scholarly journals Production and characterization of specific antibodies for evaluation of glycated insulin in plasma and biological tissues

2000 ◽  
Vol 167 (1) ◽  
pp. 153-163 ◽  
Author(s):  
AM McKillop ◽  
JT McCluskey ◽  
AC Boyd ◽  
MH Mooney ◽  
PR Flatt ◽  
...  
Keyword(s):  
Guinea Pigs ◽  
Pancreatic Beta Cells ◽  
Biological Tissues ◽  
Cross Reactivity ◽  
Diabetic Rats ◽  
High Titre ◽  
Keyhole Limpet Haemocyanin ◽  
Specific Antibodies ◽  
Titration Curves ◽  
Insulin In Plasma

Previous studies have shown that glycation of insulin occurs in pancreatic beta-cells under conditions of hyperglycaemia and that the site of glycation is the N-terminal Phe(1) of the insulin B-chain. To enable evaluation of glycated insulin in diabetes, specific antibodies were raised in rabbits and guinea-pigs by using two synthetic peptides (A: Phe-Val-Asn-Gln-His-Leu-Cys-Tyr, and B: Phe-Val-Asn-Gln-His-Leu-Tyr-Lys) modified by N-terminal glycation and corresponding closely to the N-terminal sequence of the glycated human insulin B-chain. For immunization, the glycated peptides were conjugated either to keyhole limpet haemocyanin or ovalbumin using glutaraldehyde, m-maleimidobenzoyl-N-hydroxysuccinimide ester or 1-ethyl-3-(3-dimethylamino propyl) carbodiimide hydrochloride. Antibody titration curves, obtained using I(125)-tyrosylated tracer prepared from glycated peptide A, revealed high-titre antisera in five groups of animals immunized for 8-28 weeks. The highest titres were observed in rabbits and guinea-pigs immunized with peptide B coupled to ovalbumin using glutaraldehyde. Under radioimmunoassay conditions, these antisera exhibited effective dose (median) (ED(50)) values for glycated insulin of 0.3-15 ng/ml and 0.9-2.5 ng/ml respectively, with negligible cross-reactivity against insulin or other islet peptides. The degree of cross-reaction with glycated proinsulin was approximately 50%. Glycated insulin in plasma of control and hydrocortisone-treated diabetic rats measured using rabbit 3 antiserum (1:10 000 dilution; sensitivity <19 pg/ml) was 0. 08+/-0.01 and 1.5+/-0.6 ng/ml (P<0.01), corresponding to 4 and 16% of total circulating insulin concentration respectively. Immunocytochemistry studies of the pancreas of streptozotocin-treated diabetic rats using a 1:1000 dilution of guinea-pig 2 antiserum revealed clusters of fluorescent positively stained cells in islets. These studies document the successful production of polyclonal antisera specific for glycated insulin and their usefulness in radioimmunoassays and immunocytochemistry. The demonstration of glycated insulin in plasma and islets of animal models of diabetes supports the view that glycation of insulin is involved in the pathogenesis of this disease.

scholarly journals Direct Determination of Theophylline in Serum by Fluoroimmunoassay Using Highly Specific Antibodies

1985 ◽  
Vol 22 (5) ◽  
pp. 519-525 ◽  
Author(s):  
A J Hodgkinson ◽  
A M Sidki ◽  
J Landon ◽  
F J Rowell ◽  
S M Mahmod
Keyword(s):  
Serum Sample ◽  
Solid Phase ◽  
Direct Determination ◽  
Cross Reactivity ◽  
High Titre ◽  
Specific Antibodies ◽  
Endogenous Fluorophores ◽  
Separation Step ◽  
Related Compounds

Described is the development of a fluoroimmunoassay for theophylline using a fluorescein labelled derivative of theophylline as tracer and antibodies coupled to magnetisable solid-phase particles. Three approaches are described for the preparation of antibodies for theophylline, of which one produced highly specific, high titre antibodies. The fluoroimmunoassay using these antibodies required a 10 μL sample, reached equilibrium within 5 min, and the results correlated closely with those of an established enzymoimmunoassay method. Potentially interfering endogenous fluorophores from the serum sample were reliably removed at the separation step of the bound and free fractions. There was no significant cross-reactivity with all other structurally related compounds.

