Genuine and apparent cross-reaction of polyclonal antibodies to proteins and peptides

Worm infections are found in livestock and can be transmitted to humans. Toxocara vitulorum is a worm species which commonly infected people. Cross-reaction among worms can generate false positive to establish helminthiasis diagnosis through antibody inspection. This study aimed to determine specific proteins that caused cross-reaction between Toxocara vitulorum antigen and anti-M. digitatus serum by using the western blot technique. In the present study, the whole worms extracted of T. vitulorum and M. digitatus have been used to make polyclonal antibodies from M. digitatus with Wistar rats as hosts. The cross-reaction between whole worm extract of T. vitulorum protein and anti-M. digitatus serum obtained 12 protein bands that each relative molecular mass (Mr) valued of 176, 124, 92, 68, 59, 47, 31, 29, 26, 16, 12, and 10 kDa. Cross-reaction occurred between T. vitulorum protein and anti-M. digitatus.

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Measurement of the free alpha subunit of human glycoprotein hormones by a monoclonal antibody-based immunoradiometric assay, and further exploration of antigenic sites on the choriogonadotropin molecule.

Clinical Chemistry ◽

10.1093/clinchem/33.7.1147 ◽

1987 ◽

Vol 33 (7) ◽

pp. 1147-1151 ◽

Cited By ~ 7

Author(s):

R J Norman ◽

R Haneef ◽

R H Buck ◽

S M Joubert

Keyword(s):

Solid Phase ◽

Polyclonal Antibodies ◽

Antigenic Site ◽

Reference Interval ◽

Cross Reaction ◽

Beta Subunit ◽

Alpha Subunit ◽

Immunoradiometric Assay ◽

Glycoprotein Hormones ◽

Antigenic Sites

Abstract Three monoclonal antibodies were raised against the free alpha subunit of choriogonadotropin (hCG); each recognized a different antigenic site on the molecule. One (antibody 42) preferentially bound to the alpha subunit when it was coupled to the beta subunit as dimeric choriogonadotropin (hCG), thyrotropin (TSH), lutropin (LH), or follitropin (FSH). Antibody 71 showed some cross-reaction with intact FSH; antibody 75 was more specific for the alpha subunit. All were of low affinity (10(-7) to 10(-8) mol/L), but when combined in immunoradiometric assays (IRMAS) they proved to be as sensitive as current radioimmunoassays involving polyclonal antibodies. Advantages of the combination of antibody 75 bound to the solid phase and antibody 71 as the radiolabeled antibody were: detection limit of at least 0.1 micrograms/L; linear dilution of serum and urine; insignificant cross-reaction with intact hCG, allowing direct assay in pregnancy fluids; and a coefficient of variation less than 3% over the reference interval for nonpregnant women. There was 4% cross-reaction with intact FSH, suggesting that the epitopes recognized by nos. 71 and 75 are more exposed in FSH and that perhaps there is less folding in this molecule than in intact hCG.

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Chapter 2 Production of Polyclonal Antibodies against Proteins and Peptides

Methods in Cell Biology - Methods in Cell Biology Volume 37 ◽

10.1016/s0091-679x(08)60242-3 ◽

1993 ◽

pp. 7-56 ◽

Cited By ~ 22

Author(s):

Detlev Drenckhahn ◽

Thomas Jöns ◽

Frank Schmitz

Keyword(s):

Polyclonal Antibodies ◽

Proteins And Peptides

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Immunohistochemical localization of the metalloproteinase meprin in salivary glands of male and female mice.

Journal of Histochemistry & Cytochemistry ◽

10.1177/39.1.1701182 ◽

1991 ◽

Vol 39 (1) ◽

pp. 123-129 ◽

Cited By ~ 18

Author(s):

S S Craig ◽

C Mader ◽

J S Bond

Keyword(s):

Salivary Glands ◽

Polyclonal Antibodies ◽

Inbred Strains ◽

Immunohistochemical Localization ◽

Mouse Strains ◽

Parotid Glands ◽

Inbred Strains Of Mice ◽

Membrane Bound ◽

Enzyme Functions ◽

Proteins And Peptides

Meprin is a membrane-bound metalloproteinase which is expressed at high levels in the brush border membrane of proximal tubules of kidneys of some mouse strains (referred to as high meprin-activity mice). The mature active proteinase is not present in kidneys of many inbred strains of mice; however, these low meprin-activity mice possess a kidney protein that crossreacts with polyclonal antibodies prepared against meprin. In the present studies, immunohistochemical methods were used to determine the presence of meprin in liver, pancreas, spleen, testis, thymus, kidney, salivary glands, stomach, duodenum, and skin. Meprin crossreactivity was observed only in kidney and salivary glands. In salivary glands, the enzyme was found on the luminal surface of intercalated and striated ducts of submandibular and parotid glands and on interlobular ducts of the latter. In both kidney and salivary glands, the intensity of immunochemical staining was greater in males compared with females. For both sexes, immunoreactivity was markedly greater in the high meprin-activity mice compared to the low meprin-activity mice. These studies indicate that meprin has a limited tissue distribution, and that genetic and hormonal factors that regulate the proteinase are similar in kidney and salivary glands. The localization of the proteinase implies that the enzyme functions in modifying proteins and peptides that are secreted or re-absorbed in the ducts of these tissues.

