scholarly journals Developing Monoclonal Antibodies for Immunohistochemistry

Cells ◽  
2022 ◽  
Vol 11 (2) ◽  
pp. 243
Author(s):  
Jacqueline Cordell
Keyword(s):  
Alkaline Phosphatase ◽  
Monoclonal Antibodies ◽  
Recombinant Proteins ◽  
Synthetic Peptides ◽  
Polyclonal Antibodies ◽  
Fixed Tissue ◽  
Direct Production ◽  
Endogenous Peroxidase ◽  
Key Steps ◽  
And Training

The experiences of a laboratory which pioneered the application of monoclonal antibodies to diagnostic histochemistry is described. This was achieved in four key steps: (1) Monoclonal antibodies were successfully produced to replace the difficult-to-produce and limited polyclonal antibodies available for immunohistochemistry. (2) Monoclonal antibodies were produced to improve the immunoenzymatic detection of bound antibodies, using immunoperoxidase or alkaline phosphatase, increasing sensitivity and allowing the use of two chromogens when applied together. The availability of a reliable alkaline phosphatase-based detection allowed the detection of antigens in tissues with high endogenous peroxidase. (3) Methodologies were developed to unmask antigens not detected in routinely processed paraffin-embedded tissue. (4) Synthetic peptides were used as immunising antigens for the direct production of specific molecules of diagnostic interest. This was expanded to include recombinant proteins. Many reacted with fixed tissue and recognised homologous molecules in other species. In addition to these developments, the laboratory promoted the collaboration and training of researchers to spread the expertise of monoclonal production for diagnosis.

scholarly journals Monoclonal antibodies to different epitopes on a cell-surface enzyme, human placental alkaline phosphatase, effect different patterns of labeling with protein A-colloidal gold.

1987 ◽  
Vol 35 (11) ◽  
pp. 1277-1284 ◽  
Author(s):  
R Jemmerson ◽  
M Agre
Keyword(s):  
Alkaline Phosphatase ◽  
Monoclonal Antibodies ◽  
Cell Surface ◽  
Colloidal Gold ◽  
Polyclonal Antibodies ◽  
Protein A ◽  
Placental Alkaline Phosphatase ◽  
Gold Particles ◽  
Human Placental Alkaline Phosphatase ◽  
Membrane Components

Two monoclonal antibodies (mAbs) to different epitopes on human placental alkaline phosphatase (PLAP), both of the immunoglobulin G2a heavy-chain class and having similar affinities for PLAP, were compared for their ability to label the enzyme on the HeLa cell surface. In one type of experiment employing [125I]-labeled mAbs, the results demonstrated quantitative differences in binding of the mAbs to the cells. At saturating levels, the number of molecules of mAb E5 bound to the cells was almost eight times the number of mAb B10 molecules bound. In another type of experiment, mAbs were indirectly visualized on the cell surface using protein A tagged with colloidal gold particles in transmission electron microscopy. Only one of the antibodies (E5) displayed a clustered distribution of PLAP that previously had been observed with rabbit polyclonal antibodies and goat anti-rabbit IgG-labeled gold (J Histochem Cytochem 33:1227, 1985). The other antibody (B10) showed less frequent and more scattered labeling; three to four times more gold particles were visualized in each cluster on cells bound by mAb E5 compared to cells bound by B10. These results are consistent with the idea that not all epitopes on a membrane-bound antigen may be equally accessible for antibody binding. Even identical epitopes on different PLAP molecules are not equally hindered by other membrane components, since at least some of the PLAP molecules are labeled by the more sterically hindered mAb B10. Quantification of the number of gold particles employing the more abundantly bound mAb E5 provides an average estimate of seven to eight molecules of PLAP in each cluster. Because of inefficiencies in labeling, however, this value is probably lower than the real number.

