Kinetic ELISA Research Articles

Development, certification, and use of a pharmacopoeial standard for the content of immunoglobulin A in human immunoglobulins for parenteral administration

Scientific relevance. The immunoglobulin A (IgA) impurity content in parenteral human immunoglobulins should be determined in accordance with the State Pharmacopoeia of the Russian Federation by kinetic nephelometry, radial immunodiffusion, or enzyme immunoassay (ELISA) with a reference standard. The International Standard (IS) for the content of IgA is certified using gravimetry and radial immunodiffusion. However, neither of the existing standards for the content of IgA in human immunoglobulins is currently certified using all three compendial methods. This prevents analysts from comparing test results obtained by different methods and may lead to an underestimation of the IgA content in human immunoglobulins.Aim. This study aimed to determine the procedure for the development, certification, and use of a pharmacopoeial reference standard (RS) for the content of IgA in human immunoglobulins.Materials and methods. The authors studied candidate RSs for the IgA content derived from human plasma for fractionation. The IgA content determination involved kinetic nephelometry, radial immunodiffusion, and ELISA, as well as commercial test kits and the IS. The authors quantified the IgA impurity in samples of commercial human immunoglobulins from various manufacturers. The data analysis involved descriptive statistics and variance analysis using Microsoft Excel and Statistica 10.Results. The authors established a pharmacopoeial standard with a certified IgA content of 1.98 mg/mL (expanded uncertainty, 0.44 mg/mL; coverage coefficient, k=2; confidence level, 95%) for IgA impurity quantification in human immunoglobulins by radial immunodiffusion and ELISA and that of 1.31–2.64 mg/mL (expanded uncertainty, 0.67 mg/mL; coverage ratio, k=3; confidence level, 99%) for intralaboratory quality control of IgA impurity quantification by kinetic nephelometry, radial immunodiffusion, and ELISA.Conclusions. The pharmacopoeial standard developed in the study has been included in the register of standards of the State Pharmacopoeia of the Russian Federation as the Reference Standard for the Content of Immunoglobulin Class A (IgA) (Registry No. 3.1.00454). The pharmacopoeial standard is intended for the standardisation of analytical methods for the-determination of the IgA impurity content in parenteral human immunoglobulins.

Development of a Kinetic ELISA and Reactive B Cell Frequency Assay to Detect Respiratory Syncytial Virus Pre-Fusion F Protein-Specific Immune Responses in Infants.

Respiratory syncytial virus (RSV) is a major cause of respiratory disease in infants, making vaccination an attractive preventive strategy. Due to earlier reports of vaccine-enhanced disease in RSV-naive children, assessing prior RSV infection is critical for determining eligibility for future infant vaccine trials. However, this is complicated by the presence of maternally transferred maternal antibodies. We sought to develop assays that measure immune responses to RSV pre-fusion (F) protein that discriminates between maternal and infant responses. We measured RSV-specific responses in two groups of children <3 years of age; those with laboratory-confirmed RSV (RSV-infected) and those enrolled prior to their first RSV season (RSV-uninfected). Serial blood samples were obtained and recent infections with RSV and other respiratory viruses were assessed during follow-up. An RSV pre-F-specific kinetic enzyme-linked immunosorbent assay (kELISA) and an F-specific reactive B cell frequency (RBF) assay were developed. One hundred two young children were enrolled between July 2015 and April 2017; 74 were in the RSV-uninfected group and 28 were in the RSV-infected group. Participants were asked to provide sequential blood samples over time, but only 53 participants in the RSV-uninfected group and 22 participants in the RSV-infected groups provided multiple samples. In the RSV-infected group, most had positive kELISA and RBF during the study. In the RSV-uninfected group, two patterns emerged: declining kELISA values without reactive B cells, due to maternal transplacental antibody transfer, and persistently positive kELISA with reactive B cells, due to asymptomatic undiagnosed RSV infection. A kELISA targeting RSV pre-F epitopes and an RBF assay targeting RSV F-specific B cells generally allow discrimination between maternally and infant-derived antibodies.

