Development of a Sandwich ELISA to Detect Hepcidin in Human Serum.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2000-2000 ◽  
Author(s):  
Tara L Arvedson ◽  
George Doellgast ◽  
Hossein Salimi-Moosavi ◽  
Chadwick King ◽  
Ian Foltz ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Amino Acid ◽  
Polyclonal Antibody ◽  
Limit Of Detection ◽  
Sandwich Elisa ◽  
Alkaline Treatment ◽  
Detection Range ◽  
Antibody Preparation ◽  
Amino Acid Peptide ◽  
Class 1

Abstract Abstract 2000 Poster Board I-1022 Hepcidin is a 25 amino acid peptide that is the central mediator of iron metabolism. Iron excess, deficiency and maldistribution have been implicated in the etiology of many diseases including atherosclerosis, diabetes, neurodegeneration and the anemia of inflammation. Determination of hepcidin levels may be useful in diagnosis and treatment decisions for some or all of these diseases. Serum hepcidin measurement has so far been limited to a prohepcidin (60 amino acid hepcidin precursor) ELISA, mass spectrometry (MS)-based assays or competition ELISAs using polyclonal anti-hepcidin antibodies. The current work describes the generation of a sandwich ELISA using monoclonal antibodies to detect human hepcidin (hHepc) and optimization of assay conditions to resolve inconsistencies between MS- and ELISA-based detection. The ability of two anti-hHepc antibodies to sandwich (bind simultaneously) with hHepc was demonstrated using a rabbit polyclonal antibody preparation from hHepc-immunized animals. The same polyclonal antibody preparation was used for both hHepc capture and detection. The limit of detection achieved with this assay was O.D.450<1, suggesting that only a small proportion of the total antibodies could bind concurrently. To improve hHepc detection, a panel of monoclonal antibodies was screened for the ability to sandwich. Antibody epitope characterization studies using purified antibodies and >1000 hybridoma supernatants identified three classes of antibodies: classes 1 and 2 each recognized epitopes found in both full length mature hHepc (hHepc 25) and a shorter version (hHepc 20); class 3 bound a different epitope and demonstrated an increased affinity for hHepc 25 over hHepc 20. The majority of antibodies characterized were in class 1 while antibodies in classes 2 and 3 were rare (∼1% of antibody panel) highlighting the difficulty in achieving a sandwiching event. Antibodies 19D12 (class 1) and 23F11 (class 2) were identified as the optimal sandwich pair with a detection range of approximately 0.2-1000 ng/ml using synthetic hHepc. Initial comparisons of data generated using the sandwich ELISA and a fully-quantitative MS-based assay demonstrated a lack of consistent agreement. This issue was somewhat addressed by introduction of an alkaline treatment step to dissociate any protein/hHepc complexes in serum. Subsequent comparison of the two assays using sera from several different patient populations (anemia of cancer, chemotherapy-induced anemia, kidney disease) as well as healthy donors demonstrated good correlation (R2 range = 0.83-0.92; n=237). This sandwich ELISA may represent a tool for aligning the MS and ELISA-generated results in a format that has the potential to be high throughput and widely available. Disclosures: Arvedson: Amgen: Employment. Doellgast:Amgen: Employment. Salimi-Moosavi:Amgen: Employment. King:Amgen: Employment. Foltz:Amgen: Employment. Chen:Amgen: Employment. Li:Amgen: Employment. Sasu:Amgen: Employment.

scholarly journals Development of Vasoinhibin-Specific Monoclonal Antibodies

2021 ◽  
Vol 12 ◽  
Author(s):  
Nils Müller ◽  
Juan Pablo Robles ◽  
Magdalena Zamora ◽  
Johannes Ebnet ◽  
Hülya Markl-Hahn ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Serum Proteins ◽  
Limit Of Detection ◽  
Sandwich Elisa ◽  
Vascular Diseases ◽  
High Specificity ◽  
Cross Reactivity ◽  
Dimensional Structure ◽  
Serum Samples ◽  
Limit Of Quantitation

