scholarly journals Systematic comparison of monoclonal versus polyclonal antibodies for mapping histone modifications by ChIP-seq

2016 ◽  
Author(s):  
Michele Busby ◽  
Catherine Xue ◽  
Catherine Li ◽  
Yossi Farjoun ◽  
Elizabeth Gienger ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Monoclonal Antibodies ◽  
Histone Modifications ◽  
Polyclonal Antibodies ◽  
Post Translational Modifications ◽  
Systematic Comparison ◽  
Mouse Cells ◽  
Overall Performance ◽  
Consistent Performance ◽  
Human And Mouse

AbstractBackgroundThe robustness of ChIP-seq datasets is highly dependent upon the antibodies used. Currently, polyclonal antibodies are the standard despite several limitations: they are non-renewable, vary in performance between lots, and need to be validated with each new lot. In contrast, monoclonal antibody lots are renewable and provide consistent performance. To increase ChIP-seq standardization, we investigated whether monoclonal antibodies could replace polyclonal antibodies. We compared monoclonal antibodies that target five key histone modifications (H3K4me1, H3K4me3, H3K9me3, H3K27ac and H3K27me3) to their polyclonal counterparts in both human and mouse cells.ResultsOverall performance was highly similar for four monoclonal/polyclonal pairs, including when we used two distinct lots of the same monoclonal antibody. In contrast, the binding patterns for H3K27ac differed substantially between polyclonal and monoclonal antibodies. However, this was most likely due to the distinct immunogen used rather than the clonality of the antibody.ConclusionsAltogether, we found that monoclonal antibodies as a class perform as well as polyclonal antibodies for the detection of histone post-translational modifications in both human and mouse. Accordingly, we recommend the use of monoclonal antibodies in ChIP-seq experiments.

scholarly journals Circumsporozoite proteins of malaria parasites contain a single immunodominant region with two or more identical epitopes.

1983 ◽  
Vol 157 (6) ◽  
pp. 1947-1957 ◽  
Author(s):  
F Zavala ◽  
A H Cochrane ◽  
E H Nardin ◽  
R S Nussenzweig ◽  
V Nussenzweig
Keyword(s):  
Monoclonal Antibody ◽  
Monoclonal Antibodies ◽  
Specific Binding ◽  
Polyclonal Antibodies ◽  
Binding Assays ◽  
Malaria Parasites ◽  
Immunodominant Region ◽  
Additional Finding ◽  
Single Region ◽  
Competition Binding

We have used panels of monoclonal antibodies to circumsporozoite (CS) proteins of Plasmodium falciparium, P. vivax, and P. knowlesi to determine the number of topographically independent epitopes of these antigens. The results of competition binding assays indicated that single regions of the CS molecules were recognized by the homologous monoclonal antibodies. Competition binding assays were also used to study the specificity of antibodies contained in the sera of humans and monkeys that had developed sterile immunity after immunization with irradiated, intact sporozoites. We found that single monoclonal antibodies inhibited 70-95% of the specific binding of the polyclonal antibodies to crude extracts of sporozoites. It appears, therefore, that CS proteins are among the most immunogenic constituents of sporozoites, and that a single region of these molecules contains most of the immunogenic activity. An additional finding was that the immunodominant region of CS molecules is multivalent with regard to the expression of a single epitope. This was demonstrated by the ability of monomers of CS proteins to bind simultaneously two or more molecules of the same monoclonal antibody.

Abstract 14697: Novel Assays for Quantification of Lipoprotein-Associated (PCSK9-apoB, PCSK9-Lp(a)) Proprotein Covertase Subtilisin/Kexin Type 9 (PCKS9)

Circulation ◽  
2015 ◽  
Vol 132 (suppl_3) ◽  
Author(s):  
Calvin Yeang ◽  
Yun-Seok Choi ◽  
Sang-Rok Lee ◽  
Monica L Bertoia ◽  
Eric B Rimm ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Monoclonal Antibodies ◽  
Human Plasma ◽  
Polyclonal Antibodies ◽  
Human Monoclonal Antibody ◽  
Ldl Receptor ◽  
Plasma Lipoproteins ◽  
Health Study ◽  
Clinical Samples ◽  
Ldl Particles

