scholarly journals Performance of soluble Klotho assays in clinical samples of kidney disease

2019 ◽  
Vol 13 (2) ◽  
pp. 235-244 ◽  
Author(s):  
Javier A Neyra ◽  
Orson W Moe ◽  
Johanne Pastor ◽  
Fabiola Gianella ◽  
Sachdev S Sidhu ◽  
...  
Keyword(s):  
Kidney Disease ◽  
Protease Inhibitor ◽  
Human Serum ◽  
Kidney Function ◽  
Enzyme Linked Immunosorbent Assay ◽  
Serum Samples ◽  
Freeze Thaw ◽  
Human Serum Samples ◽  
Thaw Cycle ◽  
Freeze Thaw Cycle

Abstract Background Soluble Klotho has multiple systemic salutary effects. In animals, both acute and chronic kidney disease models display systemic Klotho deficiency. As such, there is considerable interest in investigating soluble Klotho as a biomarker in patients with different types and severity of kidney diseases. Unfortunately, there remains uncertainty regarding the best method to measure soluble Klotho in human serum samples. Methods Using human serum samples obtained from several clinical cohorts with a wide range of kidney function, we measured soluble Klotho using a commercial enzyme-linked immunosorbent assay (ELISA) as well as with an immunoprecipitation–immunoblot (IP–IB) assay utilizing a synthetic antibody with high affinity and specificity for Klotho. Recovery of spiking with a known amount of exogenous Klotho was tested. A subset of samples was analyzed with and without the addition of a protease inhibitor cocktail at the time of collection or after the first freeze–thaw cycle to determine if these maneuvers influenced performance. Results The IP–IB assay was superior to the ELISA at recovery of exogenous Klotho (81–115% versus 60–81%) across the spectrum of kidney function. Klotho measurements by IP–IB were highly correlated with estimated glomerular filtration rate (eGFR) (R = 0.80, P < 0.001) in comparison with the commercial ELISA, which exhibited minimal correlation with eGFR (R = 0.18, P = 0.12). Use of a protease inhibitor cocktail neither improved nor impaired performance of the IP–IB assay; however, subsequent freeze–thaw cycle resulted in a significant reduction in Klotho recovery and dissipated the correlation between Klotho levels and eGFR. With the ELISA, the use of protease inhibitor cocktail resulted in an increase in intrasubject variability. Conclusions The IP–IB assay is preferable to the commercial ELISA to measure soluble Klotho concentrations in never-thawed serum samples of humans with varying severity of kidney disease. However, due to the labor-intensive nature of the IP–IB assay, further research is needed to secure an assay suitable for high-throughput work.

Effect of Freezing and Thawing of Serum on the Immunoassay of Lipoprotein(a)

1992 ◽  
Vol 38 (9) ◽  
pp. 1873-1877 ◽  
Author(s):  
D S Sgoutas ◽  
T Tuten
Keyword(s):  
Room Temperature ◽  
Enzyme Linked Immunosorbent Assay ◽  
Freezing And Thawing ◽  
Serum Samples ◽  
Lipoprotein A ◽  
Freeze Thaw ◽  
Thaw Cycle ◽  
Quick Freezing ◽  
Freeze Thaw Cycle

Abstract We studied the effect of freezing and thawing of serum on the determination of lipoprotein(a) [Lp(a)] with a commercial enzyme-linked immunosorbent assay (ELISA) and an immunoturbidimetric assay (ITA). Portions of sera from 11 apparently healthy persons and pooled sera, from an additional 10 subjects were frozen at either -20 or -70 degrees C and thawed at room temperature. Cycles of freezing and thawing were repeated during the experiments (1 month). Samples were assayed for Lp(a) after thawing. Pooled sera were subjected to quick freezing at -70 degrees C and thawing at room temperature in cycles. Results show a significant (P less than 0.05) decrease in Lp(a) concentration in sera subjected to freezing and thawing. Samples thawed from -20 degrees C gave concentrations by ELISA that were significantly lower than those of fresh samples after one freeze-thaw cycle. By ITA the decrease was significant only after two cycles. In specimens frozen at -70 degrees C, Lp(a) concentrations determined by ELISA decreased after two cycles, and by ITA after three freeze-thaw cycles. Serum samples subjected to quick freezing at -70 degrees C and thawing did not show significant decreases in Lp(a) immunoreactivity during four cycles. Immunoreactivity of Lp(a) in samples stored at 4 degrees C decreased after 6 days but fell faster in serum samples subjected to freezing and thawing before storage at 4 degrees C.