RECOMBINANT AND PLASMA FVIII SHARE EPITOPES RECOGNIZED BY A PANEL OF MONOCLONAL AND POLYCLONAL ANTIBODIES

1987 ◽  
Author(s):  
N Pancham ◽  
M Dumas ◽  
M S Madant ◽  
P C Esmon ◽  
V Voehrinqer
Keyword(s):  
Monoclonal Antibodies ◽  
Human Plasma ◽  
Western Blotting ◽  
Synthetic Peptides ◽  
Polyclonal Antibodies ◽  
Functional Characteristics ◽  
Activity Inhibition ◽  
One Stage ◽  
Recombinant Fviii

A panel of neutralizing and nonneutralizing monoclonal and polyclonal antibodies raised against human plasma FVIII and human recombinant FVIII was employed in Western blotting, activity inhibition and two-site ELISA studies to investigate structural similarities between recombinant FVIII (r-FVIII) and plasma FVIII (p-FVIII). The monoclonal antibodies were obtained either commercially or produced in-house and react with either the 90kd, 80kd or B-region polypeptides. Some antibodies were raised to FVIII synthetic peptides. Rabbit polyclonal antibodies were obtained from either r-FVIII or p-FVIII immunizations. Recombinant FVIII and p-FVIII were compared either as intact molecules or following thrombin digestion by immunoblots after SDS gradient PAGE. Combinations of antibodies were used in two-site ELISA. Activity inhibition was evaluated by one-stage clotting or in FXa generation assays. Recombinant FVIII and p-FVIII responded similarly to these antibodies in all of the immunotechniques used. Collectively, these results indicate that using this approach r-FVIII and p-FVIII display similar structural and functional characteristics.

An Immunoradiometric Assay for Factor VIII Related Antigen (VIIIRAg) Using Two Monoclonal Antibodies-Comparison with Polyclonal Rabbit Antibodies for Use in von Willebrand’s Disease Diagnosis

1984 ◽  
Vol 52 (03) ◽  
pp. 250-252 ◽  
Author(s):  
Y Sultan ◽  
Ph Avner ◽  
P Maisonneuve ◽  
D Arnaud ◽  
Ch Jeanneau
Keyword(s):  
Monoclonal Antibodies ◽  
Structural Changes ◽  
Polyclonal Antibodies ◽  
Disease Diagnosis ◽  
Immunoradiometric Assay ◽  
Von Willebrand's Disease ◽  
Von Willebrand’S Disease ◽  
Structural Abnormalities ◽  
Antigenic Material ◽  
Von Willebrand

SummaryTwo monoclonal antibodies raised against FVIII/von Willebrand protein were used in an immunoradiometric assay (IRMA) to measure this antigen in normal plasma and plasma of patients with different forms of von Willebrand’s disease. The first antibody, an IgG1 was used to coat polystyrene tubes, the second one, an IgG2a, iodinated and used in the second step. Both antibodies inhibit ristocetin induced platelet agglutination and react strongly with platelets, megacaryocytes and endothelial cells. The IRMA test using these antibodies showed greater sensitivity than that using rabbit polyclonal anti VIIIRAg antibodies. A good correlation between the two tests was nevertheless found when VIIIRAg was measured in the majority of patient’s plasma. However 5 patients from 3 different families showed more antigenic material in the rabbit antibody IRMA than in the monoclonal antibody IRMA. It is suggested therefore that the monoclonal antibodies identify part of the VIIIR:Ag molecule showing structural abnormalities in these vWd patients, these structural changes remaining undetected by the polyclonal antibodies.

Combinations of nonlabeled, 125I-Labeled, and Anti-Idiotypic Antiplacental Alkaline Phosphatase Monoclonal Antibodies at Experimental Radioimmunotargeting

1997 ◽  
Vol 38 (6) ◽  
pp. 1087-1093 ◽  
Author(s):  
R. Rossi Norrlund ◽  
D. Holback ◽  
L. Johansson ◽  
S. -O. Hietala ◽  
K. Riklund Åhlstrom
Keyword(s):  
Alkaline Phosphatase ◽  
Monoclonal Antibodies

Use of monoclonal and polyclonal antibodies to detect prostatic acid phosphatase by immunoblotting.

1986 ◽  
Vol 32 (10) ◽  
pp. 1832-1835 ◽  
Author(s):  
P C Patel ◽  
L Aubin ◽  
J Côte
Keyword(s):  
Monoclonal Antibodies ◽  
Acid Phosphatase ◽  
Western Blot ◽  
Polyclonal Antibodies ◽  
Prostatic Acid Phosphatase ◽  
The Other ◽  
Dot Blot ◽  
Western Blots ◽  
Dot Blot Assay ◽  
Polyclonal Antisera

