Development and Validation of an Immunoenzymatic Assay for Pig IgG Quantification in Serum Samples

2021 ◽  
Author(s):  
Yelieny Machin ◽  
Keyword(s):  
Polyclonal Antibody ◽  
Immune Status ◽  
Enzyme Linked Immunosorbent Assay ◽  
Elisa Assay ◽  
Serum Samples ◽  
Humoral Immune ◽  
Porcine Serum ◽  
Sandwich Type ◽  
Development And Validation ◽  
Immunoenzymatic Assay

This research describes the development and validation of a sandwich-type Enzyme-Linked-Immunosorbent Assay (ELISA) assay based on a sheep polyclonal antibody developed to determine porcine immunoglobulin G (IgG) concentration in serum samples. The coating and blocking conditions were established. The immunoassay demonstrated the ability to specifically quantify pig IgG in serum samples without reacts with rabbit or mouse IgG, evaluated in serum and ascites, respectively. Given its precision, specificity and accuracy, this ELISA is a tool for the measurement of IgG in porcine serum samples, to evaluate the humoral immune status of pigs.

Quantification of human apolipoprotein A-IV by "sandwich"-type enzyme-linked immunosorbent assay.

1988 ◽  
Vol 34 (4) ◽  
pp. 739-743 ◽  
Author(s):  
M Rosseneu ◽  
G Michiels ◽  
W De Keersgieter ◽  
J Bury ◽  
J P De Slypere ◽  
...  
Keyword(s):  
Immunosorbent Assay ◽  
Enzyme Linked Immunosorbent Assay ◽  
Elisa Assay ◽  
Chromatographic Fractionation ◽  
Human Apolipoprotein ◽  
Apolipoprotein A ◽  
Sandwich Type ◽  
Antibody Conjugate ◽  
The Mean

Abstract A specific and sensitive "sandwich"-type enzyme-linked immunosorbent assay (ELISA) has been developed for quantifying human apo A-IV. Using apo A-IV immunosorbent columns, we isolated monospecific anti-apo A-IV antibodies for coating the ELISA plates and for preparing peroxidase-antibody conjugate. The assay can detect as little as 0.20 ng of apo A-IV, with mean intra- and interassay CVs of 3.6% and 8.2%, respectively. The apoA-IV concentrations in normolipemic and hyperlipemic plasma were unaffected by either delipidation or treatment with detergents or urea. To validate the ELISA assay we compared it with an immunoelectrophoretic technique. ApoA-IV concentrations in plasma from normo- and dyslipemic subjects compared well by the two assays (r = 0.89). The mean apo A-IV concentration, measured by ELISA in plasma from 50 normolipemic subjects, was 143 (SD 52) mg/L; values for dyslipemic subjects were not significantly different. We also used this new assay to monitor apo A-IV profiles of normolipemic and hypertriglyceridemic plasma after chromatographic fractionation.

scholarly journals Use of GRA6-Derived Synthetic Polymorphic Peptides in an Immunoenzymatic Assay To Serotype Toxoplasma gondii in Human Serum Samples Collected from Three Continents

2008 ◽  
Vol 15 (9) ◽  
pp. 1380-1386 ◽  
Author(s):  
Susana Sousa ◽  
Daniel Ajzenberg ◽  
Manuel Vilanova ◽  
José Costa ◽  
Marie-Laure Dardé
Keyword(s):  
Toxoplasma Gondii ◽  
Latin American ◽  
Enzyme Linked Immunosorbent Assay ◽  
Clear Correlation ◽  
Type I ◽  
Serum Samples ◽  
Typing Method ◽  
Microsatellite Genotype ◽  
Human Infections ◽  
Immunoenzymatic Assay

ABSTRACT Serotyping is a simple typing method that consists of an immunoenzymatic assay (enzyme-linked immunosorbent assay [ELISA]) using synthetic polymorphic peptides derived from Toxoplasma gondii antigens. We developed a new ELISA based on GRA6 C-terminal polymorphic peptides. Serum samples from 41 human infections due to 23 archetypal (type I, II, or III) and 18 nonarchetypal strains were selected in order to validate this approach. For 20 out of the 23 archetypal infections, there was a clear correlation between microsatellite genotype and GRA6 serotyping. All infections due to nonarchetypal strains were misclassified as archetypal strain infections. The GRA6 C-terminal peptides from these strains were analyzed to explain this misclassification. A second group of 455 patients with acute and chronic toxoplasmosis due to unknown genotypes from different European, African, and Latin American countries were included in this study, and the strain type predicted by this method. The results suggest that serotyping is a promising method for typing strains, although limitations exist for African and South American strains as a consequence of higher peptide polymorphism. Other peptides from different markers must be studied in order to discriminate archetypal from nonarchetypal strains.

