scholarly journals Cirrhosis of liver: Comparative cross reactivity for quantification of SERPINA4/Kallistatin

2021 ◽  
Vol 2 (1) ◽  
pp. 01-05
Author(s):  
Krishna Nallagangula
Keyword(s):  
Liver Dysfunction ◽  
Healthy Subjects ◽  
Polyclonal Antibodies ◽  
Cross Reactivity ◽  
Cirrhotic Liver ◽  
Research Centre ◽  
Pvdf Membrane ◽  
Sds Page ◽  
Cirrhosis Of Liver ◽  
Monospecific Antibodies

Cirrhosis of liver is an end stage of chronic liver insults from varied etiologies which leads to impaired liver functions. Proteins expressed from liver and enters into circulation reflects degree of liver dysfunction. Serpins (Serine protease inhibitors) are class of plasma proteins expressed from liver; SERPINA4/Kallistatin is a multifunctional serpin clade A protein expressed from liver and concentration in serum is the reflection of extent of liver dysfunction. The present study aimed to compare cross reactivity of serpins for polyclonal and monospecific antibodies in both cirrhotic liver and healthy subjects. Blood samples were collected from 20 subjects (10 cirrhotic liver, 10 healthy) from R. L. Jalappa Hospital and Research Centre, Kolar, Karnataka, India. Separation of proteins was carried out by SDS-PAGE. Cross reactivity study was analyzed using western blot. Proteins present in cirrhotic liver and healthy subject’s serum were separated by SDS PAGE. There was no band detection on both (cirrhotic liver and healthy) PVDF (polyvinylidene diflouride) membranes with polyclonal antibodies. However, a significant band was observed with protein of interest in healthy PVDF membrane with monospecific antibodies. There was no band in cirrhotic liver PVDF membrane even with monospecific antibodies. Comparative cross reactivity analysis of serpins in quantification of SERPINA4/Kallistatin in the present study demonstrated that there will not be any immunological cross reactivity between serpins and SERPINA4/Kallistatin due to the absence of identical epitope in cirrhotic liver and healthy subjects.

scholarly journals Development of monospecific polyclonal antibodies against hypervirulent Klebsiella pneumoniae

2020 ◽  
Vol 22 (1) ◽  
Author(s):  
Darniati Darniati ◽  
Surachmi Setiyaningsih ◽  
Dewi Ratih Agungpriyono ◽  
Ekowati Handharyani
Keyword(s):  
Klebsiella Pneumoniae ◽  
Polyclonal Antibodies ◽  
Pcr Amplification ◽  
Cross Reactivity ◽  
Klebsiella Pneumonia ◽  
Purification Method ◽  
Sds Page ◽  
New Zealand White Rabbits ◽  
Cluster Gene ◽  
Monospecific Antibodies

Abstract. Darniati, Setiyaningsih S, Agungpriyono DR, Handharyani E. 2021. Development of monospecific polyclonal antibodies against hypervirulent Klebsiella pneumoniae. Biodiversitas 22: 99-105. The aim of the research was to produce monospecific antibodies against Klebsiella pneumoniae serotype K1 and K2. Klebsiella pneumonia isolates were recovered from pneumonic lungs of Aceh cattle. The bacteria were identified by rpoB (RNA polymerase β subunit) PCR amplification specific for K. pneumoniae. The presence of  capsule cluster gene magA  and k2A  was used to characterize capsular serotype K1 and K2, respectively. Antisera were produced using serial immunization of New Zealand White rabbits with K. pneumoniae serotypes K1 and K2. The presence of antibodies was determined by using immunodiffusion test and purification was performed by immunoaffinity purification method. K. pneumoniae antibodies began to appear on the 14th day after the first injection and progressively intensified after the 1st and 2nd booster. The cross-reactivity between the antigen was eliminated by absorbing the antisera with the opposite antigen and several serotypes of non-K1/K2. After purification, serum protein concentrations were substantially decreased from 58.48 µg/µL and 53.99 µg/µL to 2.38 µg / µL and 3.72 µg / µL for K1 and K2, respectively). The SDS-PAGE analysis showed two bands with molecular weight at 54 kDa and 25 kDa representing the heavy and light chains of immunoglobulin G, respectively. Purified polyclonal antiseras against K. pneumoniae serotypes K1 and K2 showed high affinity and specificity for homologous antigens in immunodiffusion and direct agglutination tests, and immunohistochemical staining.