Abstract We investigated two techniques of immunoblotting--the Western blot and the dot blot--for use in detecting prostatic acid phosphatase (PAP, EC 3.1.3.2). We used polyclonal antisera to human PAP, produced in rabbits by hyperimmunization with purified PAP, and PAP-specific monoclonal antibodies in the immunoenzymatic protocols. We conclude that PAP can be readily detected by Western blots with use of polyclonal antisera, but not with monoclonal antibodies. On the other hand, using a dot blot assay, we could easily detect PAP with both polyclonal and monoclonal antibodies.

scholarly journals A molecularly defined skin test reagent for the diagnosis of bovine tuberculosis compatible with vaccination against Johne’s Disease

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Sonya Middleton ◽  
Sabine Steinbach ◽  
Michael Coad ◽  
Kevina McGill ◽  
Colm Brady ◽  
...  
Keyword(s):  
Skin Test ◽  
Recombinant Proteins ◽  
Bovine Tuberculosis ◽  
Synthetic Peptides ◽  
Blood Test ◽  
High Specificity ◽  
Active Components ◽  
Interferon Gamma Release Assays ◽  
Skin Responses

AbstractTuberculin Purified Protein Derivatives (PPDs) exhibit multiple limitations: they are crude extracts from mycobacterial cultures with largely unknown active components; their production depends on culture of mycobacteria requiring expensive BCL3 production facilities; and their potency depends on the technically demanding guinea pig assay. To overcome these limitations, we developed a molecularly defined tuberculin (MDT) by adding further antigens to our prototype reagent composed of ESAT-6, CFP-10 and Rv3615c (DIVA skin test, DST). In vitro screening using PBMC from infected and uninfected cattle shortlisted four antigens from a literature-based list of 18 to formulate the MDT. These four antigens plus the previously identified Rv3020c protein, produced as recombinant proteins or overlapping synthetic peptides, were formulated together with the three DST antigens into the MDT to test cattle experimentally and naturally infected with M. bovis, uninfected cattle and MAP vaccinated calves. We demonstrated significant increases in MDT-induced skin responses compared to DST in infected animals, whilst maintaining high specificity in unvaccinated or MAP vaccinated calves. Further, MDT can also be applied in in vitro blood-based interferon-gamma release assays. Thus, MDT promises to be a robust diagnostic skin and blood test reagent overcoming some of the limitations of PPDs and warrants full validation.

scholarly journals Development and characterization of a novel mAb against bilitranslocase - a new biomarker of renal carcinoma

2013 ◽  
Vol 47 (2) ◽  
pp. 128-137 ◽  
Author(s):  
Sendi Montanic ◽  
Michela Terdoslavich ◽  
Uros Rajcevic ◽  
Luigina De Leo ◽  
Serena Department of Medical Sciences, Uni ◽  
...  
Keyword(s):  
Renal Cell Carcinoma ◽  
Monoclonal Antibodies ◽  
Cell Carcinoma ◽  
Renal Cell ◽  
Renal Carcinoma ◽  
Vascular Endothelial Cells ◽  
Polyclonal Antibodies ◽  
Functional Characterization ◽  
Immunological Approach

Background. Bilitranslocase (TC 2.A.65.1.1) is a bilirubin-specific membrane transporter, found on absorptive (stomach and intestine) and excretory (kidney and liver) epithelia and in vascular endothelium. Polyclonal antibodies have been raised in rabbits in the past, using a synthetic peptide corresponding to AA65-77 of rat liver bilitranslocase, as an antigen. Affinity-purified antibodies from immune sera have been found to inhibit various membrane transport functions, including the bilirubin uptake into human hepatocytes and the uptake of some flavonoids into human vascular endothelial cells. It was described by means of immunohistochemistry using polyclonal antibodies that bilitranslocase expression is severely down-regulated in clear cell renal carcinoma. The aim of our work was development and characterization of high-affinity, specific mAbs against bilitranslocase, which can be used as a potential diagnostic tool in renal cell carcinoma as well as in a wide variety of biological assays on different human tissues. Materials and methods. Mice were immunized with a multi-antigen peptide corresponding to segment 65-75 of predicted primary structure of the bilitranslocase protein. By a sequence of cloning, immune- and functional tests, we aimed at obtaining a specific monoclonal antibody which recognizes a 37 kDa membrane protein, and influences the transport activity of bilitranslocase. Results. On the basis of previous results, specific IgM monoclonal antibodies were produced in BALB/c mice, in order to further improve and extend the immunological approach to the study of bilitranslocase in renal cancer cells as well as to develop its potential diagnostics use. Conclusions. In this article we show an immunological approach, based on newly developed monoclonal antibodies, to a detailed biochemical and functional characterization of a protein whose gene and protein structure is still unknown. We were able to demonstrate our novel mAb as a tumor marker candidate of renal cell carcinoma, which may prove useful in the diagnostic procedures.