A Retrospective Study with a Commercial Vaccine against Lyme Borreliosis in Dogs Using Two Different Vaccination Schedules: Characterization of the Humoral Immune Response

Lyme borreliosis, a multisystemic disease caused by spirochetes of the genus Borrelia, is the most common tick-borne disease in the northern hemisphere. Differently from human medicine, several vaccines are available for dogs. To provide the best protection possible, vaccination schemes should be adapted regularly to meet the needs resulting from an increased tick exposure risk due to an inescapable climate change. In this retrospective study, a total of 183 vaccinations were performed with a commercial, multivalent vaccine against Lyme borreliosis, and vaccinated dogs were monitored over an observation period of 13 months. Dogs were either vaccinated on days 0 and 21 and a booster on day 365 (standard vaccination schedule), or with an additional booster vaccination on day 180. Canine serum samples were then tested for their borrelia-specific antibody levels using a two-tiered test system consisting of a kinetic ELISA followed by a line immunoassay. Dogs vaccinated with the standard vaccination schedule displayed decreasing antibody levels between days 120 and 360, which is probably insufficient to prevent an infection with borreliae. In contrast, the additional booster vaccination received on day 180 intercepts this decline in antibody levels between days 225 and 360, providing a sufficient immunity to prevent infection. The results from this retrospective study allow us to recommend a basic vaccination schedule with an additional booster vaccination on day 180 to ensure the best possible protection for dogs against Lyme borreliosis.

An Overview of ELISA-Based Initial Velocity Methods to Measure the Immunoreactive Fraction, Association Rate, And Equilibrium Constants of Monoclonal Antibodies

Three biochemical parameters—the on-rate (k+1), the dissociation rate (k-1), and the equilibrium (Ka) constants—describe the antibody interaction with a ligand. In addition, the antibody immune reactive fraction (IRF) is important because it affects k+1 and Ka constant values. The on-rate and dissociation constants are kinetic, but the equilibrium constant is a thermodynamic parameter, and the methods for their determination are different. Among the available methods to determine these parameters, only surface plasmon resonance (SPR)-based techniques allow calculation of the three antibody constants in a single experiment. However, conventional immunoassays are also suitable to determine antibody constants. Here, we describe some enzyme immunoassays (ELISA) methods developed in our laboratory to, i) quantitate mAb in culture supernatants from the initial velocity of the progress curve, ii) determine mAb IRF by a two-step ELISA method, iii) calculate mAb on-rate constant by kinetic ELISA based on time-course data of ligand binding to capture mAb, and, iv) describe a competitive-inhibition ELISA to measure the inhibition constant (Ki) of mAb binding to plate-bound ligand, in the presence of competing concentrations of mAb and soluble ligand.

Monoclonal antibody on-rate constant determined from time-course data of ligand binding by capture ELISA: Evaluation of eight data analysis methods

We describe an ELISA method with which to determine monoclonal antibody (mAb) on-rate constants (k+1) based on time-course data of ligand (L) binding to plate-bound mAb. The assay was performed in pseudo-first order kinetic conditions ([L] > > [mAb]) and at various starting ligand concentrations. Time-course initial velocity was analyzed by several methods to derive the pseudo-first order (kobs) and second order (k+1) association rate constants of the antibody; the methods included i) an exponential first order rate equation, ii) reaction half-time from the Michaelis-Menten relationship, iii) the Vmax/Km tangent of the time-course curve, iv) Boeker's extrapolated-vo method, v-vi) modified Hanes-Woolf and Lineweaver-Burk linear plots, vii) a LOS plot, and viii) initial velocity gradient. Due to k+1 value dispersion associated with the methods of analysis, the on-rate constant of mAb SIM 253–19 anti-cholera toxin was estimated as an average value of 1.79 ± 0.11 × 106 M−1 s−1, 95% CL (1.68–1.89) and 5.8 (%CV [coefficient of variation]), which is similar to the k+1 obtained by surface plasmon resonance, 1.60 ± 0.17 × 106 M−1 s −1 (mean ± half range). This kinetic ELISA is a sensitive, quantitative method by which to determine antibody association rate constants.