scholarly journals Diversity of Antigen Recognition by Serum Antibodies in Experimental Bovine Tuberculosis

1998 ◽  
Vol 66 (11) ◽  
pp. 5344-5349 ◽  
Author(s):  
Konstantin P. Lyashchenko ◽  
John M. Pollock ◽  
Roberto Colangeli ◽  
Maria Laura Gennaro
Keyword(s):  
Antibody Production ◽  
Bovine Tuberculosis ◽  
Tuberculin Test ◽  
Economic Problem ◽  
Antigen Recognition ◽  
Enzyme Linked Immunosorbent Assay ◽  
Serum Antibodies ◽  
Serum Samples ◽  
Humoral Immune ◽  
Humoral Immune Responses

ABSTRACT Tuberculosis in cattle remains a major zoonotic and economic problem in many countries. The standard diagnostic assay for bovine tuberculosis, the intradermal tuberculin test, has low accuracy. Therefore, alternative immunodiagnostic methods, such as serological assays, are needed for detection of infected animals. Development of an accurate serodiagnostic test requires a detailed understanding of the humoral immune responses during bovine tuberculosis and, in particular, identification of the key antigens of Mycobacterium bovisinvolved in antibody production. In this study, we characterized antibody responses in cattle experimentally infected with M. bovis. Sequential serum samples were collected every 3 to 4 weeks for up to 27 months postinfection. Circulating immunoglobulin G antibody levels were measured by an enzyme-linked immunosorbent assay using 12 highly purified recombinant proteins of M. bovis. Six proteins, ESAT-6, 14-kDa protein, MPT63, MPT70, MPT51, and MPT32, were identified as major seroreactive antigens in bovine tuberculosis. A remarkable animal-to-animal variation of antigen recognition by serum antibodies was observed. Kinetic analyses of the antibody production to individual antigens during infection revealed that the heterogeneous antigen recognition profile changed markedly in a given infected animal as disease progressed.

scholarly journals Serological Discrimination of Dogs Infected with Gastric Helicobacter spp. and Uninfected Dogs

1999 ◽  
Vol 37 (5) ◽  
pp. 1280-1287 ◽  
Author(s):  
Dalit Strauss-Ayali ◽  
Kenneth W. Simpson ◽  
Amy H. Schein ◽  
Patrick L. McDonough ◽  
Richard H. Jacobson ◽  
...  
Keyword(s):  
Molecular Mass ◽  
Bacterial Colonization ◽  
Enzyme Linked Immunosorbent Assay ◽  
Major Protein ◽  
Serum Samples ◽  
Lymphoid Follicles ◽  
Humoral Immune ◽  
Colonization Density ◽  
Antigenic Homology ◽  
H Pylori

Characterization of the humoral immune responses of people toHelicobacter pylori infection has facilitated the investigation of the host response to bacterial virulence factors and the development of sensitive and specific diagnostic tests. Dogs are commonly infected with gastric Helicobacter spp., but the presence of multiple Helicobacter spp. and possible coinfection in individual dogs have complicated serological evaluation. Evaluation of the antigenic homology of Helicobacter spp. revealed that the major protein bands of Helicobacter felisand Helicobacter bizzozeronii, two Helicobacterspp. that infect dogs, were very similar to UreA (29 to 31 kDa), UreB (63 to 66 kDa), and HSP (58 to 60 kDa) of H. pylori, and sera from infected and uninfected dogs bound in a similar way to each antigen. Immunoblotting and an enzyme-linked immunosorbent assay (ELISA) with H. felis ATCC 49179 antigen were performed with 101 serum samples (from 78 infected dogs and 23 uninfected dogs). Samples from uninfected dogs (median = 8) had fewer bands on immunoblotting than samples from infected dogs (median = 16) (P < 0.05). Combinations of the presence of any two of the low-molecular-mass bands (19, 25, 30, 32, and 37 kDa) or the high-molecular-mass bands (86 and 94 kDa) were found almost solely in samples from infected dogs (P < 0.0001). Kinetic ELISA results were significantly higher for samples from infected dogs (median = 0.0802 optical density unit [OD]/min) than for samples from uninfected dogs (median = 0.01428 OD/min). The combination of ELISA and immunoblotting results gave a specificity of 95.6% and a sensitivity of 79.8%. No correlation between ELISA results, colonization density, degree of inflammation, and presence of lymphoid follicles was observed. The results indicate substantial antigenic homology between H. felis, H. pylori, andH. bizzozeronii. The combination of ELISA and immunoblotting was a highly specific and moderately sensitive indicator of infection. The degree of seropositivity assessed by ELISA was not related to bacterial colonization density, the degree of gastric inflammation, or the presence of lymphoid follicles.