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TdT expression in germ cell tumours: a possible immunohistochemical cross-reaction and diagnostic pitfall

Journal of Clinical Pathology ◽

10.1136/jclinpath-2019-205713 ◽

2019 ◽

Vol 72 (8) ◽

pp. 536-541 ◽

Cited By ~ 1

Author(s):

Marta Jaconi ◽

Fulvio Magni ◽

Francesca Raimondo ◽

Maurilio Ponzoni ◽

Clizia Chinello ◽

...

Keyword(s):

Germ Cell ◽

Polyclonal Antibodies ◽

Mature Teratoma ◽

Staining Intensity ◽

Cross Reaction ◽

Terminal Deoxynucleotidyl Transferase ◽

Routine Practice ◽

Multicentric Study ◽

Clear Cut ◽

Significant Difference

AimsVery recent papers proposed a possible role for the expression of terminal deoxynucleotidyl transferase (TdT) in the tumourigenesis of gonadal and extragonadal germ cell-derived tumours (GCTs). Our multicentric study evaluated the magnitude of the immunoreactivity for TdT in GCTs, encompassing seminoma, dysgerminoma, mature teratoma and mixed GCTs.Methods and resultsThe histological series was stained with both monoclonal and polyclonal antibodies, yielding a positivity of 80% of cases with well-defined nuclear reactivity. A significant difference in staining intensity between monoclonal and polyclonal antibodies was observed (p=0.005). However, exploiting western blot and more innovative proteomic approaches, no clear-cut evidence of the TdT protein was observed in the neoplastic tissues of the series.ConclusionsAlternatively to the pathogenetic link between TdT expression and GCTs tumourigenesis, we hypothesised the occurrence of a spurious immunohistochemical nuclear cross-reaction, a well-known phenomenon with important implications and a possible source of diagnostic pitfalls in routine practice for pathologists.

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Production of anti-Cryptosporidium polyclonal antibodies and standardization of direct immunofluorescence for detecting oocysts in water

Revista da Sociedade Brasileira de Medicina Tropical ◽

10.1590/s0037-86822011000500011 ◽

2011 ◽

Vol 44 (5) ◽

pp. 587-590 ◽

Cited By ~ 2

Author(s):

Silvia Cristina Osaki ◽

Adriana Oliveira Costa ◽

Ludmilla Della Coletta Troiano ◽

Ernesto Renato Kruger ◽

Juliana Tracz Pereira ◽

...

Keyword(s):

Immunoglobulin G ◽

Water Samples ◽

Polyclonal Antibodies ◽

Giardia Duodenalis ◽

Fluorescein Isothiocyanate ◽

Immunofluorescence Assay ◽

Cross Reaction ◽

Direct Immunofluorescence ◽

Cryptosporidium Oocysts ◽

Direct Immunofluorescence Assay

INTRODUCTION: The production of anti-Cryptosporidium polyclonal antibodies and its use in direct immunofluorescence assays to determine the presence of Cryptosporidium in water are described in the present work. METHODS: Two rabbits were immunized with soluble and particulate antigens from purified Cryptosporidium oocysts. The sera produced were prepared for immunoglobulin G extraction, which were then purified and conjugated with fluorescein isothiocyanate (FITC). Slides containing known amounts of oocysts were prepared to determine the sensitivity of the technique. To test the specificity, slides containing Giardia duodenalis cysts were prepared. RESULTS: The conjugate was successfully used in water samples experimentally contaminated with Cryptosporidium oocysts, and it was possible to detect up to five oocysts/spot, corresponding to contamination of 250 oocysts/mL. CONCLUSIONS: The three immunizations performed in the rabbits were enough to produce antibodies against Cryptosporidium, the standard direct immunofluorescence assay permitted the detection of five oocysts in 20% of the samples, and no cross-reaction with Giardia duodenalis cysts occurred.

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Sensitive time-resolved fluoroimmunoassay of salmon calcitonin

Clinical Chemistry ◽

10.1093/clinchem/40.9.1774 ◽

1994 ◽

Vol 40 (9) ◽

pp. 1774-1777 ◽

Cited By ~ 6

Author(s):

H Rong ◽

O Tørring ◽

M Sääf ◽

U Sjöstedt ◽

H E Sjöberg ◽

...