Granulocyte microvesicles with a high plasmin generation capacity promote clot lysis and improve outcome in septic shock

AbstractMicrovesicles (MVs) have previously been shown to exert profibrinolytic capacity, which is increased in patients with septic shock (SS) with a favorable outcome. We, therefore, hypothesized that the plasmin generation capacity (PGC) could confer to MVs a protective effect supported by their capacity to lyse a thrombus, and we investigated the mechanisms involved. Using an MV-PGC kinetic assay, ELISA, and flow cytometry, we found that granulocyte MVs (Gran-MVs) from SS patients display a heterogeneous PGC profile driven by the uPA (urokinase)/uPAR system. In vitro, these MVs lyse a thrombus according to their MV-PGC levels in a uPA/uPAR-dependent manner, as shown in a fluorescent clot lysis test and a lysis front retraction assay. Fibrinolytic activators conveyed by MVs contribute to approximately 30% of the plasma plasminogenolytic capacity of SS patients. In a murine model of SS, the injection of high PGC Gran-MVs significantly improved mouse survival and reduced the number of thrombi in vital organs. This was associated with a modification of the mouse coagulation and fibrinolysis properties toward a more fibrinolytic profile. Interestingly, mouse survival was not improved when soluble uPA was injected. Finally, using a multiplex array on plasma from SS patients, we found that neutrophil elastase correlates with the effect of high-PGC-capacity plasma and modulates the Gran-MV plasmin generation capacity by cleaving uPA-PAI-1 complexes. In conclusion, we show that the high PGC level displayed by Gran-MVs reduces thrombus formation and improves survival, conferring to Gran-MVs a protective role in a murine model of sepsis.

Development of a competitive inhibition kinetic ELISA to determine the inhibition constant (Ki) of monoclonal antibodies

Antibody-antigen interactions are mediated by the same molecular recognition mechanisms as those of an enzyme and its substrate. On this basis, we developed a competitive inhibition kinetic ELISA to measure monoclonal antibody (mAb) inhibition constants. Serially diluted samples of ligand (mAb) and inhibitor (soluble antigen) were incubated to equilibrium in ELISA plates coated with a fixed concentration of antigen (receptor). Plates were washed, and bound mAb measured with antiglobulin-peroxidase. Initial velocity data of receptor-bound mAb at various ligand and inhibitor concentrations were analyzed with enzyme linear competitive inhibition methods by non-linear regression (NLR), linear transformations (Cornish-Bowden, Lineweaver-Burk, Hanes-Woolf, Dixon, Cortés [1/i0.5 vs. Vi/Vmax], Ascenzi [Ks/Vmax/Ks,0/Vmax vs. [I]]) and NLR IC50 plots, to derive mAb inhibition constants (Ki). We obtained similar mAb Ki and Kd values by ELISA and surface plasmon resonance, which confirmed the accuracy of the ELISA method. This competitive inhibition ELISA is a simple (it requires no labeling or prior knowledge of antibody concentration), sensitive (it detects Ki values in the low nanomolar range by conventional colorimetry), and reproducible method with which to calculate mAb inhibition constants.

Effect of sodium hypochlorite solution and gel with/without passive ultrasonic irrigation on Enterococcus faecalis, Escherichia coli and their endotoxins

Background: Sodium hypochlorite (NaOCl) is the most commonly used irrigant in endodontics . The purpose of this study was to evaluate the effect of NaOCl solution (2.5%) and gel (3%) with/without passive ultrasonic irrigation (PUI) on Enterococcus faecalis, Escherichia coli, and their endotoxins, lipopolysaccharide (LPS) and lipoteichoic acid (LTA). Methods: 40 human lower premolars were contaminated with E. coli (ATCC 25922) for 28 days and E. faecalis (ATCC 29212) for 21 days. Specimens were randomly divided into four groups: (1) 2.5% NaOCl irrigating the canals without PUI activation; (2) 2.5% NaOCl with PUI; (3) 3% NaOCl gel irrigating the canals without PUI; and (4) 3% NaOCl gel with PUI. 40 mL of irrigant was used for each group. PUI activation was carried out using E1-Irrisonic stainless-steel tip at 10% frequency. After treatment, all specimens were filled with 3mL of 17% ethylenediaminetetraacetic acid (EDTA) for 3min and then washed with nonpyrogenic saline solution. Three samples were collected from the canals: S1, at baseline to confirm biofilm formation; S2 after treatment; and S3 after EDTA. Samples were assessed for E. coli and E. faecalis colony forming units, and LPS and LTA were assessed using chromogenic kinetic LAL assay and ELISA, respectively. Data were analyzed by Kruskal-Wallis, Friedmann and Dunn tests with α≤0.05. Results: All groups were effective in reducing the microbial load of E. coli and E. faecalis after treatment without a significant difference among the groups. NaOCl and NaOCl gel groups had no significant difference in reducing LPS and LTA. Statistically increased reduction was seen for NaOCL + PUI and NaOCl gel + PUI compared for groups without PUI. Conclusions: NaOCl gel has the same antimicrobial action of NaOCl solution and can partially detoxify endotoxins. PUI improves NaOCl (gel or solution) action over E. faecalis and E. coli and their endotoxins.

