The optimal concentrations of the components of the medium were found, which make it possible to increase the level of biomass formation by 10% compared to the standard formulas, and the CPS — by 1.5—2 times.

Immunoregulatory proteins and cytokines in the blood of patients with Graves' disease

Immunoregulatory proteins (alpha-2-macroglobulin, lactoferrin) actively participate in inflammatory and autoimmune processes, affect synthesis and transport of hormones and cytokines, and control cell proliferation and apoptosis. However, the role of these proteins in the pathogenesis of Graves' disease (GD) is poorly understood. Objective - the study objective was to determine blood levels of alpha-2-macroglobulin (α2-MG), lactoferrin (LF), and cytokines (TNF-α, IL-6, IL-8, and IFN-γ) in GD. We determined blood levels of TSH, free T4, TSH receptor antibodies, TNF-α, IL-6, IL-8, IFN-γ, and LF by ELISA as well as α2-MG by quantitative rocket immunoelectrophoresis in 50 patients with decompensated and compensated (4-6 months and 1.5-2 years after treatment onset) GD and 25 healthy females (control group). GD clinically manifested by body weight l in 84% of patients, sinus tachycardia in almost all patients, paroxysmal atrial fibrillation in 18% of cases, endocrine ophthalmopathy in 12% of patients, and neurological changes. In decompensated GD, there was a statistically significant increase in levels of IFN-γ, IL-6, and α2-MG and an especially significant increase in levels of IL-8 and LF. At 4-6 months after treatment onset, clinical manifestations were stopped in all patients, levels of IL-6 and α2-MG decreased, but the concentrations of TSH receptor antibodies (TSHR-Abs), IL-8, IFN-γ, and LF remained elevated. At 1.5-2 years, levels of the studied proteins and cytokines did not differ from those in the control group. An increase in blood levels of IL-8, LF, IL-6, and α2-MG in incident or recurrent GD and a decrease in the levels during treatment confirm involvement of immunoregulatory proteins in pathogenesis of the disease.

Human MOSPD2: A bacterial Lmb mimicked auto-antigen is involved in immune infertility

Autoantibody production is one of the leading factors of immune infertility, an autoimmune disease of the male reproductive system. The potential involvement of MHC-class II derived self-peptides against bacterial proteins in the antisperm antibody (ASA) production has been reported previously. Apparently, Streptococcus agalactiae has been considered as an important pathogen to impart infection-induced infertility in a bacteriospermia associated leukocytospermia (LCS/BS) state. Hence, the present study attempts to confirm S. agalactiae specific Laminin binding protein (Lmb) derived self-peptide (‘KDSYTKKAKAFKKEA’) namely human Motile Sperm domain-containing protein 2 (MOSPD2) as an auto-antigen in LCS/BS condition. Semen samples were collected from infertile men with LCS/BS (n ​= ​17) and their fertile counterparts (n ​= ​10). Gram-positive bacteria were predominantly identified in the entire 17 LCS samples using culture method followed by 16S rDNA sequencing technique. TLRs 2 and 4 expression used as markers of immune response in spermatozoa and sperm dysfunction were elevated in the LCS/BS spermatozoa as compared to their fertile counterparts. A significant increase in oxidative stress indices i.e., protein carbonylation, lipid peroxidation and acridine orange test (AOT), was also observed in the LCS/BS spermatozoa. Spermatozoa lysate (both auto and heterologous), bacterial lysate (control) and synthesized MOSPD2 self-peptide were used to test their antigenicity against the autoantibodies by rocket immunoelectrophoresis (RIEP) assay. Seminal plasma from LCS/BS patients with S. agalactiae was used as the source of autoantibodies. Spermatozoa and bacteria lysate; and MOSPD2 self-peptide were able to bind autoantibodies in the seminal plasma. Besides, the self-peptide showed a dose dependent increase in the precipitation of antibody. T-cell epitope mapping of 48 Enterococcus faecalis and 91Staphylococcus aureus surface proteins confirmed MOSPD2 as a global auto-antigen. Thus, augmentation of TLR expression in LCS/BS spermatozoa inferred MOSPD2 to be a putative immunogen. Altogether, these findings will delineate the significance of MOSPD2 auto-antigen in a bacteria derived immune infertility condition.