scholarly journals Design of antigen synthesis and preparation and characterization of specific and eurytopic antibodies against B-group aflatoxins

2020 ◽  
pp. 52-60
Author(s):  
Yanang Wang ◽  
Hanna Fotina
Keyword(s):  
Indirect Elisa ◽  
Polyclonal Antibodies ◽  
High Titer ◽  
Cross Reactivity ◽  
Synthesis Methods ◽  
Inhibition Concentration ◽  
Artificial Antigen ◽  
Sds Page ◽  
Sensitivity Specificity ◽  
Immune Efficacy

The aim of this study was to prepare B-group aflatoxins(BGAFs) antibody with strong specificity and good eurytopicity. According to the molecular structure and active site of aflatoxin B1 (AFB1), the BGAFs artificial antigen AFB1-BSA was prepared by 6 methods such as oxime active ester(OAE)，methylation of ammonia(MOA)，mixed anhydride(MA)，semi acetal(SA)，epoxide(EP) and enol ether derivative(EED) and identified by UV and SDS-PAGE. Polyclonal antibodies against AFB1(AFB1 pAb) were prepared by immunizing New Zealand rabbits with AFB1-BSA, and the titers of AFB1 pAb was detected by indirect ELISA, the sensitivity of AFB1 pAb was analyzed by indirect competitive ELISA(icELISA) and the specificity and eurytopicity of AFB1 pAb was analyzed by cross-reactivity(CR) test. The results showed that AFB1-BSA was synthesized successfully and the best one was OAE method among 6 synthesis methods of BGAFs artificial antigen and its conjugation ratio of AFB1 to BSA was about 8.46∶1. The immune efficacy of OAE method was the best, its AFB1 pAb had high titers of 1∶（1.28×104） by indirect ELISA, a good sensitivity with the 50% inhibition concentration(IC50) of 10.32 μg/L to AFB1 by icELISA and a high CR to AFB2 of 75.21%, AFG1 of 44.13%, AFG2 of 14.72%, AFM1 of 16.36% and AFM2 of 1.44%, respectively. In this study, AFB1 pAbs with high titer, sensitivity, specificity and eurytopicity were prepared, which laid a matter and technical foundation for the establishment of BGAFs immunoassay.

scholarly journals Study on Insulin Like Growth Factor-1 As a Marker of Severity of Liver Dysfunction in Patients with Liver Cirrhosis

2019 ◽  
Vol 18 (1) ◽  
pp. 3-7
Author(s):  
Aloke Kumar Raha ◽  
Mohammad Izazul Hoque ◽  
Mamun Al Mahtab ◽  
Nooruddin Ahmad ◽  
Salimar Rahman ◽  
...  
Keyword(s):  
Growth Factor ◽  
Hepatitis B ◽  
Patient Group ◽  
Liver Dysfunction ◽  
Healthy Subjects ◽  
Clinical Stage ◽  
Insulin Like Growth Factor ◽  
Healthy Individuals ◽  
Cirrhotic Patients ◽  
Cirrhosis Of Liver

Background: Insulin Like Growth Factor-1 (IGF-1) is a polypeptide hormone predominantly synthesized in liver. It has been reported that serum IGF-1 concentrations low in hypopitutuitarism, malnutrition and various diseases particularly in patients with chronic liver disease. The aim of the study was to see the level of IGF-1 in patients with cirrhosis and its relation with severity of liver dysfunction. Materials and Methods : This study was carried out in the Department of Hepatology, Bangabandhu Sheikh Mujib Medical University (BSMMU) Dhaka, Bangladesh. 40 persons (30 patients of cirrhosis of liver and 10 healthy individuals) were selected and grouped as Group-1 : 10 Child grade A patient, Group - 2 : 10 Child grade B patient, Group - 3 : 10 Child grade C cirrhotic patients and Group - 4 : 10 healthy subjects. Patients with recent antiviral therapy, acute illness, hepatocellular carcinoma and spontaneous bacterial peritonitis were excluded from the study. IGF-1 was measured using IMMULITE, DPC, USA which employs automated chemiluminescent immunoassays. Results : Among 30 cirrhotic patients 26 were male and 4 were female. Mean age was 38.83 ± 14.08 years. Etiology of cirrhosis was hepatitis B virus in 23 patient, hepatitis C virus in 1 patient, Cryptogenic in 6 patients. Healthy subjects were hepatitis B and C virus negative with normal liver function test and mean age was 36.40 ± 7.76 years, Mean serum IGF-1 was 82 ± 10.85 ng/ml in Child grade A, 50.77 ± 7.47 ng/ml in Child grade B, 29.69 ± 3.17 ng/ml in Child grade C patients and 130.96 ± 2.43 ng/ml in healthy individuals. Conclusion : Serum level of IGF -1 is low in patients with cirrhosis of liver than healthy individuals and reflects the severity of liver dysfunction in different clinical stage. Chatt Maa Shi Hosp Med Coll J; Vol.18 (1); Jan 2019; Page 3-7