scholarly journals Seroprevalence of Jamestown Canyon virus in the Japanese general population

2020 ◽  
Vol 20 (1) ◽  
Author(s):  
Hirofumi Kato ◽  
Masaaki Satoh ◽  
Madoka Kawahara ◽  
Satoshi Kitaura ◽  
Tomoki Yoshikawa ◽  
...  
Keyword(s):  
General Population ◽  
Human Serum ◽  
Health Agency ◽  
Febrile Illness ◽  
Central Nervous System Disease ◽  
Enzyme Linked Immunosorbent Assay ◽  
Antibody Assay ◽  
Serum Samples ◽  
Human Serum Samples ◽  
Jamestown Canyon Virus

Abstract Background Jamestown Canyon virus (JCV) is a mosquito-borne orthobunyavirus that causes acute febrile illness, meningitis, and meningoencephalitis, mainly among adults. JCV is widely distributed in North America and the number of JCV cases in the U.S. has increased in recent years. Therefore, the central nervous system disease caused by JCV can be considered a potentially re-emerging viral disease. However, the seroprevalence of JCV is unknown in Japan. The purpose of this study is to evaluate the seroprevalence of JCV in the Japanese population. Methods We used an IgG enzyme-linked immunosorbent assay (IgG-ELISA) with JCV-infected cell-lysates and/or a neutralizing (NT) antibody assay. The cut-off value of IgG-ELISA was determined using IgG-ELISA to analyze serum specimens from 37 healthy Japanese donors. IgG-ELISA was validated by assessing its sensitivity and specificity, using 38 human serum samples previously tested for the presence or absence of antibodies against JCV and snowshoe hare virus (SSHV), in an in-house NT antibody assay conducted by the Public Health Agency of Canada. The seroepidemiological study was performed using IgG-ELISA and NT antibody assay to analyze 246 human serum samples from the serum bank of the National Institute of Infectious Diseases (NIID) in Japan. Results The cut-off value of IgG-ELISA was determined at 0.20, based on the mean (− 0.075) and standard deviation (0.092) values using Japanese donors’ sera. The sensitivity and the specificity of IgG-ELISA determined using 25 JCV-positive and 4 JCV-negative serum samples were 96 and 100%, respectively. Analysis of the 246 Japanese serum samples revealed that no specimen showed a higher value than the cut-off value of IgG-ELISA, and no sample tested positive by the NT antibody assay. Conclusions Our results showed that JCV is not circulating significantly in Japan. To the best of our knowledge, this is the first report to demonstrate the seroprevalence of JCV in the general population in Japan.

scholarly journals The Effects of a Single Freeze-Thaw Cycle on Concentrations of Nutritional, Noncommunicable Disease, and Inflammatory Biomarkers in Serum Samples

2021 ◽  
Author(s):  
Ransi Ann Abraham ◽  
Garima Rana ◽  
Praween K. Agrawal ◽  
Robert Johnston ◽  
Avina Sarna ◽  
...  
Keyword(s):  
Large Scale ◽  
Low Density Lipoprotein ◽  
Density Lipoprotein ◽  
Noncommunicable Disease ◽  
Noncommunicable Diseases ◽  
Serum Samples ◽  
Freeze Thaw ◽  
Thaw Cycle ◽  
Freeze Thaw Cycle ◽  
The Stability

Abstract Background The stability of biological samples is vital for reliable measurements of biomarkers in large-scale survey settings, which may be affected by freeze-thaw procedures. We examined the effect of a single freeze-thaw cycle on 13 nutritional, noncommunicable diseases (NCD), and inflammatory bioanalytes in serum samples. Method Blood samples were collected from 70 subjects centrifuged after 30 minutes and aliquoted immediately. After a baseline analysis of the analytes, the samples were stored at − 70°C for 1 month and reanalyzed for all the parameters. Mean percentage differences between baseline (fresh blood) and freeze-thaw concentrations were calculated using paired sample t-tests and evaluated according to total allowable error (TEa) limits (desirable bias). Results Freeze-thaw concentrations differed significantly (p < 0.05) from baseline concentrations for soluble transferrin receptor (sTfR) (− 5.49%), vitamin D (− 12.51%), vitamin B12 (− 3.74%), plasma glucose (1.93%), C-reactive protein (CRP) (3.45%), high-density lipoprotein (HDL) (7.98%), and cholesterol (9.76%), but they were within respective TEa limits. Low-density lipoprotein (LDL) (− 0.67%), creatinine (0.94%), albumin (0.87%), total protein (1.00%), ferritin (− 0.58%), and triglycerides (TAG) (2.82%) concentrations remained stable following the freeze-thaw cycle. In conclusion, single freeze-thaw cycle of the biomarkers in serum/plasma samples after storage at − 70°C for 1 month had minimal effect on stability of the studied analytes, and the changes in concentration were within acceptable limit for all analytes.