RADIOIMMUNOASSAY FOR 2-METHOXYOESTRONE IN HUMAN PLASMA

1979 ◽  
Vol 91 (1) ◽  
pp. 158-166 ◽  
Author(s):  
Günter Emons ◽  
Peter Ball ◽  
Gertrud v. Postel ◽  
Rudolf Knuppen
Keyword(s):  
Human Plasma ◽  
Plasma Concentrations ◽  
Cross Reactivity ◽  
Elderly Men ◽  
Specific Antibodies ◽  
Assay Procedure ◽  
Menopausal Women ◽  
Bovine Serum Albumin Conjugate ◽  
Post Menopausal

ABSTRACT A bovine serum albumin conjugate of 2-methoxyoestrone was used for the preparation of highly specific antibodies in rabbits. Cross-reactivity for catecholoestrogens and monophenolic steroids was below 0.3 %. Only 2-methoxyoestradiol cross-reacted with 44 %. An assay procedure for the determination of unconjugated and conjugated 2-methoxyoestrone in human plasma is described. The following mean plasma concentrations (pg/ml) were found (unconjugated/conjugated): children 61/1130, young men 74/1320, elderly men 109/1260, cycling women 131/1040, post-menopausal women 102/1420, and pregnant women 3980/5850.

Reactivity and Specificity of Trichinella spiralis Fractions in Cutaneous and Serological Tests

1977 ◽  
Vol 6 (3) ◽  
pp. 274-279
Author(s):  
Omar O. Barriga
Keyword(s):  
Guinea Pigs ◽  
Trichinella Spiralis ◽  
Chronic Phase ◽  
Epidemiological Studies ◽  
Cross Reactivity ◽  
Serological Tests ◽  
Specific Diagnosis ◽  
Diethylaminoethyl Cellulose ◽  
Cutaneous Reactions ◽  
Healthy Persons

Six diethylaminoethyl-cellulose fractions of a larval Trichinella spiralis extract, an Ascaris suum extract, and a nonrelated protein were used for cutaneous tests in guinea pigs with 8-, 14-, and 73-day-old T. spiralis infections, in guinea pigs with 13-day-old A. suum infections, and in normal guinea pigs. A selected T. spiralis fraction was used in hemagglutination (HA) tests with sera of 8 T. spiralis -infected rabbits, 41 sera of trichinellosis patients positive by bentonite agglutination tests, and 50 sera of clinically healthy persons. Immediate-type cutaneous reactions revealed extensive cross-reactivity between both parasites, although the establishment of conventional limits for considering a reaction positive allowed the specific diagnosis of acute or chronic trichinellosis with different fractions. Delayed-type reactions were specific with all fractions except one, and different fractions reacted during either the acute or the chronic phase of trichinellosis. HA detected anti- Trichinella antibodies in all the rabbits 9 to 10 days postinfection, in all trichinellosis patients, and in none of the healthy people. Correlation between HA and bentonite agglutination titers and other considerations suggest that HA with the selected fraction detects early antibodies. HA inhibition tests with A. suum extract suggest lack of HA cross-reactivity between the A. suum - and T. spiralis -selected fractions. The use of different fractions in diverse tests for clinical or epidemiological studies is suggested.

Fluorescent antibody techniques have allowed for the direct identification and enumeration of individual bacteria in environmental samples without requiring prior growth in culture media (Bahlool and Schmidt 1980, Cloete and Steyn 1988, Macario et al. 1989). The technique involves the use of specific antibodies raised against surface markers of defined pure cultures that are either labelled directly with fluorescent dye molecules or via a fluorescent secondary antibody. This approach has yielded important insights into the spatial distribution of microorganisms, but it suffers from a number of disadvantages. For example, expression of the antigen may be influenced by environmental factors; false-positive and false-negative results may be obtained due to cross-reactivity or lack of reaction; non-specific binding of antibodies may result in high levels of background fluorescence; and production of specific antibodies requires a pure culture of the organism of interest (Cloete and de Bruyn Various recombinant DNA techniques have subsequently been developed that are independent of cultivation methods (Fig. 1). These techniques provide ways of detecting and quantifying specific phylogenetic groups of microbes on 16S rDNA sequences, and relevant structural genes provide ways of monitoring microbial populations of environmental and industrial systems. In addition to these tools, a number of emerging technologies such as the use of biomarker genes are being increasingly used to monitor with great precision and accuracy the behaviour of microbes in the environment.