Expression and Clinical Significance of Sperm-Associated Antigen 9 in Patients With Endometrial Carcinoma

2012 ◽  
Vol 22 (1) ◽  
pp. 87-93 ◽  
Author(s):  
Peng Yu ◽  
Lei Yan ◽  
Hui Zhang ◽  
Xiaoyan Lin ◽  
Xingbo Zhao
Keyword(s):  
Immune Response ◽  
Endometrial Cancer ◽  
Endometrial Carcinoma ◽  
Humoral Immune Response ◽  
Enzyme Linked Immunosorbent Assay ◽  
Serum Samples ◽  
Humoral Immune ◽  
Significant Difference ◽  
New Type ◽  
Grade 3

ObjectiveTo investigate the expression and humoral immune response of sperm-associated antigen 9 (SPAG9) in endometri al carcinoma.MethodsSperm-associated antigen 9 gene expression levels were evaluated in endometrial carcinoma, endometrial hyperplasia, adjacent tissues, and normal endometrial tissues by reverse transcriptase-polymerase chain reaction, immunohistochemistry, and Western blot. Sperm-associated antigen 9 concentration in serum samples from 10 healthy women, 20 women with benign diseases, and 50 women with endometrial carcinoma was detected by enzyme-linked immunosorbent assay.Results(1) Sperm-associated antigen 9 antibodies were detected in approximately 72% of patients with endometrial cancer but not in healthy controls. (2) A significant difference has been found among pathological types and degrees (P < 0.05), and it was also found to be expressed in transferred lymph nodes. (3) Sperm-associated antigen 9 serum concentration (ng/mL) of patients with endometrial carcinoma is significantly higher than those of the healthy group (P < 0.05). Patients harboring grade 3 endometrial carcinoma were found to have significantly higher SPAG9 concentrations than those of grade 1/grade 2 (P = 0.003).ConclusionsSPAG9 is positively expressed in endometrial cancer, and with a high humoral immune response in patients. It may serve as a new type of endometrial cancer markers for early detection, diagnosis and treatment.

scholarly journals Evaluation of the immune status of birds and domestic and companion animals for the influenza A virus in Eastern Saudi Arabia

2020 ◽  
Vol 13 (9) ◽  
pp. 1966-1969
Author(s):  
Abdelmohsen Abduallah Alnaeem ◽  
Abdulkareem Al-Shabeb ◽  
Maged Gomaa Hemida
Keyword(s):  
Saudi Arabia ◽  
Influenza A ◽  
Large Scale ◽  
Immune Status ◽  
Companion Animals ◽  
Type A ◽  
Influenza Viruses ◽  
Enzyme Linked Immunosorbent Assay ◽  
Serum Samples ◽  
Influenza Type