Keyword(s):

Detection Limit ◽

Salmon Calcitonin ◽

Polyclonal Antibodies ◽

Affinity Purification ◽

Cross Reaction ◽

Human Calcitonin ◽

Sensitive Assay ◽

Time Resolved ◽

Analytical Recovery

Abstract A two-site assay was developed by use of the "dissociation and enhancement lanthanide fluoroimmunoassay" (DELFIA) technique for determination of salmon calcitonin (SCT) in serum after administration to osteoporotic patients. Polyclonal antibodies were produced in rabbits immunized with SCT coupled to ovalbumin. After affinity purification, the antibodies were used both as immobilized capture antibodies and as Eu-chelate-labeled signal antibodies. A sensitive assay with a detection limit of 1.1 pmol/L was achieved, and no cross-reaction with human calcitonin was observed. The intra- and interassay CVs were < 12% (n = 10) and < 15% (n = 4), respectively. Analytical recovery of SCT added to serum was 91% +/- 3% (mean +/- SD, n = 4). SCT was measurable in all the samples from eight osteoporotic patients after subcutaneous SCT administration. We conclude that this new sensitive and specific two-site DELFIA can reliably measure SCT in serum.

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Immunocytochemical methodology for the localization of neuronal antigens at the ultrastructural level

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100161941 ◽

1990 ◽

Vol 48 (3) ◽

pp. 876-877

Author(s):

Jorge Pecci Saavedra ◽

Mark Connaughton ◽

Juan José López ◽

Alicia Brusco

Keyword(s):

Spinal Cord ◽

Substantia Nigra ◽

Polyclonal Antibodies ◽

Ultrastructural Level ◽

Point Of View ◽

Nerve Tissue ◽

Tissue Fixation ◽

Optimal Conditions ◽

Interpeduncular Nucleus ◽

Technical Point

The use of antibodies as labels for the localization of specific molecules in the nervous systan has been extensively applied in recent years. Both monoand polyclonal antibodies or antisera have been employed. The knowledge of the organization of neuronal connectivities, gliovascular relationships, glioneuronal relationships and other features of nerve tissue has greatly increased.A number of areas of the nervous systan have been analyzed in our laboratory, including the nuclei of the raphe system, the reticular formation, interpeduncular nucleus, substantia nigra, caudate nucleus, putamen, pallidum, spinal cord, pineal gland and others.From a technical point of view, a number of variables needed to be taken into account in order to obtain reliable and reproducible results. The design of the optimal conditions of tissue fixation, embedding, sectioning, dilution of antibodies, and adaptation of Sternberger PAP technique were sane of the parameters taken into account to optimize the results. It is critical that each step of the technique be defined for each particular case.

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Sequencing of Proteins and Peptides

10.1016/s0075-7535(08)x7013-5 ◽

1981 ◽

Keyword(s):

Proteins And Peptides

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Immunological Relationship Between Plasminogen Activator Inhibitors from Different Sources

Thrombosis and Haemostasis ◽

10.1055/s-0038-1651056 ◽

1987 ◽

Vol 57 (01) ◽

pp. 029-034 ◽

Cited By ~ 18

Author(s):

Göran Urdén ◽

Joanna Chmielewska ◽

Tomas Carlsson ◽

Björn Wiman

Keyword(s):

Plasminogen Activator ◽

Polyclonal Antibodies ◽

Active Form ◽

Polyclonal Antiserum ◽

The Other ◽

Hep G2 ◽

Inhibitor Activity ◽

Tissue Plasminogen ◽

Immunological Relationship ◽

Different Sources

SummaryPolyclonal antibodies have been raised against the inhibitor moiety in the purified complex between tissue plasminogen activator and its fast inhibitor (PA-inhibitor) in human plasma/ serum. A radioimmunoassay for quantitation of PA-inhibitor antigen was developed. The polyclonal antiserum and a previously described monoclonal antibody against the PA-inhibitor (14) have been used to study the immunological relationship between PA-inhibitors from plasma, serum, platelets, placenta extract and conditioned media from Hep G2 and HT 1080 cells. It was demonstrated that the ratio between PA-inhibitor activity and antigen varied considerably between the different sources. In the plasma samples studied, similar activity and antigen concentrations were found, suggesting that the PA-inhibitor in these samples mainly was in an active form. On the other hand the other sources seemed to contain variable amounts of inactive PA-inhibitor forms. Immunoadsorption experiments revealed that the PA-inhibitor (activity and antigen) from all the sources were specifically bound to the insolubilized antibodies (polyclonal and monoclonal). In no case, however, could active PA-inhibitor be eluted from the immunoadsorption columns. Also the competitive radioimmunoassays suggested that the PA-inhibitors from the different sources studied, were closely immunologically related.

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