2003 ◽  
pp. 218-219
Keyword(s):  
Recombinant Dna ◽  
Culture Media ◽  
Specific Binding ◽  
Fluorescent Antibody ◽  
False Negative ◽  
Cross Reactivity ◽  
Phylogenetic Groups ◽  
Specific Antibodies ◽  
Negative Results ◽  
Recombinant Dna Techniques

scholarly journals Physical activity as a non-pharmacological and useful strategy in controlling of the apoptotic symptoms in diabetic model rats

2021 ◽  
Vol 16 (8) ◽  
pp. 602-606
Author(s):  
M. Bostani ◽  
S.A. Noaein
Keyword(s):  
Aerobic Exercise ◽  
Pancreatic Beta Cells ◽  
Diabetic Control ◽  
Pancreatic Tissue ◽  
Diabetic Rats ◽  
Aerobic Exercise Training ◽  
Bax Protein ◽  
Male Wistar Rats ◽  
Healthy Control ◽  
Induced Apoptosis

Background. In recent years, diabetes has become a global health problem. Apoptosis of pancreatic beta cells plays an important role in the pathogenesis of type 1 diabetes. Exercise as a non-pharmacological strategy to reduce the diabetic-induced complications has always been of interest to researchers. Therefore, the purpose of this study was to investigate the effect of aerobic exercise on levels of Bax, Bcl-2 and Bax/Bcl-2 ratio in pan­creatic tissue of streptozotocin (STZ)-induced diabetic rats. Materials and methods. A total number of 40 male Wistar rats (10 weeks old, 200–250 gr weight) were randomly divided into healthy control (HC), healthy trained (HT), diabetic control (DC), and diabetic trained (DT) groups. Diabetes was also induced by a single intraperitoneally injection of streptozocin (45 mg/kg). The training groups performed the exercise on the treadmill for five consecutive days within six weeks. The pancreatic tissue levels of the Bax and the Bcl-2 proteins were further determined via ELISA method. Results. The results showed that the induction of diabetes had significantly decreased the levels of Bcl-2 protein and increased the levels of Bax protein and Bax/Bcl-2 ratio in the pancreatic tissue (p < 0.05). As well, the findings showed that six weeks of aerobic exercise training had significantly increased the levels of Bcl-2 and significantly decreased the levels of Bax protein in DT group. Also, the Bax/Bcl-2 ratio reduced significantly in DT group (p < 0.05). The increase in displacement and transmission of apoptosis inducing factor (AIF) that have seen in oxidative stress status, is reduced in the tissues of trained individuals which indica­ting of the inhibition in the apoptotic signaling. Conclusions. According to the results of this study, exercise can be considered as an effective strategy to reduce the rate of diabetic-induced apoptosis and control its complications.

scholarly journals Desmosomal glycoproteins 2 and 3 (desmocollins) show N-terminal similarity to calcium-dependent cell-cell adhesion molecules

1990 ◽  
Vol 97 (2) ◽  
pp. 239-246
Author(s):  
J.L. Holton ◽  
T.P. Kenny ◽  
P.K. Legan ◽  
J.E. Collins ◽  
J.N. Keen ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Cell Adhesion ◽  
Intercellular Space ◽  
Solid Phase ◽  
Rabbit Antiserum ◽  
High Titre ◽  
Polyclonal Antiserum ◽  
Keyhole Limpet Haemocyanin ◽  
Calcium Dependent ◽  
Dependent Cell