scholarly journals Examination of the Efficacy and Cross-Reactivity of a Novel Polyclonal Antibody Targeting the Disintegrin Domain in SVMPs to Neutralize Snake Venom

Toxins ◽  
2021 ◽  
Vol 13 (4) ◽  
pp. 254
Author(s):  
Shelby S. Szteiter ◽  
Ilse N. Diego ◽  
Jonathan Ortegon ◽  
Eliana Salinas ◽  
Abcde Cirilo ◽  
...  
Keyword(s):  
Snake Venom ◽  
Polyclonal Antibody ◽  
Polyclonal Antibodies ◽  
Cross Reactivity ◽  
Snake Envenomation ◽  
Disintegrin Domain ◽  
The Common ◽  
Neutralization Ability ◽  
New Strategies

Snake envenomation can result in hemorrhage, local necrosis, swelling, and if not treated properly can lead to adverse systemic effects such as coagulopathy, nephrotoxicity, neurotoxicity, and cardiotoxicity, which can result in death. As such, snake venom metalloproteinases (SVMPs) and disintegrins are two toxic components that contribute to hemorrhage and interfere with the hemostatic system. Administration of a commercial antivenom is the common antidote to treat snake envenomation, but the high-cost, lack of efficacy, side effects, and limited availability, necessitates the development of new strategies and approaches for therapeutic treatments. Herein, we describe the neutralization ability of anti-disintegrin polyclonal antibody on the activities of isolated disintegrins, P-II/P-III SVMPs, and crude venoms. Our results show disintegrin activity on platelet aggregation in whole blood and the migration of the SK-Mel-28 cells that can be neutralized with anti-disintegrin polyclonal antibody. We characterized a SVMP and found that anti-disintegrin was also able to inhibit its activity in an in vitro proteolytic assay. Moreover, we found that anti-disintegrin could neutralize the proteolytic and hemorrhagic activities from crude Crotalus atrox venom. Our results suggest that anti-disintegrin polyclonal antibodies have the potential for a targeted approach to neutralize SVMPs in the treatment of snakebite envenomations.

scholarly journals Nitrotyrosine Density of Rabbit Urinary Bladder Muscle and Mucosa Measured via Western Blotting and 96-Well Plate Analysis

ISRN Urology ◽  
2012 ◽  
Vol 2012 ◽  
pp. 1-5
Author(s):  
Brittany Fitzpatrick ◽  
Catherine Schuler ◽  
Robert E. Leggett ◽  
Robert M. Levin
Keyword(s):  
Quantitative Analysis ◽  
Western Blotting ◽  
Optical Density ◽  
Tissue Concentration ◽  
Detrusor Muscle ◽  
Pvdf Membrane ◽  
Sds Page ◽  
Wash Buffer ◽  
Rabbit Bladder ◽  
Plate Analysis

Purpose. Nitrotyrosine was quantitated in rabbit bladder muscle and mucosa using two analytical systems: Western blotting analyses and a 96-well plate quantitative analysis kit. Materials and Methods. Rabbit bladder muscle and mucosa were obtained from control rabbits. For the Western analysis, the samples were loaded into a SDS page gel and then transferred to a PVDF membrane. The optical density was measured using a Kodak Scanner. Using the 96-well plate, the samples and standards were loaded, incubated with primary and secondary antibody, washed and vacuumed with 10x wash buffer three times between each incubation period. Stop buffer was added to the plate and the results were quantified via the plate reader. Results. For both muscle and mucosa tissue, the optical density readings were linear with tissue concentration; the concentration of nitrotyrosine in the mucosa was significantly higher than in the muscle. However, whereas the Western blot analysis is based on relative optical densities, the 96-well plate kit provides a truly quantitative analysis. Discussion. Mucosa tissue displayed a higher density of nitrotyrosine than did detrusor muscle tissue. This may well be due to the significantly higher metabolic activity of the mucosa compared to the muscle.