POEMS syndrome coexisting with Castleman disease and negative results of hematological examination: report of one case and review of literature

Objective To strengthen the recognition of atypical POEMS syndrome in order to improve diagnosis rate of rare cases of POEMS syndrome. Methods The diagnosis and treatment of a rare case of POEMS syndrome coexisting with Castleman disease but without M protein in serum, urine and bone marrow who was admitted to Xuanwu Hospital, Capital Medical University in November 2017 were retrospectively analyzed with review of the literature. Results The elder male was suspected with a diagnosis of POMES syndrome, but absence of monoclonal plasma cell disease that made it difficult to diagnose. Systemic PET-CT found an active metabolic lesion in left iliac bone. Although the lymph node biopsy had been performed for a diagnosis of Castleman disease, a bone biopsy was also done for a definite diagnosis. Pathological result indicated a plasmacytoma which confirmed a diagnosis of POEMS syndrome without M protein in serum, urine and bone marrow. Literature review suggested that the application of immunofixation electrophoresis was helpful to improve the diagnostic rate of POEMS syndrome. For patients with a suspected diagnosis of POEMS syndrome, bone biopsy, flow cytometry and systemic PET-CT may assist in the search for monoclonal plasma cell. Periphery neuropathy, bone lesion and treatment response were helpful in distinguishing Castleman disease coexisting with POEMS syndrome from Castleman disease without POEMS syndrome. Conclusions When a mandatory major criterion of POEMS syndrome is not sufficient, it should be actively sought, especially for patient with a suspected diagnosis of POEMS syndrome. For patients with multiple lesions, multi-site biopsies are necessary to assist in diagnosis. Key words: POEMS syndrome; Castleman disease; Plasmacytoma; Immunoelectrophoresis; Biopsy

Immunoelectrophoresis: A Method with Many Faces.

Immunoelectrophoresis (IEP) was the first practical method that combined electrophoresis and immunoprecipitation for identifying and characterizing proteins within complex mixtures. Over the years, IEP has been extended to include a variety of techniques and, as a general name, has been applied to virtually any technique that involves electrophoresis and antigen-antibody precipitin reaction for proteins. Because of the diversity in technical details of different IEP versions, the method described here deals only with classic IEP. Although it requires some manual expertise, IEP is versatile, relatively easy to customize, and economical with no need for expensive instrumentation. Further, it can discern identity, partial identity, and nonidentity of the proteins. Any low-viscosity body fluid specimen or, possibly, culture fluid and tissue extract could be tested with IEP if proper antibodies are available. With these attributes, classic IEP remains a valuable tool for clinical diagnostic testing, purity checking of biochemical and pharmaceutical products, and research.

Use of Serological Interactions to Differentiate between Trichoderma harzianum and Trichoderma longbrachiatum Isolated from Cotton Roots

Double diffusion (DD) and immunoelectrophoresis (IE) techniques were used to differentiate among Trichoderma isolates. Each of T. longbrachiatum and T. harzianum isolates tended to group together based on their antigenic composition, which indicated a remarkable overall serological similarity among isolates of each species. However, the serological differences among the isolates did not always reflect the taxonomic differences because some isolates of T. longbrachiatum and T. harzianum showed considerable serological similarity. Thus, DD and IE were not useful techniques in identification of Trichoderma isolates.

Acute-phase response and its biomarkers in acute and chronic urticaria.