Effect of sodium hypochlorite solution and gel with/without passive ultrasonic irrigation on Enterococcus faecalis, Escherichia coli and their endotoxins.

Background: Sodium hypochlorite (NaOCl) is the most commonly used irrigant in endodontics . The purpose of this study was to evaluate the effect of NaOCl solution (2.5%) and gel (3%) with/without passive ultrasonic irrigation (PUI) on Enterococcus faecalis, Escherichia coli, and their endotoxins, lipopolysaccharide (LPS) and lipoteichoic acid (LTA). Methods: 40 human lower premolars were contaminated with E. coli (ATCC 25922) for 28 days and E. faecalis (ATCC 29212) for 21 days. Specimens were randomly divided into four groups: (1) 2.5% NaOCl irrigating the canals without PUI activation; (2) 2.5% NaOCl with PUI; (3) 3% NaOCl gel irrigating the canals without PUI; and (4) 3% NaOCl gel with PUI. 40 mL of irrigant was used for each group. PUI activation was carried out using E1-Irrisonic stainless-steel tip at 10% frequency. After treatment, all specimens were filled with 3mL of 17% ethylenediaminetetraacetic acid (EDTA) for 3min and then washed with nonpyrogenic saline solution. Three samples were collected from the canals: S1, at baseline to confirm biofilm formation; S2 after treatment; and S3 after EDTA. Samples were assessed for E. coli and E. faecalis colony forming units, and LPS and LTA were assessed using chromogenic kinetic LAL assay and ELISA, respectively. Data were analyzed by Kruskal-Wallis, Friedmann and Dunn tests with α≤0.05. Results: All groups were effective in reducing the microbial load of E. coli and E. faecalis after treatment without a significant difference among the groups. NaOCl and NaOCl gel groups had no significant difference in reducing LPS and LTA. Statistically increased reduction was seen for NaOCL + PUI and NaOCl gel + PUI compared for groups without PUI. Conclusions: NaOCl gel has the same antimicrobial action of NaOCl solution and can partially detoxify endotoxins. PUI improves NaOCl (gel or solution) action over E. faecalis and E. coli and their endotoxins.

A kinetic ELISA to determine the immunoreactive fraction of monoclonal antibodies

We developed a two-step ELISA to determine the immunoreactive fraction of monoclonal antibodies in conditions of antigen excess. An antibody aliquot at limiting dilution was incubated in wells coated with increasing amounts of antigen up to concentrations that bind 100% of antibody. At equilibrium, a supernatant aliquot was transferred to a second plate coated with excess of antiglobulin, and the captured antibody was incubated with peroxidase-conjugated anti-IgG. Antibody was quantitated from the enzyme velocity gradient in a kinetic ELISA, and the immunoreactive fraction calculated as (1 - gradienti/gradientT) x 100, where i and T are the gradients for the free and total antibody fractions. For four distinct monoclonal antibodies (anti-diphtheria toxoid, −cholera toxin, −bovine serum albumin (BSA), and -trinitrophenyl-BSA), measurement of inter-assay variability yielded values ranging from 3.1 to 7.4 (% coefficient of variation), which supports method repeatability. This ELISA is simple, precise, and applicable to mono- and polyclonal antibodies.

Development of an in vivo model for Toxoplasma gondii infections in chickens and turkeys simulating natural routes of infection

Turkeys and chickens were orally infected with tissue cysts (one mouse brain) or oocysts (103, 105 or 106 oocysts) of three T. gondii strains of the clonal types II and III (ME49, CZ-Tiger, NED) to investigate the influence of the applied T. gondii strain and infective doses on the distribution of T. gondii in several organs and tissues and the serologic response of chickens and turkeys. Organ samples from 16 different tissues, including heart, brain, muscles and gizzard were analyzed by PCR. Brain and heart were found most frequently positive for T. gondii DNA in both species, followed by gizzard. Serological analysis with kinetic ELISA for turkey samples and IFAT for chicken samples were performed once a week. In both species a dose-depending serological response was found. Turkeys seroconverted one week after infection with CZ-Tiger strain and medium and high doses of ME49 oocysts. In chickens, infection with medium and high doses of CZ-Tiger led to seroconversion one week p.i. Frequency of T. gondii positive organs showed a trend of a dose-effect in both species after infection with the type II strains. The NED strain showed low virulence in chickens and turkeys, demonstrated by clearly less T. gondii positive organs. Infection with tissue cysts of all three strains revealed T. gondii stages in tissues of turkeys and chickens. In conclusion, our data show a risk for human infection with T. gondii due to consumption of chicken and turkey meat.