Insulin-induced phosphorylation of ENaC correlates with increased sodium channel function in A6 cells

2005 ◽  
Vol 288 (1) ◽  
pp. C141-C147 ◽  
Author(s):  
Yu-Hua Zhang ◽  
Diego Alvarez de la Rosa ◽  
Cecilia M. Canessa ◽  
John P. Hayslett
Keyword(s):  
Sodium Channel ◽  
Sodium Transport ◽  
Polyclonal Antibodies ◽  
Hormonal Stimulation ◽  
Α Subunit ◽  
A6 Cells ◽  
Kinase C ◽  
Sds Page ◽  
Protein Kinase C Inhibitor ◽  
Synergistic Increase

The purpose of this study was to determine whether there is a correlation between phosphorylation and activity of the epithelial sodium channel (ENaC). The three subunits that form the channel were immunoprecipitated from A6 cells by using specific polyclonal antibodies after labeling cells with 35S or 32P. When immune complexes were resolved on SDS-PAGE, the α-subunit migrated at 85 and 65 kDa, the β-subunit at 115 and 100 kDa, and the γ-subunit at 90 kDa. In the resting state all three subunits were phosphorylated. The α-subunit was phosphorylated only in the 65-kDa band, suggesting that the posttranslational modification that gives rise to the rapidly migrating form of α is a requirement for phosphorylation. Stimulation with 100 nM insulin for 30 min increased phosphorylation of α-, β-, and γ-subunits approximately twofold. Exposure to 1 μM aldosterone for 16 h increased protein abundance and phosphorylation proportionately in the three subunits. When insulin was applied to cells pretreated with aldosterone, phosphorylation was also increased approximately twofold, but the total amount of phosphorylated substrate was larger than in control conditions because of the action of aldosterone. This result might explain the synergistic increase in sodium transport under the same conditions. The protein kinase C inhibitor chelerythrine abolished insulin effects and decreased sodium transport and subunit phosphorylation. Together, our findings suggest that ENaC activity is controlled by subunit phosphorylation in cells that endogenously express the channel and the machinery for hormonal stimulation of sodium transport.

scholarly journals Role of maltase in the utilization of sucrose by Candida albicans

1993 ◽  
Vol 291 (3) ◽  
pp. 765-771 ◽  
Author(s):  
P R Williamson ◽  
M A Huber ◽  
J E Bennett
Keyword(s):  
Candida Albicans ◽  
Intracellular Localization ◽  
Cross Reactivity ◽  
Neutral Sugar ◽  
Sequence Specificity ◽  
Western Blots ◽  
Sds Page ◽  
Molecular Masses ◽  
Kinetics Of

Two isoenzymes of maltase (EC 3.2.1.20) were purified to homogeneity from Candida albicans. Isoenzymes I and II were found to have apparent molecular masses of 63 and 66 kDa on SDS/PAGE with isoelectric points of 5.0 and 4.6 respectively. Both isoenzymes resembled each other in similar N-terminal sequence, specificity for the alpha(1-−>4) glycosidic linkage and immune cross-reactivity on Western blots using a maltase II antigen-purified rabbit antibody. Maltase was induced by growth on sucrose whereas beta-fructofuranosidase activity could not be detected under similar conditions. Maltase I and II were shown to be unglycosylated enzymes by neutral sugar assay, and more than 90% of alpha-glucosidase activity was recoverable from spheroplasts. These data, in combination with other results from this laboratory [Geber, Williamson, Rex, Sweeney and Bennett (1992) J. Bacteriol. 174, 6992-6996] showing lack of a plausible leader sequence in genomic or mRNA transcripts, suggest an intracellular localization of the enzyme. To establish further the mechanism of sucrose assimilation by maltase, the existence of a sucrose-inducible H+/sucrose syn-transporter was demonstrated by (1) the kinetics of sucrose-induced [14C]sucrose uptake, (2) recovery of intact [14C]sucrose from ground cells by t.l.c. and (3) transport of 0.83 mol of H+/mol of [14C]sucrose. In total, the above is consistent with a mechanism whereby sucrose is transported into C. albicans to be hydrolysed by an intracellular maltase.