Vasoinhibin is a protein hormone with antiangiogenic, antivasodilatatory, and antivasopermeability effects generated by the proteolytic cleavage of prolactin. The discovery of its role in diabetic retinopathy and peripartum cardiomyopathy led to the evaluation of new pharmacological treatments in clinical interventional trials. However, the quantitative evaluation of vasoinhibin in biological samples from patients has not been possible due to the lack of vasoinhibin-specific antibodies. Recently, loop 1 of vasoinhibin was identified to have a different three-dimensional structure compared to PRL, and thus to contain vasoinhibin-specific epitopes. Here, we report the development of two sets of vasoinhibin-specific monoclonal antibodies against two neighboring regions of the vasoinhibin loop 1. An experimental sandwich ELISA with two monoclonal anti-vasoinhibin antibodies was developed, which had no cross-reactivity to recombinant human full-length prolactin. The ELISA had a quantitation limit of 100 ng/ml, and intra-assay- and inter-assay coefficients of variation of 12.5% and 14%, respectively. The evaluation of 15 human serum samples demonstrated concentrations of below limit of detection (n=3), below limit of quantitation (n=1) and between 0.23 µg/ml (230 ng/ml) to 605 µg/ml (n=12) in the quantifiable range. Despite the high specificity of the monoclonal-monoclonal antibody sandwiches which discriminate vasoinhibin from PRL, there might be cross-reactivities by serum proteins other than vasoinhibin. A fully established vasoinhibin ELISA may support diagnostic and therapeutic measures in vascular diseases.

Evidence for heterogeneity in the 160/165 × 10(3) Mr glycoprotein components of desmosomes

1987 ◽  
Vol 88 (4) ◽  
pp. 513-520
Author(s):  
J.C. Jones ◽  
K.L. Vikstrom ◽  
R.D. Goldman
Keyword(s):  
Monoclonal Antibodies ◽  
Polyclonal Antibody ◽  
Polyclonal Antibodies ◽  
Mouse Skin ◽  
Basal Cells ◽  
Antibody Preparation ◽  
Plaque Component ◽  
Mouse Tissues ◽  
Double Label ◽  
Cryostat Sections

We have prepared both monoclonal and polyclonal antibody preparations directed against the 160/165 × 10(3) Mr glycoproteins (desmogleins) of bovine tongue epithelial desmosomes. The polyclonal antibody preparation recognizes desmosomes in a number of mouse tissues, e.g. mouse skin, heart, bladder and trachea, as determined by immunofluorescence microscopy. Furthermore, the polyclonal antibodies recognize polypeptide(s), present in the high salt, Triton-insoluble residues (‘cytoskeleton preparations’) of mouse skin, heart, bladder and trachea, which comigrate with the 160/165 × 10(3) Mr glycoproteins of bovine tongue epithelial desmosomes as determined by ‘Western’ immunoblotting. Conversely, the monoclonal 160/165 × 10(3) Mr antibody preparation recognizes desmosomes of stratified squamous epithelial tissues but not desmosomes in other tissue types. Moreover, whereas the monoclonal antibodies recognize 160/165 × 10(3) Mr polypeptides in mouse skin cell cytoskeletons they show no immunoreactivity with the cytoskeleton preparations of mouse bladder, trachea and heart following immunoblotting. These results suggest therefore that although there are conserved epitopes of the 160/165 × 10(3) Mr glycoproteins there are also epitopes of these molecules which vary from tissue to tissue. Double label immunofluorescence observations of cryostat sections of mouse skin using the monoclonal antibodies and antibodies directed against desmoplakin, a plaque component of desmosomes, reveal that the monoclonal antibodies do not recognize certain desmosomes in basal cells which are recognized by desmoplakin antibodies. Indeed, double label observations of cryostat sections of mouse skin using the monoclonal antibodies and human autoantibodies which react with hemidesmosomal components suggest that the monoclonal antibodies stain desmosomes located along the apical surfaces of basal cells but fail to recognize desmosomes along the lateral surfaces of these same cells.(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals Development of Monoclonal Antibodies against HIV-1 p24 Protein and Its Application in Colloidal Gold Immunochromatographic Assay for HIV-1 Detection