scholarly journals Antibody Responses to Escherichia coli O157 and Other Lipopolysaccharides in Healthy Children and Adults

2003 ◽  
Vol 10 (5) ◽  
pp. 797-801 ◽  
Author(s):  
Armando Navarro ◽  
Carlos Eslava ◽  
Ulises Hernandez ◽  
Jose Luis Navarro-Henze ◽  
Magali Aviles ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Human Serum ◽  
Western Blotting ◽  
Rabbit Serum ◽  
Enzyme Linked Immunosorbent Assay ◽  
Healthy Children ◽  
Serum Samples ◽  
E Coli ◽  
Human Serum Samples ◽  
Bactericidal Activities

ABSTRACT In Mexico, diarrheal disease due to different serotypes of Escherichia coli is highly prevalent, with only sporadic isolation of O157 non-H7 strains. This could be due to exposure to the O157 or related E. coli lipopolysaccharide (LPS), such as O7 or O116, at an early age. By using enzyme-linked immunosorbent assay (ELISA) and Western blotting, the present study analyzed 605 serum samples from Mexican adults and infants without clinical symptoms of disease for the presence of antibodies to these three E. coli LPSs. The bactericidal activities of homologous and heterologous rabbit and human serum samples against O7, O116, and O157 E. coli LPSs were also determined. By using a cutoff point of 0.7, it was found by the ELISAs that 28 of 562 (5%) of the serum samples from adolescents and adults and 2 of 43 (5%) of the serum samples from infants less than 1 year of age reacted with the O157 LPS. By using cutoff points between 0.4 and 0.699, the proportion of serum samples from both age groups that reacted with the O157 LPS increased to 20%. Western blotting analysis of selected serum samples that showed an intermediate response against the O157 LPS by the ELISAs showed that 61 of 88 (69%) reacted with the same LPS. A similar result was observed for maternal milk samples. The bactericidal activities of rabbit serum samples against the O7, O116, and O157 LPSs showed that they were positive for both homologous and heterologous antigens. Similar results were observed with the human serum samples. O157 non-H7 strains were identified in only 10% of the E. coli strains isolated from 263 Mexican children with and without diarrhea over the past 15 years. This absence of O157:H7 strains in Mexico may be associated with the presence of antibodies against O157 or related E. coli LPSs.

scholarly journals PSIV-25 Effect of Various Handling Methods of Plasma and Serum Samples on Pregnancy Associated Glycoprotein Concentrations

2020 ◽  
Vol 98 (Supplement_4) ◽  
pp. 289-290
Author(s):  
Grace M Wesson ◽  
Lohana Fernandez ◽  
Rebecca K Poole ◽  
Gessica A Franco ◽  
Sydney T Reese ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Plasma Samples ◽  
Freezing And Thawing ◽  
Serum Samples ◽  
Freeze Thaw ◽  
Thaw Cycle ◽  
Freeze Thaw Cycle ◽  
Repeated Freezing ◽  
Pregnancy Status ◽  
Handling Methods

