Quantification of human serum apolipoprotein AI by enzyme immunoassay.

1985 ◽  
Vol 31 (2) ◽  
pp. 247-251 ◽  
Author(s):  
J Bury ◽  
M Rosseneu
Keyword(s):  
Heat Treatment ◽  
Human Serum ◽  
Enzyme Linked Immunosorbent Assay ◽  
Apolipoprotein Ai ◽  
Serum Samples ◽  
Assay Sensitivity ◽  
Microtiter Plates ◽  
Sandwich Type ◽  
Apo Ai ◽  
Assay Conditions

Abstract We developed a quantitative assay for apolipoprotein AI (apo AI) in human serum, using a "sandwich"-type enzyme-linked immunosorbent assay. Diluted serum samples were pipetted into the wells of polystyrene microtiter plates that had been previously coated with purified rabbit anti-human apo AI antibodies. After incubation for 2 h and washing, antibodies conjugated to horseradish peroxidase (EC 1.11.1.7) were added and incubated for 2 h; after further washing, the bound enzyme was assayed by oxidation of o-phenylenediamine. Assay conditions were optimized for the incubation time and the amounts of coating antibodies and conjugate. Assay sensitivity is about 0.5 ng of apo AI, with a working range of 1 to 14 ng, similar to that of radioimmunoassays for human apo AI. The standard curves for apo AI in serum or HDL and for purified apo AI were parallel. Delipidation, heat treatment, or addition of detergents did not affect the amount of immunoassayable apo AI in human serum. The intra- and interassay CVs were 4 and 8%, respectively. Results for 100 serum samples compared well with those by immunonephelometry (r = 0.94).

scholarly journals Management of Factors for Improving Antigen–Antibody Interaction in Lateral flow Immunoassay of Tetracycline in Human Serum Samples

2019 ◽  
Vol 12 (1) ◽  
pp. 17-24
Author(s):  
Anna N. Berlina ◽  
Anastasia V. Bartosh ◽  
Anatoly V. Zherdev ◽  
Sergei A. Eremin ◽  
Boris B. Dzantiev
Keyword(s):  
Human Serum ◽  
Lateral Flow ◽  
Lateral Flow Immunoassay ◽  
Serum Samples ◽  
Assay Sensitivity ◽  
Detection Techniques ◽  
Human Serum Samples ◽  
Antigen Antibody ◽  
Site Control ◽  
Assay Conditions

Detection of antibiotics in the blood is necessary for characterizing their common or individual pharmacokinetics. This has increased the need in rapid detection techniques, such as lateral flow immunoassay, for the on-site control of antibiotics. The present study characterized factors influencing the analytical parameters of lateral flow immunoassay to increase its sensitivity for detecting tetracycline in human serum samples. Assay sensitivity was increased by altering the concentrations of immunoreagents and surfactant and the number of interaction stages in the assay with indirect labeling a specific antibody. The optimal assay conditions reduced the limit of visual detection of tetracycline from 100 to 10 ng/mL. The developed assay allowed us to detect tetracycline in both two-fold diluted and undiluted human serum samples within 15 min. Our results suggest that the developed assay can be used to screen patients under antibiotic treatment.

scholarly journals C-Terminal Invariable Domain of VlsE Is Immunodominant but Its Antigenicity Is Scarcely Conserved among Strains of Lyme Disease Spirochetes

2001 ◽  
Vol 69 (5) ◽  
pp. 3224-3231 ◽  
Author(s):  
Fang Ting Liang ◽  
Lisa C. Bowers ◽  
Mario T. Philipp
Keyword(s):  
Lyme Disease ◽  
Antibody Response ◽  
Human Serum ◽  
Synthetic Peptide ◽  
Species Complex ◽  
Enzyme Linked Immunosorbent Assay ◽  
Serum Samples ◽  
Variable Surface ◽  
Entire Sequence ◽  
Carboxyl Terminal