Alcohol Abstinence Does Not Fully Reverse Abnormalities of Mucosal-Associated Invariant T Cells in the Blood of Patients With Alcoholic Hepatitis.

OBJECTIVES:Alcoholic hepatitis (AH) develops in approximately 30% of chronic heavy drinkers. The immune system of patients with AH is hyperactivated, yet ineffective against infectious diseases. Mucosal-associated invariant T (MAIT) cells are innate-like lymphocytes that are highly enriched in liver, mucosa, and peripheral blood and contribute to antimicrobial immunity. We aimed to determine whether MAIT cells were dysregulated in heavy drinkers with and without AH and the effects of alcohol abstinence on MAIT cell recovery.METHODS:MR1 tetramers loaded with a potent MAIT cell ligand 5-(2-oxopropylideneamino)-6-d-ribitylaminouracil were used in multiparameter flow cytometry to analyze peripheral blood MAIT cells in 59 healthy controls (HC), 56 patients with AH, and 45 heavy drinkers without overt liver disease (HDC) at baseline and 6- and 12-month follow-ups. Multiplex immunoassays were used to quantify plasma levels of cytokines related to MAIT cell activation. Kinetic Turbidimetric Limulus Amebocyte Lysate Assay and ELISA were performed to measure circulating levels of 2 surrogate markers for bacterial translocation (lipopolysaccharide and CD14), respectively.RESULTS:At baseline, patients with AH had a significantly lower frequency of MAIT cells than HDC and HC. HDC also had less MAIT cells than HC (median 0.16% in AH, 0.56% in HDC, and 1.25% in HC). Further, the residual MAIT cells in patients with AH expressed higher levels of activation markers (CD69, CD38, and human leukocyte antigen [HLA]-DR), the effector molecule granzyme B, and the immune exhaustion molecule PD-1. Plasma levels of lipopolysaccharide and CD14 and several cytokines related to MAIT cell activation were elevated in patients with AH (interferon [IFN]-α, interleukin [IL]-7, IL-15, IL-17, IL-18, IL-23, IFN-γ, and tumor necrosis factor α). Decreased MAIT cell frequency and upregulated CD38, CD69, and HLA-DR correlated negatively and positively, respectively, with aspartate aminotransferase level. MAIT cell frequency negatively correlated with IL-18. HLA-DR and CD38 levels correlated with several cytokines. At follow-ups, abstinent patients with AH had increased MAIT cell frequency and decreased MAIT cell activation. However, MAIT cell frequency was not fully normalized in patients with AH (median 0.31%).DISCUSSION:We showed that HDC had a reduction of blood MAIT cells despite showing little evidence of immune activation, whereas patients with AH had a severe depletion of blood MAIT cells and the residual cells were highly activated. Alcohol abstinence partially reversed those abnormalities.

How Working Tasks Influence Biocontamination in an Animal Facility

The exposure to biocontaminants in animal facilities represents a risk for developing infectious, allergic and toxic diseases. The aim of this study was to determine what factors could be associated with a high level of exposure to biological agents through the measure and characterization of airborne fungi, bacteria, endotoxin, (1,3)-β-d-glucan and animal allergens. Airborne microorganisms were collected with an air sampler and identified by microscopic and biochemical methods. Endotoxin, (1,3)-β-d-glucan, Mus m 1, Rat n 1, Can f 1, Fel d 1, Equ c 4 allergens were detected on inhalable dust samples by Kinetic LAL, Glucatell, and ELISA assays, respectively. Our data evidenced that changing cages is a determinant factor in increasing the concentration of the airborne biocontaminants; the preparation of bedding and distribution of feed, performed in the storage area, is another critical working task in terms of exposure to endotoxins (210.7 EU/m3) and (1,3)-β-d-glucans (4.3 ng/m3). The highest concentration of Mus m 1 allergen (61.5 ng/m3) was observed in the dirty washing area. The detection of expositive peaks at risk of sensitization (&gt;2 μg/g) by Fel d 1 in animal rooms shows passive transport by operators themselves, highlighting their role as vehicle between occupational and living environments.