2016 ◽  
Vol 2016 ◽  
pp. 1-6 ◽  
Author(s):  
Yi Ma ◽  
Chao Ni ◽  
Emmanuel E. Dzakah ◽  
Haiying Wang ◽  
Keren Kang ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Early Diagnosis ◽  
Colloidal Gold ◽  
Limit Of Detection ◽  
Sandwich Elisa ◽  
Immunochromatographic Assay ◽  
Suitable Alternative ◽  
P24 Protein ◽  
Hybridoma Cell Lines ◽  
Hiv 1

Human immunodeficiency virus type 1 (HIV-1) p24 protein is the most abundant viral protein of HIV-1. This protein is secreted in blood serum at high levels during the early stages of HIV-1 infection, making it a biomarker for early diagnosis. In this study, a colloidal gold immunochromatographic assay (GICA) was established for detecting p24 protein using mouse monoclonal antibodies (mAbs). The HIV-1 p24 protein was expressed inE. colistrain BL21 and the purified protein was used to immunize mice. Stable hybridoma cell lines secreting anti-p24 monoclonal antibodies were obtained after ELISA screening and subcloning by limiting dilution. 34 different capture and labeling mAb pairs were selected by a novel antibody-capture indirect sandwich ELISA and then applied in GICA to detect p24 protein. The GICA method has a limit of detection (LOD) of 25 pg/mL and could detect p24 protein in all 10 positive samples obtained from the National Reference of HIV-1 p24 antigen. Out of 153 negative samples tested, 3 false positives results were obtained. The overall specificity of this test was 98.03%. The good sensitivity and specificity of this method make it a suitable alternative to provide a more convenient and efficient tool for early diagnosis of HIV infection.

scholarly journals Rationally Designed Synthetic Haptens to Generate Anti-Ciguatoxin Monoclonal Antibodies, and Development of a Practical Sandwich ELISA to Detect Ciguatoxins

Toxins ◽  
2019 ◽  
Vol 11 (9) ◽  
pp. 533 ◽  
Author(s):  
Takeshi Tsumuraya ◽  
Masahiro Hirama
Keyword(s):  
Monoclonal Antibodies ◽  
Limit Of Detection ◽  
Sandwich Elisa ◽  
Food Poisoning ◽  
Enzyme Linked Immunosorbent Assay ◽  
Fda Guidance ◽  
Fish Poisoning ◽  
Highly Sensitive ◽  
Taste And Smell ◽  
Reliable Methods

“Ciguatera” fish poisoning (CFP) is one of the well-known food poisoning caused by the ingestion of fish that have accumulated trace amounts of ciguatoxins (CTXs). CFP affects more than 50,000 individuals annually. The difficulty in preventing CFP comes from the lack of reliable methods for analysis of CTXs in contaminated fish, together with the normal appearance, taste, and smell of CTX-contaminated fish. Thus, a sensitive, accurate, routine, and portable analytical method to detect CTXs is urgently required. Monoclonal antibodies (mAbs) specific against either wing of major CTX congeners (CTX1B, 54-deoxyCTX1B, CTX3C, and 51-hydroxyCTX3C) were generated by immunizing mice with rationally designed synthetic haptens-KLH conjugates instead of the CTXs. Haptenic groups with a surface area greater than 400 Å2 are required to produce mAbs that can strongly bind to CTXs. Furthermore, a highly sensitive fluorescence-based sandwich enzyme-linked immunosorbent assay (ELISA) was developed. This protocol can detect and quantify four major CTX congeners (CTX1B, 54-deoxyCTX1B, CTX3C, and 51-hydroxyCTX3C) with a limit of detection (LOD) of less than 1 pg/mL. The LOD determined for this sandwich ELISA is sufficient to detect CTX1B-contaminated fish at the FDA guidance level of 0.01 ppb.