ABSTRACT VlsE, the variable surface antigen of Borrelia burgdorferi, contains two invariable domains located at the amino and carboxyl terminal ends, respectively, and a central variable domain. In this study, both immunogenicity and antigenic conservation of the C-terminal invariable domain were assessed. Mouse antiserum to a 51-mer synthetic peptide (Ct) which reproduced the entire sequence of the C-terminal invariable domain of VlsE from B. burgdorferi strain B31 was reacted on immunoblots with whole-cell lysates extracted from spirochetes of 12 strains from the B. burgdorferi sensu lato species complex. The antiserum recognized only VlsE from strain B31, indicating that epitopes of this domain differed among these strains. When Ct was used as enzyme-linked immunosorbent assay (ELISA) antigen, all of the seven monkeys and six mice that were infected with B31 spirochetes produced a strong antibody response to this peptide, indicating that the C-terminal invariable domain is immunodominant. None of 12 monkeys and only 11 of 26 mice that were infected with strains other than B31 produced a detectable anti-Ct response, indicating a limited antigenic conservation of this domain among these strains. Twenty-six of 33 dogs that were experimentally infected by tick inoculation were positive by the Ct ELISA, while only 5 of 18 serum samples from dogs clinically diagnosed with Lyme disease contained detectable anti-Ct antibody. Fifty-seven of 64 serum specimens that were collected from American patients with Lyme disease were positive by the Ct ELISA, while only 12 of 21 European samples contained detectable anti-Ct antibody. In contrast, antibody to the more conserved invariable region IR6 of VlsE was present in all of these dog and human serum samples.

A rat granulosa cell plasminogen activator bioassay for FSH in human serum

1990 ◽  
Vol 126 (1) ◽  
pp. 159-168 ◽  
Author(s):  
A. N. Thakur ◽  
R. Coles ◽  
A. Sesay ◽  
B. Earley ◽  
H. S. Jacobs ◽  
...  
Keyword(s):  
Human Serum ◽  
Plasminogen Activator ◽  
Granulosa Cell ◽  
Serum Samples ◽  
Glycoprotein Hormone ◽  
Assay Sensitivity ◽  
Serum Factors ◽  
Human Serum Samples ◽  
Heat Treated ◽  
Fsh Bioactivity

ABSTRACT A previously described in-vitro rat granulosa cell plasminogen activator bioassay for FSH has been modified and applied in the assay of human serum. This modified method consists of exposing the diethylstilboestrol-stimulated granulosa cells from 25- to 26-day-old rats to FSH or test substance for 3·5 h in wells coated with 125I-labelled fibrinogen and treated with thrombin. Following stimulation with FSH, the dose-related production of plasminogen activator was measured as the degree of 125I-labelled fibrinolysis in the presence of added plasminogen. Using the urinary FSH/LH bioassay reference preparation as the assay standard, the useful range of the assay was 0·3–15IU/l, with an assay sensitivity of 0·3 IU/l. As determined using purified glycoprotein hormone preparations, the assay was highly specific for FSH. The minor degree of FSH bioactivity measured in some of the hormone preparations was accounted for by the amount of FSH contamination in these preparations. To abolish interference caused by unknown serum factors, we heat-treated the serum samples for 15 min at 56 °C before the assay. The results indicated that neither immunoreactivity nor bioactivity was affected by this treatment. Furthermore, heat-treated human sera gave responses parallel to the standard curve at the three dose levels (2, 4 and 8 μl) studied. We used this bioassay to estimate the FSH-like bioactivity in 15 human serum samples. The estimates of immunoreactive FSH in these samples correlated well with the corresponding FSH bioactivity (r = 0·745, n = 15 and P < 0·05). The results indicate that with this sensitive and rapid (completed within 24 h) bioassay, it should be possible to measure FSH bioactivity in heat-treated human serum samples. Journal of Endocrinology (1990) 126, 159–168

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 Hemoglobin-Specific Antibody in a Multiply Transfused Patient With Sickle Cell Disease