Using Synthetic Peptides for Exploring Protein-Protein Interactions in the Assembly of the NADPH Oxidase Complex.

The NADPH oxidase complex, responsible for reactive oxygen species (ROS) generation by phagocytes, consists of a membrane-associated flavocytochrome b 558 (a heterodimer of NOX2 and p22phox) and the cytosolic components p47phox, p67phox, Rac(1 or 2), and p40phox. NOX2 carries all redox stations through which electrons flow from NADPH to molecular oxygen, to generate the primary ROS, superoxide. For the electron flow to start, a conformational change in NOX2 is required. The dominant hypothesis is that this change is the result of the interaction of NOX2 with one or more of the cytosolic components (NADPH oxidase assembly). At the most basic level, assembly is the sum of several protein-protein interactions among oxidase components. This chapter describes a reductionist approach to the identification of regions in oxidase components involved in assembly. This approach consists of "transforming" one component in an array of overlapping synthetic peptides and assessing binding to the peptides of another component, represented by a recombinant protein. The peptides are tagged with biotin, at the N- or C-terminus, and immobilized on streptavidin-coated 96-well plates. The protein partners are expressed with a 6His tag and added to the plates in the fluid phase. Binding of the protein to the peptides is quantified by a kinetic ELISA , using a peroxidase-conjugated anti-polyhistidine antibody. Protein-peptide binding assays were applied successfully to (a) identifying the binding site on one component (represented by peptides) for another component (proteins), (b) precisely defining the "binding sequence," (c) acquiring information on the binding site in the partner protein, (d) investigating the effect of conformational changes in proteins on binding to peptides, (e) determining the effect of physicochemical modification of peptides on binding of proteins, and (f) identifying epitopes recognized by anti-oxidase component antibodies by binding of antibody to peptide arrays derived from the component.

Searching for Lyme borreliosis in Australia: results of a canine sentinel study

BackgroundLyme borreliosis is a common tick-borne disease of the northern hemisphere that is caused by bacterial spirochaetes of the Borrelia burgdorferi (sensu lato) (Bbsl) complex. To date, there has been no convincing evidence for locally-acquired Lyme borreliosis on the Australian continent and there is currently a national debate concerning the nature and distributions of zoonotic tick-transmitted infectious disease in Australia. In studies conducted in Europe and the United States, dogs have been used as sentinels for tick-associated illness in people since they readily contact ticks that may harbour zoonotic pathogens. Applying this principle, we used a combination of serological assays to test dogs living in tick ‘hot spots’ and exposed to the Australian paralysis tick, Ixodes holocyclus, for evidence of exposure to B. burgdorferi (s.l.) antigens and other vector-borne pathogens.ResultsAltogether, 555 dogs from four demographic groups were recruited into this study. One dog had evidence of exposure to Anaplasma spp. but no other dog was positive in screening tests. A total of 122 dogs (22.0%) had a kinetic ELISA (KELA) unit value > 100, and one dog with a high titre (399.9 KELA units) had been vaccinated against B. burgdorferi (sensu stricto) before travelling to Australia. Older dogs and those with a history of tick paralysis were significantly more likely to have a KELA unit value > 100. Line immunoassay analysis revealed moderate-to-weak (equivocal) bands in 27 (4.9%) dogs.ConclusionsExcept for a single dog presumed to have been exposed to Anaplasma platys, infection with Anaplasma spp. B. burgdorferi (s.l.), Ehrlichia spp., and Dirofilaria immitis, was not detected in the cohort of Australian dogs evaluated in this study. These results provide further evidence that Lyme borreliosis does not exist in Australia but that cross-reacting antibodies (false positive results) are common and may be caused by the transmission of other tick-associated organisms.