IntroductionSince urticaria is a persisting inflammatory disease it is important to establish the prognostic factors for the duration and severity of the disease.AimTo evaluate serum concentrations of selected acute-phase proteins (APP) in patients with various forms of urticaria as compared to healthy volunteers and also to analyze these concentrations in different types of urticaria. Additionally, to evaluate the correlation between serum levels of selected APP and disease activity.Material and methodsSerum concentrations of C-reactive protein (CRP), α1-acid glycoprotein (AGP), α1-antichymotrypsin (ACT), α1-antitrypsin (AT), ceruloplasmin (Cp), transferrin (Tf), α2-macroglobulin (α2M) and haptoglobin (Hp) were measured. Quantitative measurement was conducted using the rocket immunoelectrophoresis. Disease activity was assessed with the use of total symptom score.ResultsAnalysis of serum APP concentrations revealed statistically higher serum concentrations of CRP, AGP and ACT in the entire group of patients with urticaria in comparison with the control group. In the entire group of patients with urticaria, CRP, AGP, ACT, Cp and Hp correlated positively with disease activity, intensity of pruritus and the number and size of urticarial wheals. Statistically lower serum concentrations of CRP, ACT, Cp and Hp were detected in the group of patients with acute urticaria (AU) and angioedema together, compared to the patients suffering from AU only.ConclusionsPatients with symptoms of various forms of urticaria present a distinct profile of serum APP concentrations. A significant correlation observed between CRP, AGP, ACT, Cp, Hp and clinical activity score points to the potential role of APP as markers of the urticarial activity.

Association of serum levels of lipoprotein A-I and lipoprotein A-I/A-II with high on-treatment platelet reactivity in patients with ST-segment elevation myocardial infarction.

ObjectiveHigh-density lipoproteins (HDLs) are a very heterogeneous group of particles. Little is known about the impact of their subfractions including lipoprotein A-I (LpA-I) and lipoprotein A-I/A-II (LpA-I/A-II) on platelet function and high on-treatment platelet reactivity (HPR), particularly in the acute phase of ST-segment elevation myocardial infarction (STEMI). The aim of the study was to evaluate the relationship between serum levels of LpA-I and LpA-I/A-II and HPR in STEMI patients.MethodsFifty-two consecutive STEMI patients (26.9% women, mean age 60.6±9.1 years) were enrolled into this study. Clinical and demographic data were collected and HDL subfractions were measured by rocket immunoelectrophoresis. Platelet reactivity was assessed using light transmission aggregometry and quantitative flow cytometry.ResultsWe found a positive correlation between platelet aggregation after both ADP-5 and ADP-20 stimulation and serum level of LpA-I. Compared with subjects with satisfactory platelet response to clopidogrel, patients with HPR had 32.44% higher serum level of LpA-I (p=0.021). On the other hand, patients with HPR assessed by ADP-5 stimulation had 22.13% lower serum level of LpA-I/A-II (p=0.040). Regression analysis showed that LpA-I [odds ratio (OR) 1.03; 95% confidence interval (CI) 1-1.07; p=0.049] and current smoking (OR 0.18; 95% CI 0.04-0.81; p=0.025) were independent predictors of HPR. With receiver operating characteristic (ROC) curve analysis, we designated the cut-off point at serum level of 57.52 mg/dL for LpA-I for predicting HPR (AUC=0.71, p=0.010).ConclusionThis study showed that higher serum level of LpA-I measured in the acute phase of STEMI is an independent risk factor for HPR. Our study is the first to demonstrate an important and distinct activity of LpA-I and LpA-I/A-II that can prove pleiotropic and different functions of HDL subfractions in acute STEMI.

Characterization of equine vitamin D-binding protein, development of an assay, and assessment of plasma concentrations of the protein in healthy horses and horses with gastrointestinal disease.