Blood ◽  
1997 ◽  
Vol 89 (6) ◽  
pp. 2155-2158 ◽  
Author(s):  
Peter A. Noronha ◽  
Loyda N. Vida ◽  
C. Lucy Park ◽  
George R. Honig
Keyword(s):  
Sickle Cell Disease ◽  
Sickle Cell ◽  
Solid Phase ◽  
Enzyme Linked Immunosorbent Assay ◽  
Antibody Activity ◽  
Cell Disease ◽  
Serum Samples ◽  
Igg Antibody ◽  
Microtiter Plates ◽  
Level Of Activity

Abstract Human hemoglobins (Hbs) are known to be immunogenic, and both normal and variant forms of Hb have been shown to stimulate antibody formation in a variety of animal species. In patients who are homozygous for the sickle Hb (HbS) mutation, transfusion of normal, HbA-containing erythrocytes provides a potential stimulus for HbA alloimmunization. We tested serum samples for the presence of anti-Hb antibody by a solid-phase enzyme-linked immunosorbent assay (ELISA) using Hb-coated polystyrene microtiter plates. Hb-bound antibody was identified using an antihuman IgG antibody. Serum samples from 89 patients with sickle cell disease were initially tested for evidence of Hb antibody. The serum from three individuals exhibited antibody activity against HbA with little or no activity against HbS. Only one of them, a multiply transfused adult with HbSS, was available for further study. When this patient's antibody was tested against a variety of normal and mutant Hbs using antibody either to human IgG or to κ chains, the anti-Hb antibody demonstrated specificity for the region of the Hb β chain corresponding to the site of the amino acid substitution of HbS. The level of activity of the patient's anti-HbA showed no significant change over 1.5 years of observation. The transfusion of erythrocytes containing Hb structurally different from that of the recipient appeared to be capable of stimulating the production of Hb-specific alloimmune antibody.

Sandwich enzyme immunoassay of osteocalcin in serum with use of an antibody against human osteocalcin

1993 ◽  
Vol 39 (6) ◽  
pp. 942-947 ◽  
Author(s):  
D A Monaghan ◽  
M J Power ◽  
P F Fottrell
Keyword(s):  
Monoclonal Antibody ◽  
Enzyme Immunoassay ◽  
Solid Phase ◽  
Sandwich Elisa ◽  
Normal Subjects ◽  
Enzyme Linked Immunosorbent Assay ◽  
Serum Samples ◽  
Standard Curve ◽  
Microtiter Plates ◽  
Human Osteocalcin

Abstract We have developed and thoroughly validated a solid-phase sandwich enzyme-linked immunosorbent assay (ELISA) on microtiter plates for osteocalcin in human serum with use of an antibody raised against human osteocalcin. We used a monoclonal antibody against bovine osteocalcin as the capture antibody; the second antibody was a polyclonal antibody against human osteocalcin. The amount of bound second antibody was determined with use of swine anti-rabbit antibody labeled with horseradish peroxidase. We demonstrated independence of volume and determined the recovery of added standard and within- and between-assay precision. The minimal detection limit for osteocalcin was between 1.0 and 1.5 micrograms/L and the midpoint of the standard curve ranged from 14 to 17 micrograms/L. The intraassay CV was &lt; or = 8% in the range 2.7-52 micrograms/L; the interassay CV was usually &lt; or = 15% in the same range. Analytical recovery of human osteocalcin standard added to serum samples was consistently &gt; 90%. Values for osteocalcin measured in serum from 44 normal subjects were similar to those obtained with a competitive enzyme immunoassay (EIA) that used a monoclonal antibody against bovine osteocalcin. There was a good correlation between the two assays [r2 = 0.877, slope and intercept (+/- SE) = 0.88(+/- 0.051) and 0.316(+/- 0.523), respectively]. The range and mean (+/- SD) for the sandwich ELISA and the competitive EIA were 1.7-18.1 micrograms/L [8.7(+/- 4.4) micrograms/L] and 1.9-22.8 micrograms/L [9.1(+/- 4.4) micrograms/L], respectively.

scholarly journals Development of the ELISA Test System for Quantitative Determination of IgG to the Varizella zoster virus in Human Serum and Assessment of its Diagnostic Efficiency