Background: PCSK9 is a major regulator of plasma LDL-C. Monoclonal antibodies to PCSK9 lower LDL-C by 45-65% and Lp(a) by 9-38%. The canonical function of PCSK9 is binding of LDL-receptor (LDLR) via its extracellular EGF-A domain, and subsequently mediating LDLR degradation. However, PCSK9 also weakly associates with plasma lipoproteins, with 20-40% of total plasma PCSK9 found on LDL. However, most LDL particles do not contain PCSK9. Whether PCSK9 also associates with other lipoproteins such as Lp(a) are not well described. Methods: Sensitive and quantitative sandwich-based ELISA assays were developed to measure PCSK9 associated plasma lipoproteins in both mouse and human plasma. For human plasma, commercial rabbit polyclonal antibodies binding to the C-terminal region of PCSK9 (Abgent, ThermoFisher) or REGN727 human monoclonal antibody were bound to microtiter well plates. Plasma was added and monoclonal antibodies MB47 and LPA4, binding to apoB-100 and apo(a) respectively, were used to detect PCSK9-apoB-100 and PCSK9-Lp(a) complexes with a chemiluminescent ELISA. For mouse assays, REGN727 was used as the capture antibody as it detects mouse PCSK9 and monoclonal antibody LF3 was used to detect mouse apoB. Results: PCSK9-apoB and PCSK9-Lp(a) complexes could be detected in both human plasma and in various mouse models expressing apo(a) or Lp(a). The signal to noise ratio was ~20 fold in various clinical samples, including in healthy subjects and in patients with cardiovascular disease. In 536 clinical samples from the Health Professional Follow-Up Study, PCSK9-Lp(a) correlated strongly with Lp(a) (r=0.59, p<0.001, age-adjusted) but not other lipid variables. PCSK9-apoB correlated weakly with PCSK9-Lp(a) (r=0.30, p<0.001, age-adjusted) and LDL-C (r=0.22, p<0.001, age-adjusted). These associations were virtually the same in 526 women in the Nurses’ Health Study. Conclusions: Novel ELISAs were generated to quantitate lipoprotein-associated PCSK9 in transgenic mouse and human plasma, including on apoB and Lp(a). Changes in PCSK9-Lp(a) complexes may provide insights into the Lp(a)-lowering effect of PCSK9 antibodies. Whether these assays will predict CVD outcomes waits to be determined in PCSK9 antibody and epidemiological studies.

scholarly journals Identification of an immunodominant functional domain on human CD36 antigen using human-mouse chimaeric proteins and homologue-replacement mutagenesis

1995 ◽  
Vol 305 (1) ◽  
pp. 221-224 ◽  
Author(s):  
L Daviet ◽  
R Buckland ◽  
M D Puente Navazo ◽  
J L McGregor
Keyword(s):  
Amino Acids ◽  
Monoclonal Antibody ◽  
Monoclonal Antibodies ◽  
Low Density Lipoprotein ◽  
Density Lipoprotein ◽  
Functional Domain ◽  
Oxidized Low Density Lipoprotein ◽  
Key Residues ◽  
Antibody Epitopes ◽  
Human And Mouse

The human CD36 antigen is a multifunctional membrane glycoprotein that acts as a receptor for thrombospondin, malaria-infected erythrocytes and oxidized low-density lipoprotein, as well as being implicated in the recognition of apoptotic neutrophils by macrophages. OKM5 and other anti-CD36 monoclonal antibodies have been shown to inhibit these CD36 adhesive functions, suggesting that the monoclonal-antibody epitopes and the domains that mediate these events are closely related. Analysis of a series of chimaeric exchanges between human and mouse CD36 shows that six anti-CD36 monoclonal antibodies (OKM5, FA6-152, L103, 5F1, SM phi and 10/5) recognize epitopes within the domain comprising amino acids 155-183. A seventh monoclonal antibody (13/10) binds to another domain that spans amino acids 30-76. Homologue-replacement mutagenesis performed within the human 155-183 immunodominant sequence identifies key residues for the binding of three functional monoclonal antibodies (OKM5, FA6-152 and L103). The fact that antibodies directed against the 155-183 domain can inhibit adhesion suggests that this domain is directly involved in CD36-ligand binding.

scholarly journals A comparative study of the use of monoclonal antibodies using three different immunohistochemical methods: an evaluation of monoclonal and polyclonal antibodies against human prostatic acid phosphatase.