Characterization of Five Monoclonal Antibodies Obtained After Immunization In Vitro with a Synthetic 19-Amino Acid Peptide of Thyroglobulin

Hybridoma ◽  
1987 ◽  
Vol 6 (6) ◽  
pp. 655-662 ◽  
Author(s):  
MARK DE BOER ◽  
PETER ADMIRAAL ◽  
KARIN KOK ◽  
FERRY A. OSSENDORP ◽  
JAN J.M. DE VIJLDER ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Amino Acid ◽  
Amino Acid Peptide

scholarly journals Efficient detection of pre-proinsulin by double antibody sandwich ELISA

2020 ◽  
Author(s):  
Zhu Zhu ◽  
Guoliang Ma ◽  
Li Wang ◽  
Han Wang ◽  
Hengfang Tang ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Human Insulin ◽  
Limit Of Detection ◽  
Sandwich Elisa ◽  
Insulin Production ◽  
Recombinant Human Insulin ◽  
Elisa Method ◽  
Efficient Detection ◽  
Sds Page ◽  
Conventional Cell

Abstract ObjectiveTo generate monoclonal antibodies against pre-proinsulin (PPI), and establish sandwich ELISA method to provide a basis for PPI detection in recombinant human insulin production.MethodsThe Balb /c mice were immunized with PPI, and the hybridomas secreting anti-PPI monoclonal antibodies were obtained by conventional cell fusion technique and ELISA screening.The antibody was purified using a Protein G gel column and identified for purity by SDS-PAGE. Pairing effect was found by the sandwich ELISA, and the specificity of the paired antibody was determined. A paired antibody with better specificity was selected to establish sandwich ELISA, and construct a quantitative curve, the accuracy and sensitivity of the method were evaluated.ResultsSix anti-PPI monoclonal antibodies were obtained, named P1, P2, P3, P4, P5 and P6, of which P5 had the highest titer. The sandwich ELISA method was established with P5 for plating and P2 for detection antibodie. The linear range of the quantitative curve of PPI by sandwich ELISA was 0. 645-82.5 pg/mL, the recovery was 89%–95%, and the limit of detection was 3.06 pg/mL.ConclusionSix monoclonal antibodies against PPI were generated and the sandwich ELISA method was established to detect PPI in process control and product release control for recombinant human insulin production.

Application of Monoclonal Antibodies to Dulcitol 1-Phosphate Dehydrogenase for Rapid Detection of Salmonella

1995 ◽  
Vol 58 (8) ◽  
pp. 847-852 ◽  
Author(s):  
TAKAHISA MIYAMOTO ◽  
HUAIZE TIAN ◽  
KIYOSHI MATSUNO ◽  
RYOJI TAKATA ◽  
SHOJI HATANO
Keyword(s):  
Monoclonal Antibodies ◽  
Salmonella Typhimurium ◽  
Magnesium Chloride ◽  
Rapid Detection ◽  
Limit Of Detection ◽  
Sandwich Elisa ◽  
Enzyme Linked Immunosorbent Assay ◽  
The Novel ◽  
Inoculum Level ◽  
E Coli

Monoclonal antibodies raised against dulcitol 1-phosphate dehydrogenase of Salmonella typhimurium IFO 12529 were screened against 20 serotypes of Salmonella and 13 non-Salmonella bacteria. A sandwich-capture, enzyme-linked immunosorbent assay (sandwich ELISA) was developed for detection of Salmonella in food. The assay utilizes two monoclonal antibodies (DUI2 and DU28) which show no cross-reactions with non-Salmonella bacteria. The limit of detection of the sandwich ELISA was about 1 × 107 CPU/ml. After cultivation in a medium containing dulcitol at 37°C for 18 h followed by the sandwich ELISA. 1 CPU of Salmonella was detected. Although a high inoculum level of E. coli interfered with the detection of Salmonella, the interference was minimized by using a selective dulcitol-magnesium chloride-pyridinesulfonic acid medium for enrichment. The novel ELISA procedure detected Salmonella in chicken filtrates inoculated with 1.4 CPU/50 m1 and 1.3 × 107 CPU/50 ml of E. coli within 25 h.