2022 ◽  
Vol 20 (6) ◽  
pp. 72-80
Author(s):  
L. N. Lukhverchyk ◽  
G. L. Alatortseva ◽  
L. N. Nesterenko ◽  
V. Y. Kabargina ◽  
V. V. Dotsenko ◽  
...  
Keyword(s):  
Herpes Zoster ◽  
Blood Serum ◽  
Human Serum ◽  
Immunosorbent Assay ◽  
Test System ◽  
Enzyme Linked Immunosorbent Assay ◽  
Diagnostic Efficiency ◽  
Functional Characteristics ◽  
Serum Samples ◽  
Population Immunity

Relevance. The introduction of Varicella vaccine prophylaxis explains the need to develop a methodology for monitoring the vaccination effectiveness and the intensity of population immunity. This problem can be solved using quantitative immunoassay methods. Aim. Development of an enzyme-linked immunosorbent assay for the concentration of class G immunoglobulins (AB) to Varicella zoster virus (VZV) determining and assessing its functional characteristics and diagnostic efficiency. Materials and methods. Recombinant antigen GE VZV. WHO International Standard for Antibodies to VZV W1044. Blood serum samples from healthy people and patients with Chickenpox and Herpes zoster, blood serum samples containing IgG antibodies to herpes simplex viruses of the first and second types, cytomegalovirus, Epstein-Barr virus. Anti-VZV ELISA (IgG) reagent kit (Euroimmun, Germany). Indirect enzyme-linked immunosorbent assay. Immunization of animals with recombinant antigen GE, isolation, and purification of specific antibodies. Conjugation of monoclonal antibodies to human IgG with antibodies to antigen GE and with horseradish peroxidase. Results. An enzyme-linked immunosorbent assay in «an indirect» format has been developed to determine the specific antibodies to VZV concentration (IU/ml) in human serum/plasma. An artificial calibrator for determining the concentration of AB-VZV had been synthesized and standardized according to the International WHO-standard W1044. The main functional characteristics of the developed enzyme-linked immunosorbent assay are determined in accordance with GOST 51352-2013. The diagnostic kit was tested on blood serum samples from children with chickenpox (n = 43), adults with Herpes zoster (n = 158), healthy individuals (n = 781). The diagnostic sensitivity of the test system was 85%, the diagnostic specificity was 87% according to the ROC analysis. The absence of cross-reactivity of the test system was shown on samples with serological markers of other herpesvirus infections (n = 94). Comparative trials of the developed test system and its commercial analog, the Anti-VZV ELISA (IgG) reagent kit, did not reveal statistically significant differences between their functional characteristics. Conclusions. The developed test system for determining of the AB-VZV concentration in human serum/plasma in terms of its functional characteristics meets the GOST requirements, is characterized by high diagnostic efficiency, can be used to monitor the effectiveness of vaccine prophylaxis and strength of population immunity, as well as to assess the immune response in chickenpox and Herpes zoster.

Analysis of UK Sera for Aflatoxin by Enzyme-linked Immunosorbent Assay

1988 ◽  
Vol 7 (4) ◽  
pp. 353-356 ◽  
Author(s):  
A.P. Wilkinson ◽  
D.W. Denning ◽  
M.R.A. Morgan
Keyword(s):  
Human Serum ◽  
Direct Measurement ◽  
Immunosorbent Assay ◽  
Human Exposure ◽  
Blood Donors ◽  
Enzyme Linked Immunosorbent Assay ◽  
Fungal Metabolites ◽  
Serum Samples ◽  
Imported Food ◽  
The Uk

1 Aflatoxins are toxic, carcinogenic secondary fungal metabolites produced by certain moulds that commonly infest foods. Measurement of aflatoxins in human serum would give a direct measurement of exposure. 2 Twenty-seven serum samples from UK blood donors were found to contain aflatoxin levels not greater than 64 pmol/1 (20 pg/ml) by an enzyme-linked immunosorbent assay. 3 These findings may indicate that present UK guideline tolerances for aflatoxin in imported food are effective in limiting human exposure to toxic aflatoxins in the UK diet, though further work would be needed to confirm this. In particular, sub-populations suspected of being at higher risk may need special considerations.

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.

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