Abstract Pregnancy associated glycoproteins (PAG) can be used as a biomarker for early pregnancy diagnosis, so accurate and consistent PAG detection is critical. The objective of this study was to determine if plasma and serum PAG concentrations were altered when centrifugation occurred at different times post-collection, when subjected to repeated freezing and thawing, and when monoclonal antibodies were kept in frequently or infrequently opened containers. Plasma (n = 4) and serum (n = 4) samples were collected from two open cows and two pregnant cows 28 days after artificial insemination. Pregnancy status was determined via transrectal ultrasonography. Plasma and serum samples were evenly separated and either centrifuged on the day of collection, or placed at 4°C and centrifuged the next day. An in-house PAG ELISA was performed on all samples before freezing (NOTHAW), after being frozen for one week (INTACT), after one freeze/thaw cycle (THAW1), two freeze/thaw cycles (THAW2), and three freeze/thaw cycles (THAW3). Data were analyzed using one-way ANOVA (GLM procedure, SAS 9.4). All samples from open cows were below the baseline of the assay. For pregnant cows, plasma samples had greater PAG concentrations than serum samples (11.84 vs 3.30 ± 0.66 ng/mL, respectively, P &lt; 0.05). No differences were observed for day of centrifugation in both plasma and serum samples (P = 0.50 and P = 0.60, respectively) and in handling of monoclonal antibodies (P = 0.90). Freezing and thawing did not impact PAG concentrations in plasma samples (P = 0.19), but did alter serum concentrations (P = 0.01). Specifically, THAW1 (1.98 ng/mL) and THAW2 (1.42 ng/mL) serum PAG concentrations were lower compared to NOTHAW, THAW3, and INTACT samples (4.66, 4.85, and 3.57 ng/mL, respectively). Based on these data, plasma yields more consistent results than serum, even after several freeze-thaw cycles, and handling of monoclonal antibodies or time of centrifugation has no significant effect on measured PAG.

scholarly journals Inactivation ofZaire ebolavirusVariant Makona in Human Serum Samples Analyzed by Enzyme-Linked Immunosorbent Assay

2016 ◽  
Vol 214 (suppl 3) ◽  
pp. S218-S221 ◽  
Author(s):  
Todd Cutts ◽  
Allen Grolla ◽  
Shane Jones ◽  
Bradley W. M. Cook ◽  
Xiangguo Qiu ◽  
...  
Keyword(s):  
Human Serum ◽  
Immunosorbent Assay ◽  
Enzyme Linked Immunosorbent Assay ◽  
Serum Samples ◽  
Human Serum Samples

scholarly journals Serodiagnosis of Human Cutaneous Anthrax in India Using an Indirect Anti-Lethal Factor IgG Enzyme-Linked Immunosorbent Assay

2012 ◽  
Vol 20 (2) ◽  
pp. 282-286 ◽  
Author(s):  
N. Ghosh ◽  
I. Tomar ◽  
H. Lukka ◽  
A. K. Goel
Keyword(s):  
Early Diagnosis ◽  
Human Serum ◽  
Immunosorbent Assay ◽  
Predictive Value ◽  
Lethal Factor ◽  
Enzyme Linked Immunosorbent Assay ◽  
Serum Samples ◽  
Human Serum Samples ◽  
Cutaneous Anthrax ◽  
Predictive Rate

ABSTRACTAnthrax, caused byBacillus anthracis, is primarily a zoonotic disease. Being a public health problem also in several developing countries, its early diagnosis is very important in human cases. In this study, we describe the use of an indirect enzyme-linked immunosorbent assay (ELISA) for detection of anti-lethal factor (anti-LF) IgG in human serum samples. A panel of 203 human serum samples consisting of 50 samples from patients with confirmed cutaneous anthrax, 93 samples from healthy controls from areas of India where anthrax is nonendemic, 44 samples from controls from an area of India where anthrax is endemic, and 16 patients with a disease confirmed not to be anthrax were evaluated with an anti-LF ELISA. The combined mean anti-LF ELISA titer for the three control groups was 0.136 ELISA unit (EU), with a 95% confidence interval (CI) of 0.120 to 0.151 EU. The observed sensitivity and specificity of the ELISA were 100% (95% CI, 92.89 to 100%) and 97.39% (95% CI, 93.44 to 99.28%), respectively, at a cutoff value of 0.375 EU, as decided by receiver operating characteristic (ROC) curve analysis. The likelihood ratio was found to be 49.98. The positive predictive value (PPV), negative predictive value (NPV), efficiency, and Youden's index (J) for reliability of the assay were 92.5%, 100%, 98.02%, and 0.97, respectively. The false-positive predictive rate and false-negative predictive rate of the assay were 2.61% and 0%. The assay could be a very useful tool for early diagnosis of cutaneous anthrax cases, as antibodies against LF appear much earlier than those against other anthrax toxins in human serum samples.

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