1982 ◽  
Vol 30 (3) ◽  
pp. 253-260 ◽  
Author(s):  
W Y Naritoku ◽  
C R Taylor
Keyword(s):  
Monoclonal Antibody ◽  
Monoclonal Antibodies ◽  
Acid Phosphatase ◽  
Polyclonal Antibodies ◽  
Prostatic Acid Phosphatase ◽  
Mouse Serum ◽  
Paraffin Sections ◽  
Layer System ◽  
Significant Difference ◽  
Immunohistochemical Methods

The use of immunohistochemical methods has been advocated for the detection and localization of prostatic acid phosphatase in paraffin sections of human prostate. This article explores the possible advantages of utilizing monoclonal antibodies in this method. Monoclonal antibodies, specific for human prostatic acid phosphatase, were integrated into three different immunohistochemical procedures. In the first method, a three-layer peroxidase-antiperoxidase (PAP) system was employed; the monoclonal antibody was followed by rabbit bridge antibody directed against mouse immunoglobulin and mouse PAP complex. The second method was a three-layer system utilizing biotin-labeled horse anti-mouse antibody as "bridge" antiserum between the primary monoclonal antibody and an avidin-biotin-horseradish peroxidase complex. The third method was a four-layer system; the monoclonal antibody was followed by rabbit anti-mouse serum, swine anti-rabbit immunoglobulin as the bridge antibody and rabbit PAP complex. It was found that some, but not all, monoclonal antibodies can be used for the detection of prostatic acid phosphatase in paraffin sections. The four-layer PAP method was found to be the most sensitive method of the three systems tested; however, the avidin-biotin method required the least amount of time. No significant difference in the quality of staining was observed between monoclonal antibodies and carefully absorbed conventional antiserum.

Production of Monoclonal Antibodies Against Enamelin and Against Amelogenin Proteins of Developing Enamel Matrix

1987 ◽  
Vol 1 (2) ◽  
pp. 282-288 ◽  
Author(s):  
D. Deutsch ◽  
A. Palmon ◽  
J. Catalano-Sherman ◽  
R. Laskov
Keyword(s):  
Monoclonal Antibody ◽  
Extracellular Matrix ◽  
Monoclonal Antibodies ◽  
Structural Organization ◽  
Polyclonal Antibodies ◽  
Protein Band ◽  
Enamel Matrix ◽  
Complex Process ◽  
The One ◽  
Successful Production

The extracellular matrix of developing enamel contains two major classes of proteins, the hydrophobic proline-rich amelogenins and the acidic serine-, glycine-, and aspartic-rich enamelins. These proteins have been postulated as playing a major role in the mineralization and structural organization of developing enamel. To identify and further characterize these different proteins and their possible role in this complex process of biological mineralization, we have in recent years been concerned with the production of specific probes for these proteins. Previously, we have reported on the successful production of specific polyclonal antibodies against enamelin proteins, which did not cross-react with amelogenins, and against amelogenin proteins, which did not cross-react with enamelins (Deutsch et al., 1986, 1987). We now report the production of monoclonal antibodies against a major bovine amelogenin protein (28 kDa) and against a major bovine enamelin protein (66 kDa). One monoclonal antibody against amelogenin and one against enamelin are described. The results showed that the monoclonal antibody against the amelogenin protein reacted strongly with the 28-kDa amelogenin protein band but did not cross-react with enamelins, and the one against the enamelin protein reacted with the 66-kDa enamelin protein but did not cross-react with amelogenins. These monoclonal antibodies provide a specific and powerful tool to distinguish between and further characterize these different classes of proteins, and to improve our understanding of the process of enamel formation.

Competitive Direct Enzyme-Linked Immunosorbent Screening Assay for the Detection of Sulfamethazine Contamination of Animal Feeds

1991 ◽  
Vol 74 (5) ◽  
pp. 784-789 ◽  
Author(s):  
Dixon E Holland Deborah ◽  
Stanley E Katz
Keyword(s):  
Monoclonal Antibody ◽  
Monoclonal Antibodies ◽  
Polyclonal Antibodies ◽  
Screening Method ◽  
Screening Assay ◽  
Animal Feeds ◽  
Feed Samples