OBJECTIVE To purify and characterize equine vitamin D-binding protein (VDBP) from equine serum and to evaluate plasma concentrations of VDBP in healthy horses and horses with gastrointestinal injury or disease. ANIMALS 13 healthy laboratory animals (8 mice and 5 rabbits), 61 healthy horses, 12 horses with experimentally induced intestinal ischemia and reperfusion (IR), and 59 horses with acute gastrointestinal diseases. PROCEDURES VDBP was purified from serum of 2 healthy horses, and recombinant equine VDBP was obtained through a commercial service. Equine VDBP was characterized by mass spectrometry. Monoclonal and polyclonal antibodies were raised against equine VDBP, and a rocket immunoelectrophoresis assay for equine VDBP was established. Plasma samples from 61 healthy horses were used to establish working VDBP reference values for study purposes. Plasma VDBP concentrations were assessed at predetermined time points in horses with IR and in horses with naturally occurring gastrointestinal diseases. RESULTS The working reference range for plasma VDBP concentration in healthy horses was 531 to 1,382 mg/L. Plasma VDBP concentrations were significantly decreased after 1 hour of ischemia in horses with IR, compared with values prior to induction of ischemia, and were significantly lower in horses with naturally occurring gastrointestinal diseases with a colic duration of < 12 hours than in healthy horses. CONCLUSIONS AND CLINICAL RELEVANCE Plasma VDBP concentrations were significantly decreased in horses with acute gastrointestinal injury or disease. Further studies and the development of a clinically relevant assay are needed to establish the reliability of VDBP as a diagnostic and prognostic marker in horses.

Recombinant production of native human α-1-antitrypsin protein in the liver HepG2 cells.

Alpha-1 antitrypsin (A1AT) deficiency is associated with emphysema and liver disease. Only plasma-derived A1AT protein is available for augmentation therapy. Recombinant A1AT (recA1AT) protein expressed in various types of available hosts are either non-glycosylated or aberrantly glycosylated resulting into reduced stability and biological activity. To overcome these limitations, we have used the human liver HepG2 cell line to produce recA1AT protein. HepG2 cells were transfected by A1AT cDNA and cell populations were generated that stably overexpressed A1AT protein. Real-time RT-PCR and rocket immunoelectrophoresis of cell culture supernatants indicated that the transfection resulted more than two-fold increase in A1AT production compared to that of control parental cells. Immunoblot analysis showed that both plasma and HepG2-produced A1AT proteins have identical molecular weight in either glycosylated or deglycosylated form. Partial digestion with PNGase F indicated that the three N-glycosylation sites of recA1AT, like the native A1AT protein in plasma, are occupied. Recombinant A1AT also like the native A1AT was thermostable and could efficiently inhibit trypsin proteolytic activity against BSA and BAPNA chromogenic substrate. The recombinant HepG2 cells cultured in media containing B27 serum free supplement released recA1AT at the same level as in the serum containing media. RecA1AT production in HepG2 cells grown under serum free condition at a large scale could provide a reliable source of the native protein suitable for therapeutic use in human.

Phenotype and genotype analysis of in herited dysplasminogenemia caused by plasminogen Ala601Thr mutation