scholarly journals Prediction of Clearance of Monoclonal and Polyclonal Antibodies and Non-Antibody Proteins in Children: Application of Allometric Scaling

Antibodies ◽  
2020 ◽  
Vol 9 (3) ◽  
pp. 40
Author(s):  
Iftekhar Mahmood
Keyword(s):  
Clinical Trials ◽  
Monoclonal Antibodies ◽  
Prediction Error ◽  
Allometric Scaling ◽  
Polyclonal Antibodies ◽  
Therapeutic Proteins ◽  
Preterm Neonates ◽  
Pharmacokinetic Parameters ◽  
Pediatric Dose ◽  
Age Dependent

Allometric scaling can be used for the extrapolation of pharmacokinetic parameters from adults to children. The objective of this study was to predict clearance of therapeutic proteins (monoclonal and polyclonal antibodies and non-antibody proteins) allometrically in preterm neonates to adolescents. There were 13 monoclonal antibodies, seven polyclonal antibodies, and nine therapeutic proteins (non-antibodies) in the study. The clearance of therapeutic proteins was predicted using the age dependent exponents (ADE) model and then compared with the observed clearance values. There were in total 29 therapeutic proteins in this study with 75 observations. The number of observations with ≤30%, ≤50%, and >50% prediction error was 60 (80%), 72 (96%), and 3 (4%), respectively. Overall, the predicted clearance values of therapeutic proteins in children was good. The allometric method proposed in this manuscript can be used to select first-in-pediatric dose of therapeutic proteins in pediatric clinical trials.

scholarly journals Determination of platelet antigens and glycoproteins in the human fetus

Blood ◽  
1986 ◽  
Vol 68 (2) ◽  
pp. 488-492 ◽  
Author(s):  
Y Gruel ◽  
B Boizard ◽  
F Daffos ◽  
F Forestier ◽  
J Caen ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Human Fetus ◽  
Antenatal Diagnosis ◽  
Polyclonal Antibodies ◽  
Umbilical Vein ◽  
Membrane Surface ◽  
Platelet Suspension ◽  
Direct Puncture ◽  
Normal Human

Abstract The autosomal recessive transmission of Glanzmann's thrombasthenia (GT) and Bernard-Soulier syndrome (BSS), together with requests of families who already had children with these diseases, prompted us to investigate the feasibility of their antenatal diagnosis. The preliminary step leading to the early detection of GT or BSS was to characterize, in the normal human fetus, the platelet antigens and glycoproteins (GPs) and to define their normal amounts on the membrane surface. Blood samples from 32 fetuses between 18 to 26 weeks of gestation were collected by direct puncture of the umbilical vein using an ultrasound-guided needle. Polyclonal antibodies from human origin directed against PLA1, Leka antigens, and the GPIIb IIIa complex (IgGL), or murine monoclonal antibodies specific for GPIb (AN51, 6D1), GPIIIa (AP-3), or GPIIb IIIa (AP-2) were studied using platelet suspension immunofluorescence tests. The binding of each antibody was quantified using a cytofluorograph (Ortho 50H). PLA1 and Leka antigens were expressed in normal amounts on fetal platelets as early as 16 weeks of intrauterine life. The GPIIb IIIa complex quantified by polyclonal or monoclonal antibodies was in the same range in fetuses (IgGL = 427 +/- 23 AUF, AP-2 = 459.5 +/- 8.5; AP-3 = 536 +/- 14) and in adults (IgGL = 420 +/- 30; AP-2 = 498 +/- 11; AP-3 = 515 +/- 13). The platelet binding of antibodies that recognized GPIb was higher in fetuses (AN51 = 491.5 +/- 14; 6D1 = 479 +/- 15) than in adults (AN51 = 426.5 +/- 9; 6D1 = 449 +/- 8.7). These results suggest that immunological techniques can be applied as early as 18 weeks of gestation for the antenatal diagnosis of GT and BSS.

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