Background and Aim: Influenza type A virus infections are still one of the major concerns for the health of humans and various species of domestic and companion animals. Wild birds play an essential role in the transmission cycle of the virus. Regularly monitoring the spread of the virus is a significant step in its mitigation. Highly pathogenic avian influenza viruses, including H5N1 and H5N8, have been reported in birds in the Arabian Peninsula, including Saudi Arabia, in recent decades. This study aimed to evaluate the immune status of birds, domestic and companion animals for Influenza type A virus in Eastern Province of Saudi Arabia. Materials and Methods: We collected 195 serum samples from dromedary camels, sheep, goats, native breed chickens, doves, dogs, and cats. We tested these sera for the presence of specific antibodies against influenza type A virus using a commercially available enzyme-linked immunosorbent assay. Results: Our results show that 4% of the tested samples had antibodies in sera, including some doves, chickens, and dogs. These data suggest exposure and seroconversion of these animals or birds to the influenza type A virus. Conclusion: The presence of antibodies against influenza type A virus in sera of some animals and birds without a previous vaccination history against the virus indicates a natural exposure history regarding this virus and seroconversion. Further large-scale molecular and epidemiological studies are needed to obtain a better understanding of the dynamics of influenza type A virus among various species of animals and birds.

scholarly journals Humoral Immune Response in Chromoblastomycosis during and after Therapy

2000 ◽  
Vol 7 (3) ◽  
pp. 497-500 ◽  
Author(s):  
P. Esterre ◽  
M. Jahevitra ◽  
A. Andriantsimahavandy
Keyword(s):  
Immune Response ◽  
Fungal Species ◽  
Immunoglobulin M ◽  
Enzyme Linked Immunosorbent Assay ◽  
Antibody Concentration ◽  
Therapeutic Trial ◽  
Natural Immunity ◽  
Serum Samples ◽  
Humoral Immune ◽  
Cladophialophora Carrionii

ABSTRACT A longitudinal study was carried out in Madagascar, the most important focus of chromoblastomycosis (P. Esterre, A. Andriantsimahavandy, E. Ramarcel, and J. L. Pecarrere, Am. J. Trop. Med. Hyg. 55:45–47, 1996), to investigate natural immunity to this disease. Sequential blood samples were obtained before, during, and at the end of a successful therapeutic trial with terbinafine, a new antifungal drug. Using enzyme-linked immunosorbent assay and immunoblot methods, detailed analyses of antibody concentration and antigen mapping were conducted for 136 serum samples and tentatively correlated to epidemiological and pathobiological data. Two different cytoplasmic antigens, corresponding to the two fungal species involved (Fonsecaea pedrosoi and Cladophialophora carrionii), were used to analyze the distribution of different classes of immunoglobulins. This was done with respect to the origin of the isolates, clinical and pathobiological. Although strong individual variations were noticed, some major antigens (one of 18.5 kDa specific for F. pedrosoi and two of 23.5 and 33 kDa, respectively, specific for C. carrionii) corresponded to high antibody prevalence and concentration. As some antigenic components were also detected by immunoglobulin M (IgM) and IgA antibodies, the role that these specific antibodies could play in the immune response is discussed.

scholarly journals Seroprevalence of IgG antibodies against Anaplasma marginale in cattle from south Mozambique

2011 ◽  
Vol 20 (4) ◽  
pp. 318-324 ◽  
Author(s):  
António Amélia Mucalane Tembue ◽  
Jenevaldo Barbosa da Silva ◽  
Fábio Jorge Moreira da Silva ◽  
Marcus Sandes Pires ◽  
Cristiane Divan Baldani ◽  
...  
Keyword(s):  
Immune Status ◽  
Age Groups ◽  
Geographic Origin ◽  
Anaplasma Marginale ◽  
Enzyme Linked Immunosorbent Assay ◽  
Igg Antibodies ◽  
Serum Samples ◽  
Chi Square ◽  
Epidemiological Monitoring ◽  
Chi Square Test

The current study aimed to investigate the seroprevalence of IgG antibodies to Anaplasma marginale in cattle from Maputo, Gaza and Inhambane provinces, south Mozambique. A total of 809 serum samples from cattle were obtained and tested by indirect enzyme-linked immunosorbent assay (i-ELISA). The chi-square test at 5% significance was used to assess the association between seroprevalence and the variables gender, age and geographic origin of animals. The overall seropositivity was 76.5% (n = 619) and anti-A. marginale antibodies were detected in 89.1% (n = 156), 68.4% (n = 308) and 84.2% (n = 155) of the animals in the provinces of Maputo, Gaza and Inhambane, respectively. A significant association (p < 0.05) was found with the geographic origin of the animals, while sex had no significant relationship. The frequencies of seropositive in the age groups were 63.2% (n = 72), 80.0% (n = 92), 83.1% (n = 98) and 77.3% (n = 357) for animals <12; >12 and <24; >24 and <36; >36 months, respectively. These results indicate that in southern Mozambique there are areas of enzootic stability to A. marginale. Thus, epidemiological monitoring is required to monitor the immune status of animals in the region.