β2 Microglobulin as A Marker of Early Renal Damage In Patients With Sickle Cell Nephropathy

Introduction: Sickle cell nephropathy is a cause of significant morbidity and mortality in patients with sickle cell disease. Traditional creatinine based assay for evaluating renal function has several limitations. Hence, the need for more sensitive markers such as β2 microglobulin (β2M). This study seeks to compare β2M with creatinine as a marker of renal damage in patients with sickle cell disease. Method: This is a cross sectional study conducted at the University of Benin Teaching Hospital, Benin City, Nigeria. Serum creatinine and β2M levels were determine using kinetic Jaffe method and β2M ELISA assay (Quantikine kit) respectively in 83 sickle cell disease patients and 40 age and sex match controls. Estimated glomerular filtration rate (eGFR) was calculated using Cockcroft-Gault formular for eGFRCr estimation. Data was analyzed with SPSS version 16. Result: β2M in the SCD patients and controls were 25.0±3.9% versus 45.6±5.8% (p = 0.001), 17.4±7.5mg/dl versus 24.0±8.0mg/dl (0.001), 0.7±0.3mg/dl versus 0.9±0.3mg/dl (p = 0.003), 3.3±1.0 versus 2.3±0.7 (p = 0.001) respectively. The eGFR-Cr was higher in SCD patients than in controls (128.9±8.4ml/min/1.73m 2 versus 113.6±11.0ml/min/1.73m 2 ) but the difference was not statistically significant P = 0.316. β2M has a positive correlation with urea, creatinine and a weak negative correlation with eGFRCr. Conclusion: β2M is superior to creatinine as a marker of sickle cell nephropathy.However, caution should be applied in patients with haematological malignancies and chronic inflammatory diseases.

Immunological detection of glutamyl aminopeptidase in urine samples from cisplatin-treated rats.

The aim of this work is to demonstrate if urinary excretion of glutamyl aminopeptidase (GluAp) can be quantified by immunological methods. Urine samples from control and cisplatin-treated rats (n = 10 each group) were obtained at 1, 8, and 15 days after cisplatin injection. GluAp was analyzed by kinetic fluorimetry, ELISA, and immunoblotting. Sensitivity and specificity was studied for fluorimetric activity and ELISA 24 h after cisplatin injection. We also analyzed the predictive value over renal dysfunction at the end of the experiment. GluAp was easily detected by immunoblotting and ELISA, and its urinary excretion was increased in cisplatin-treated rats (p < 0.01). Results obtained with ELISA were strongly correlated (r = 0.8186; p < 0.0001) with fluorimetric activity. Kinetic fluorimetry was the method with the highest AUC (AUC = 1) and the highest predictive value over serum creatinine (r = 0.7630; p = 0.0001) and body weight increase (r = -0.8721; p < 0.0001). GluAp can be detected in urine samples with immunological methods, making possible the development of an antibody-based kit for its determination. Its excretion correlates with the extent of renal dysfunction in cisplatin-treated rats, confirming its value as an early marker of renal damage that can be a diagnostic aid in renal diseases.

Simultaneous assessment of rotavirus-specific memory B cells and serological memory after B cell depletion therapy with rituximab.

The mechanisms that contribute to the maintenance of serological memory are still unclear. Rotavirus (RV) memory B cells (mBc) are enriched in IgM+ and CD27- subpopulations, which are associated with autoimmune diseases pathogenesis. In patients with autoimmune diseases treated with Rituximab (RTX), some autoantibodies (auto-Abs) decrease after treatment, but other auto-Abs and pathogen-specific IgG Abs remain unchanged. Thus, maintenance of autoimmune and pathogen-specific serological memory may depend on the type of antigen and/or Ab isotype evaluated. Antigen-specific mBc and antigen-specific Abs of different isotypes have not been simultaneously assessed in patients after RTX treatment. To study the relationship between mBc subpopulations and serological memory we characterized total, RV- and tetanus toxoid (TT)-specific mBc by flow cytometry in patients with autoimmune diseases before and after treatment with RTX. We also measured total, RV- and TT-Abs, and some auto-Abs by kinetic nephelometry, ELISA, and EliA tests, respectively. Minor differences were observed between the relative frequencies of RV-mBc in healthy controls and patients with autoimmune disease. After RTX treatment, naïve Bc and total, RV- and TT-specific mBc [IgM+, switched (IgA+/IgG+), IgM+ only, IgD+ only, and CD27- (IgA+/IgG+/IgM+)] were significantly diminished. An important decrease in total plasma IgM and minor decreases in total IgG and IgA levels were also observed. IgM rheumatoid factor, IgG anti-CCP, and IgG anti-dsDNA were significantly diminished. In contrast, RV-IgA, RV-IgG and RV-IgG1, and TT-IgG titers remained stable. In conclusion, in patients with autoimmunity, serological memory against RV and TT seem to be maintained by long-lived plasma cells, unaffected by RTX, and an important proportion of total IgM and serological memory against some auto-antigens seem to be maintained by short-lived plasma cells, dependent on mBc precursors depleted by RTX.