scholarly journals Production and characterisation of monoclonal antibodies against immunoglobulins of Cirrhinus mrigala (Hamilton 1822)

2020 ◽  
Vol 67 (2) ◽  
Author(s):  
Snatashree Mohanty ◽  
M. Makesh ◽  
K. V. Rajendran ◽  
P. P. Suresh Babu ◽  
Deepika Anand ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Cross Reactivity ◽  
Catla Catla ◽  
Cirrhinus Mrigala ◽  
Indian Major Carps ◽  
Sds Page ◽  
Igg1 Isotype ◽  
Major Carps

Serum immunoglobulins (Ig) of mrigal Cirrhinus mrigala (Hamilton 1822) immunised with bovine serum albumin (BSA), were purified by affinity chromatography using BSA-CL agarose column. The purified mrigal Ig (m-Ig) was characterised under reducing condition by Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (SDS-PAGE) which revealed two bands of 85 and 26 kDa corresponding to heavy and light chain, respectively. Following fusion of splenocytes from Balb/c mice immunised with purified m-Ig with myeloma cells, three hybridomas showing reactivity with m-Ig were cloned by limiting dilution. The monoclonal antibodies (MAbs) generated by these clones were designated as 3B2-E12, 3B2-F9 and 4C3-B2 and characterised by western blotting and isotyping. Western blot analysis of the supernatant from the three clones with purified m-Ig indicated that, all the three MAbs were specific to heavy chain. Isotyping revealed that 3B2-E12 MAb was of IgG1 isotype whereas the other two MAbs were of IgG2a isotype. Cross reactivity of anti-mrigal Ig MAb (3B2-E12) was observed with serum Ig of Catla catla and Labeo rohita indicating semi-conserved nature of Ig in Indian major carps.

Concentrations of 2-hydroxyoestrogens in human sera measured by a heterologous immunoassay with an 125I-labelled ligand

1982 ◽  
Vol 100 (1) ◽  
pp. 154-160 ◽  
Author(s):  
D. Berg ◽  
F. Thaler ◽  
E. Kuss
Keyword(s):  
Standard Deviation ◽  
Binding Sites ◽  
Luteal Phase ◽  
Healthy Subjects ◽  
Cross Reactivity ◽  
Follicular Phase ◽  
Underlying Assumption ◽  
Human Sera ◽  
Tube Accuracy ◽  
Serum Components

Abstract. A heterologous immunoassay for 2-hydroxyoestrogens1 has been established in which antibodies raised against 2-hydroxyoestradiol-17-succinyl-BSA serve as binding protein and 2-hydroxyoestrone-17-cmo-[125I]iodohistamine as radioligand. Lipophilic serum components competing for binding sites in this system were defined as 'total 2-hydroxyoestrogens'. The underlying assumption of specificity was supported by the pattern of cross-reactivity evaluated with structural related steroids and o-diphenols and by the fact, that an additional chromatography of the serum extracts preceding the competing reaction had little if any effect. Sensitivity: 2.8 ± 1 pg/tube; accuracy: Y = 0.91x + 2.2; r = 0.989; precision: 5.8% intra-assay; 6.5% inter-assay. The following concentrations ( ± standard deviation) were found in the sera of healthy subjects. Young men: 29 ± 5 pg/ml (n = 11); women follicular phase: 32 ± 8 pg/ml (n = 25); luteal phase: 53 ± 13 pg/ml (n = 23); postmenopausal women: 13 ± 4 pg/ml (n = 10); pregnant women 11th–20th week: 70 ± 16 mg/ml (n = 64); 36th–40th week: 240 ± 23 pg/ml (n = 40); newborn cord blood: 604 ± 43 pg/ml (n = 48).

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