Objective To detectthe phenotype and gene mutations underlying aninherited dysplasminogenemia pedigree and search the virulence gene. Methods The peripheral venous blood samples of the proband and his family members(fourteen subjects of three generations in total)were collected, and their prothrombin time(PT), activated partial thromboplastin time(APTT), thrombin time(TT), fibrinogen(FIB), fibrinogen degradation products(FDP), D-dimmer(D-D)weretested on a STAGO analyzer, the plasminogen activity(PLG: A)and plasminogen antigen(PLG: Ag)were analyzedby thechromogenic substrate assay and rocket immunoelectrophoresis, respectively. All 19 exons, 5' and 3' untranslated regions of PLG were amplified with PCR. Direct DNA sequencing was used to analyze the amplified products, which were confirmed by backward sequencing. Three bioinformatics online softwares(SIFT, PolyPhen-2 and MutationTaster)were used to forecast the possible impact of the mutations on the protein function. At last, the model analysis of mutate site was taken on a Swiss-Pdb Viewer software. Results The PLG: Avalue of the proband and other 6 family members were decreased to the half, while the PLG: Ag was normal. The D-D and FDP value of the proband, his grandma and father were slightly higher. DNA sequencing has revealed that the proband and the other 6 members of this family had the same mutation of g. 38829G>A in exon 15, leading to the missense mutationp.Ala601Thr.The results of bioinformatics softwares showed that the mutation could affect the thePLGfunction. Protein model analysis indicated that the hydrophobic interaction force and hydrogen bond between the amino acids were changed, which might affect the stability of the PLG.In addition, all the members of this family take the heterozygous SNP of g. 2501C>A in the 5'UTR. Conclusions The p. Ala601Thr found in the inherited dysplasminogenemia pedigree in the exon 15 was responsible for the reduced PLG: A of the family, the dysplasminogenemia and this mutation were both reported for the first time in China.(Chin J Lab Med, 2016, 39: 366-371) Key words: Mutation, Missense; Plasminogen; Polymor phism, single nucleotide

Replacing Acid-Heat Precipitation with Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis Improves Detecting Bence-Jones proteinuria

Background: Detecting Bence-Jones protein in urine is essential for determining plasma cell dyscrasia and multiple myeloma. Conventionally, acid-heat precipitation assay is used for detecting Bence-jones protein in most medical laboratories; however, because of the low accuracy of this test, other more sensitive tests like urine electrophoresis are recommended. Materials and Methods: In this study, the presence of Bence-jones protein in the urine of patients suspected to monoclonal gammopathies were compared using acid-heat precipitation, capillary immunoelectrophoresis and sodium dodecyl sulphate-polyacrylamide gel electrophoresis. Moreover, the subsets of light chain (κ & λ) in capillary immunoelectrophoresis were determined. Results: Our data showed high false negative results (77.7%) using acid-heat precipitation assay in comparison with polyacrylamide gel and capillary immunoelectrophoresis (0%). Conclusion: Collectively, in spite of advantages like easy performance and low cost, acid-heat precipitation assay is not reliable for determining Bence-jones proteinuria in medical laboratories due to its low sensitivity. Therefore, it is recommended to be replaced with more sensitive assays like electrophoresis. K e y w o r ds: Bence-Jones protein, Monoclonal gammopathies, Capillary immuno-electrophoresis, Sodium dodecyl sulphate-polyacrylamide gel electrophoresis

A High-Throughput Size Exclusion Chromatography Method to Determine the Molecular Size Distribution of Meningococcal Polysaccharide Vaccine.

Molecular size distribution of meningococcal polysaccharide vaccine is a readily identifiable parameter that directly correlates with the immunogenicity. In this paper, we report a size exclusion chromatography method to determine the molecular size distribution and distribution coefficient value of meningococcal polysaccharide serogroups A, C, W, and Y in meningococcal polysaccharide (ACWY) vaccines. The analyses were performed on a XK16/70 column packed with sepharose CL-4B with six different batches of Ingovax® ACWY, a meningococcal polysaccharide vaccine produced by Incepta Vaccine Ltd., Bangladesh. A quantitative rocket immunoelectrophoresis assay was employed to determine the polysaccharide contents of each serogroup. The calculated distribution coefficient values of serogroups A, C, W, and Y were found to be 0.26 ± 0.16, 0.21 ± 0.11, 0.21 ± 0.11, and 0.14 ± 0.12, respectively, and met the requirements of British Pharmacopeia. The method was proved to be robust for determining the distribution coefficient values which is an obligatory requirement for vaccine lot release.

Assessment of Resistance in some Sugar Beet Varieties to Powdery Mildew using Crossed Immuno electrophoresis Technique

Assessment of Resistance in some Sugar Beet Varieties to Powdery Mildew using Crossed Immuno electrophoresis Technique