Plasma TIMP-1 in patients with metastatic colorectal cancer treated with first-line oxaliplatin-based therapy with or without cetuximab: Results from the Nordic VII study.

e14710 Background: Plasma Tissue Inhibitor of Metalloproteinases-1 (TIMP-1) is a biomarker in patients with metastatic colorectal cancer (mCRC). TIMP-1 inhibits matrix metalloproteinases and has tumor promoting functions. High levels of TIMP-1 associate with reduced sensitivity to irinotecan-based but not oxaliplatin-based treatment in patients with mCRC. Based on our prior studies, we here tested the hypothesis that high pretreatment plasma TIMP-1 correlate with short overall survival (OS) and short progression-free survival (PFS) but not response rate (RR) in patients with mCRC included in the NORDIC VII Study in which patients received oxaliplatin-based therapy. Methods: In the NORDIC VII Study patients were randomized to: A) Nordic FLOX; B) Nordic FLOX + cetuximab or C) Nordic FLOX intermittently + cetuximab continuously. Plasma samples for TIMP-1 analysis were available from 426 patients. Plasma TIMP-1 was determined using the MAC15 antibody in-house validated kinetic ELISA. Results: The three study populations were comparable and not different from the total intention to treat population. The median pretreatment plasma TIMP-1 was 269 ng/mL (58-1318 ng/mL). KRAS mutated in 149 patients (35%). There was no interaction between treatment arms and pretreatment plasma TIMP-1 either for PFS (p = 0.16) or OS (p = 0.64). High pretreatment plasma TIMP-1 was associated with shorter PFS (HR=1.22, 1.07-1.39, p=0.003) and shorter OS (HR=1.55, 1.36-1.80, p&lt;0.0001). RR did not correlate significantly to pretreatment plasma TIMP-1 (OR=1.16, 0.91-1.49, p=0.22). Multivariate analysis (TIMP-1, Köhne index, KRAS and BRAF status, CRP, CEA, age, gender) demonstrated that high pretreatment plasma TIMP-1 was not an independent biomarker of PFS (HR=1.06, 0.88-1.28, p=0.52) or OS (HR=1.19, 0.96-1.47, p=0.11). Conclusions: Plasma TIMP-1 is a prognostic biomarker in patients with mCRC treated with 1st line oxaliplatin-based therapy +/- cetuximab. A predictive effect of plasma TIMP-1 could only be estimated for cetuximab treatment, and no significant treatment by marker interaction was observed.

Bivalirudin Inhibits Periprocedural Platelet Function and Tissue Factor Expression of Human Smooth Muscle Cells

A major concern of stent implantation after percutaneous coronary intervention (PCI) is acute stent thrombosis. Effective inhibition of periprocedural platelet function in patients with coronary artery disease (CAD) leads to an improved outcome. In this study, we examined the periprocedural platelet reactivity after administrating bivalirudin during PCI compared to unfractionated heparin (UFH) administration. Further, the effect of bivalirudin on induced tissue factor (TF) expression in smooth muscle cells (SMC) was determined. Patients with CAD (n = 58) and double antithrombotic medication were treated intraprocedural with UFH (n = 30) or bivalirudin (n = 28). Platelet activation markers were flow cytometrically measured before and after stenting. The expression of TF in SMC was determined by real-time PCR and Western blotting. The thrombogenicity of platelet-derived microparticles and SMC was assessed via a TF activity assay. Bivalirudin significantly diminished the agonist-induced platelet reactivity post-PCI. Compared to UFH treatment, the adenosine diphosphate (ADP) and thrombin receptor-activating peptide (TRAP)-induced thrombospondin expression post-PCI was reduced when bivalirudin was administrated during intervention. In contrast to UFH, bivalirudin reduced the P-selectin expression of unstimulated and ADP-induced platelets post-PCI. Moreover, bivalirudin inhibited the thrombin-, but not FVIIa- or FVIIa/FX-induced TF expression and pro-coagulant TF activity of SMC. Moreover, bivalirudin reduced the TF activity of platelet-derived microparticles postinduction with TRAP or ADP. Bivalirudin is better than UFH in reducing periprocedural platelet activation. Moreover, thrombin-induced TF expression is inhibited by bivalirudin. Thus, bivalirudin seems to be a better anticoagulant during PCI than UFH.