scholarly journals Characterization of monoclonal antibodies against a type SAT 2 foot-and-mouth disease virus

1993 ◽  
Vol 111 (2) ◽  
pp. 391-406 ◽  
Author(s):  
J. R. Crowther ◽  
C. A. Rowe ◽  
R. Butcher
Keyword(s):  
Monoclonal Antibodies ◽  
Amino Acid ◽  
Sandwich Elisa ◽  
Foot And Mouth Disease ◽  
The Other ◽  
Linear Epitope ◽  
Field Isolates ◽  
Mouth Disease Virus ◽  
Escape Mutants

SummaryThis paper is the first to describe characterization of monoclonal antibodies (MAbs) against a South African Territories 2 (SAT 2) foot-and-mouth disease virus (isolate Rho 1/48). Twelve MAbs which neutralized homologous virus were characterized in indirect and sandwich ELISA using purified Rho 1/48 virus particles, subunits, trypsin-treated, and chemically denatured virus. All the Mabs inhibited haemagglutination by parental virus. Binding of the MAbs to 73 SAT 2 field isolates was measured in a sandwich ELISA and defined four distinct antigenic regions. Preliminary characterization of escape mutants selected with some of the MAbs using virus neutralization tests, ELISA, and amino acid sequencing is included. MAbs 2, 25, 40, 48 and 64, reacted with a linear epitope on the VP1 loop region. An amino acid change at position 149 (valine to glutamic acid) was detected in mutants selected by MAb 2 and 40 and this eliminated binding and neutralization by all the other MAb. This epitope was conformation-dependent and was conserved in all 73 isolates of SAT 2 examined. Escape mutants isolated with MAb 41 and 44, had changes at positions 156 (glycine to aspartic acid), or 158 (serine to leucine) respectively. These MAbs bound with Rho 1/48 only out of 73 field strain viruses studies and the reactions of MAbs from the other groups was unaltered. MAb 27, 28 and 37 reacted with a conformation-dependent epitope on VP1 which was not conserved in field isolates. All mutants selected by these MAbs had a single amino acid substitution at position 149 (valine to alanine). The same change was always found in field isolates which did not bind MAbs from this group. MAb 11 reacted with a linear epitope associated with amino acids 147 or 148 on VP1 and showed similar binding characteristics to a conformation dependent MAb 7, no amino acid residue changes were found within VP1 for monoclonal antibody 7 mutants.

Immunoelectron microscope detection of C-type natriuretic peptide in normal human atrial tissue

1993 ◽  
Vol 51 ◽  
pp. 388-389
Author(s):  
Chi-Ming Wei ◽  
Margaret Hukee ◽  
Christopher G.A. McGregor ◽  
John C. Burnett
Keyword(s):  
Amino Acid ◽  
Natriuretic Peptide ◽  
Vascular Tone ◽  
Cardiac Tissue ◽  
Ring Structure ◽  
Terminal Amino Acid ◽  
Amino Acid Peptide ◽  
Normal Human ◽  
Terminal Amino ◽  
The Brain

C-type natriuretic peptide (CNP) is a newly identified peptide that is structurally related to atrial (ANP) and brain natriuretic peptide (BNP). CNP exists as a 22-amino acid peptide and like ANP and BNP has a 17-amino acid ring formed by a disulfide bond. Unlike these two previously identified cardiac peptides, CNP lacks the COOH-terminal amino acid extension from the ring structure. ANP, BNP and CNP decrease cardiac preload, but unlike ANP and BNP, CNP is not natriuretic. While ANP and BNP have been localized to the heart, recent investigations have failed to detect CNP mRNA in the myocardium although small concentrations of CNP are detectable in the porcine myocardium. While originally localized to the brain, recent investigations have localized CNP to endothelial cells consistent with a paracrine role for CNP in the control of vascular tone. While CNP has been detected in cardiac tissue by radioimmunoassay, no studies have demonstrated CNP localization in normal human heart by immunoelectron microscopy.

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