Abstract A sensitive screening method has been developed for detecting sulfamethazine (SMZ) contamination of feeds by using either polyclonal or monoclonal antibodies and a direct competitive enzyme-linked immunosorbent screening assay (ELISA). Feed samples of 25.0 g are extracted with 0.5N HCI and centrifuged. The extract is adjusted to pH 7.0 with 3.0N NaOH and recentrifuged. This pH-adjusted extract is used in the EUSA. Levels as low as 0.004 μg SMZ/g feed were detected In supplemented extracts by polyclonal antibodies; levels of 0.4 μg SMZ/g feed were detected by a monoclonal antibody.

scholarly journals Systematic comparison of monoclonal versus polyclonal antibodies for mapping histone modifications by ChIP-seq

2016 ◽  
Vol 9 (1) ◽  
Author(s):  
Michele Busby ◽  
Catherine Xue ◽  
Catherine Li ◽  
Yossi Farjoun ◽  
Elizabeth Gienger ◽  
...  
Keyword(s):  
Histone Modifications ◽  
Polyclonal Antibodies ◽  
Systematic Comparison

scholarly journals A comparative study of polyclonal and monoclonal antibodies for immunocytochemical localization of cytosolic aspartate aminotransferase in rat liver.

1983 ◽  
Vol 31 (7) ◽  
pp. 920-926 ◽  
Author(s):  
C T Lin ◽  
L H Chen ◽  
T S Chan
Keyword(s):  
Monoclonal Antibody ◽  
Monoclonal Antibodies ◽  
Rat Liver ◽  
Aspartate Aminotransferase ◽  
Polyclonal Antibodies ◽  
Rabbit Antiserum ◽  
Igg Antibody ◽  
Experimental Conditions ◽  
Intense Staining ◽  
Rabbit Igg

The rabbit antiserum and mouse monoclonal hybridoma antibody against porcine cytosolic aspartate aminotransferase (c-AAT) (or cytosolic glutamic oxaloacetic transaminase (c-GOT)) were produced and compared for the localization of c-AAT in rat liver. An indirect immunocytochemical technique was performed using peroxidase-conjugated goat immunoglobulin (Ig) G anti-rabbit IgG and peroxidase-conjugated rabbit IgG anti-mouse IgG as the second antibody. Rats were perfused with paraformaldehyde-lysine-periodate fixative and the liver fragments were immersed in 4% paraformaldehyde and transferred to 10% dimethyl sulfoxide overnight and subjected to cryostat sectioning. The rabbit IgG antibody, 3 individual monoclonal antibodies, and a mixture of these 3 monoclonal antibodies were applied to the tissue sections, respectively, using the same concentration. Under the same experimental conditions, the c-AAT was localized in each individual hepatocyte by both monoclonal and polyclonal antibodies. However, a mixture of three monoclonal antibodies gave stronger staining than a single monoclonal antibody; although two antibodies yield more intense staining than just one, it was still less intense than for three. The conventional rabbit polyclonal antibody against c-AAT produced more reaction product than the combined three monoclonal antibodies. It is concluded that for immunocytochemical study, the use of a single monoclonal antibody is sensitive enough to localize its tissue antigen under the present experimental condition. To obtain a stronger reaction product, a combination of several monoclonal antibodies, at least three or more, may give better staining.

Monoclonal and polyclonal antibodies compared for radioimmunoassay of somatomedin-C in patients with acromegaly or hypopituitarism.

1987 ◽  
Vol 33 (9) ◽  
pp. 1593-1596 ◽  
Author(s):  
J M Burrin ◽  
J L Paterson ◽  
P S Sharp ◽  
T H Yeo
Keyword(s):  
Monoclonal Antibody ◽  
Monoclonal Antibodies ◽  
Polyclonal Antibody ◽  
Polyclonal Antibodies ◽  
Detection Limits ◽  
Healthy Adults ◽  
Somatomedin C ◽  
Lower Detection

Abstract We used a synthetic recombinant analog of somatomedin-C (Sm-C) to directly compare the performance of polyclonal and monoclonal antibodies for measuring Sm-C in serum of normal persons and in acromegalic or hypopituitary patients. Mean concentrations of Sm-C in healthy adults were 181 (SD 42) micrograms/L as measured with the monoclonal antibody, 194 (SD 61) micrograms/L with the polyclonal antibody. Both antisera gave excellent discrimination between acromegalics and normals. However, the assay with the polyclonal antibody was more sensitive than that with the monoclonal antibody (lower detection limits: 5 vs 100 micrograms/L) and thus better suited for quantifying Sm-C in samples from hypopituitary patients.

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