scholarly journals Development and validation of a sensitive ELISA for quantification of secretory IgA in rat saliva and faeces

2001 ◽  
Vol 35 (4) ◽  
pp. 301-306 ◽  
Author(s):  
J. Hau ◽  
E. Andersson ◽  
H.-E. Carlsson
Keyword(s):  
Immune Status ◽  
Laboratory Animals ◽  
Secretory Iga ◽  
Stress Assessment ◽  
Humoral Immune ◽  
Non Invasive ◽  
Quantitative Measurements ◽  
Immunological Markers ◽  
High Concentrations ◽  
Development And Validation

Non-invasive measures of immunological markers are an attractive means of stress assessment in laboratory animals. Salivary IgA has been used successfully as a stress marker in the human, and several reports indicate the potential of secretory IgA as a non-invasive measure of stress in animals. The present paper describes the development of an ELISA using commercially available components for the quantification of rat IgA and validation of this assay for the quantification of rat secretory IgA in saliva and faeces. The concentration of IgA in rat saliva varied significantly between duplicate samples obtained from individual rats, and the viscosity and small total volume of rat saliva gave unsatisfactory results for IgA. Faecal IgA was present in high concentrations, and duplicate samples varied by only 2-3%. However, faecal IgA seemed less stable than IgA in other biological compartments, and this finding must be taken into consideration when using quantitative measurements of IgA as a marker of mucous humoral immune status.

scholarly journals Prevalence and Genetic Analysis of Porcine Circovirus 3 in China From 2019 to 2020

2021 ◽  
Vol 8 ◽  
Author(s):  
Meng Ge ◽  
Jie Ren ◽  
Yi-Lin Xie ◽  
Dun Zhao ◽  
Fang-Cheng Fan ◽  
...  
Keyword(s):  
Potential Effect ◽  
Porcine Circovirus Type ◽  
Porcine Circovirus ◽  
Enzyme Linked Immunosorbent Assay ◽  
Serum Samples ◽  
Reading Frame ◽  
Humoral Immune ◽  
Antibody Positivity ◽  
Repeated Infections ◽  
Type 3

Porcine circovirus type 3 (PCV3), a virus belonging to the Circoviridae family, is considered to be associated with respiratory and neurological signs, cardiac and multisystemic inflammation, reproductive failure, and porcine dermatitis and nephropathy syndrome-like disease in pigs (Sus scrofa). In this study, epidemiological and serological investigations of PCV3 in clinically healthy pigs from different regions of China were performed. Overall, 42.87% (1,101/2,568) of pigs were positive for PCV3 Cap antibody via indirect enzyme-linked immunosorbent assay, with a higher prevalence of PCV3 in multiparous sows (62.22%, 881/1,416) and fattening pigs (28.96%, 159/549) than in suckling piglets (8.96%, 32/357) and nursery pigs (11.79%, 29/246). Of the 2,568 samples, 255 were further tested for PCV3 DNA using real-time polymerase chain reaction, and 63.14% of these were positive, with nearly half having &lt;10 virus copies. The PCV3 DNA and antibody positivity rates were high in the pig serum samples; however, the virus titers and antibody levels were both low, indicating that the humoral immune response of PCV3-infected pigs was weak or lagging, and persistent or repeated infections could occur. Additionally, the complete genomes of 23 PCV3 strains were sequenced and analyzed, which showed nucleotide identities of 98.5~100.0%, 98.6~100.0%, and 99.2~100.0% in the complete genome, open reading frame (ORF)2, and ORF1 sequences, respectively, and amino acid identities of 96.7~100.0% and 99.3~100.0% in the capsid and replicase proteins, respectively. Phylogenetic analysis based on ORF2 nucleotide sequences indicated that the PCV3 strains obtained in the present study could be classified into three sub-clades, with most strains clustered into clade 3c, indicating that PCV3c is the dominant subtype in the regions of China investigated. In general, the present study revealed a high prevalence and high genetic divergence of PCV3 among Chinese pig herds, and indicated that the potential effect of PCV3 on the pig industry may be a concern.

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