The N-terminal sequence of a mixture of desmosomal glycoproteins 2 and 3 (dg2/3, desmocollins) from bovine nasal epidermis, prepared by electro-elution from polyacrylamide gels, was determined by solid-phase Edman degradation. A sequence of 23 amino acids was obtained. This showed 43% identity with that of the N terminus of the calcium-dependent cell adhesion molecule, N-cadherin. A lesser degree of identity with other members of the cadherin-uvomorulin-L-CAM family was also found. In order to confirm that the sequence was derived from the dg2/3 molecules a rabbit antiserum was raised against a synthetic peptide corresponding to the sequence, conjugated to keyhole limpet haemocyanin (KLH). The antiserum obtained showed high (titre) activity against both the peptide and KLH in ELISA. Each activity could be specifically adsorbed with the appropriate ligand. The antiserum reacted specifically with both dg2 and dg3 of bovine nasal epidermis on immunoblots, this binding was blocked by the N-terminal peptide but was unaffected by KLH. The identity of dg2 and -3 in these preparations was confirmed by immunoblotting with two monoclonal antibodies and one polyclonal antiserum raised against the whole molecules. The N-terminal peptide antiserum was shown to bind to the intercellular space of desmosome profiles by immunoelectron microscopy on ultra-thin frozen sections. One of the two monoclonal antibodies (07–4D) also reacted with the desmosomal intercellular space. dg2 and -3 were shown by Staphylococcus aureus V8 protease digestion to have identical one-dimensional peptide maps. Both the N-terminal antiserum and 07–4D reacted with a V8 fragment of 19,000 Mr derived from dg2 and dg3.(ABSTRACT TRUNCATED AT 250 WORDS)

Cross -Reactivity of the V3-Specific Antibodies with the Human C1q

2001 ◽  
Vol 56 (11-12) ◽  
pp. 1135-1143 ◽  
Author(s):  
Marijana Petković ◽  
Radmila Metlaš
Keyword(s):  
Lupus Erythematosus ◽  
Sequence Similarity ◽  
Hypervariable Region ◽  
Cross Reactivity ◽  
Specific Antibodies ◽  
Immunodeficiency Virus ◽  
Human Collagen ◽  
Hiv 1 ◽  
Autoimmune Phenomena

Abstract It has been previously shown that the sequence similarity between a portion of the enve­lope glycoprotein 120 (gp120) from the human immunodeficiency virus type-1 (HIV-1) and several types of human collagen and collagen-like molecules exists. That observation led to the suggestion that the antibodies against the third hypervariable region (V3) of HIV-1 gpl20 (V3-specific antibodies) might have a role in the autoimmune phenomena observed in HIV-infected persons. In this study we have examined the cross-reactivity of the V3-specific anti­ bodies purified from sera of HIV-infected individuals, sera obtained from the rheumatoid arthritis and systemic lupus erythematosus patients, as well as from the sera of healthy volun­ teers with the separate chains of a subcomponent of the first component of the human com­ plement system, C1q. Our results show that the V3-specific antibodies are present in the sera of the HIV-infected individuals, patients suffering of the systemic autoimmune diseases as well as in the sera of healthy volunteers. Whereas these antibodies appeared in the HIV+-sera after antigen challenge, those present in the H IV --sera probably represent the antibod­ ies that are cross-reactive with the antigen. V3-reactive antibodies can be purified by affinity chromatography and they were highly specific for the V3-peptide. Additionally, they showed cross-reactivity with the separate chains of the human C1q as well as with the chicken colla­ gen type VI. Possible physiological implications are discussed.

scholarly journals Performance of an Automated Zika IgG Immunoassay in the Detection of Zika IgG Specific Antibodies—A Validation Approach in Samples from Prevalence Areas and Non-Endemic Countries

2020 ◽  
Vol 5 (2) ◽  
pp. 97
Author(s):  
Tina Laengin ◽  
Stephanie Augenstein ◽  
Elke Stadlbauer ◽  
Heike Girgnhuber ◽  
Mario Gloeck ◽  
...  
Keyword(s):  
Virus Infection ◽  
Zika Virus ◽  
Cross Reactivity ◽  
Acute Stage ◽  
Igg Antibodies ◽  
Specific Antibodies ◽  
Diagnostic Samples ◽  
Diagnostic Sensitivity And Specificity ◽  
Blood Screening ◽  
Important Marker

The diagnosis of Zika virus infection is complicated and includes testing for nucleic acids and IgM and IgG antibodies, depending on the stage of infection. Zika IgG is an important marker of infection after the acute stage; however, IgG assays can lack specificity due to the similarities between Zika and other flaviviruses. In this study, the diagnostic sensitivity and specificity of the Elecsys® Zika IgG assay were assessed in 496 samples from Zika endemic regions, and specificity only was assessed in 1685 blood screening and diagnostic samples from Zika non-endemic regions. Cross-reactivity was also assessed against a panel of 202 potentially cross-reacting samples. The performance of the Elecsys® Zika IgG assay was compared with the anti-Zika virus ELISA IgG. In the samples from the Zika endemic regions, the Elecsys® Zika IgG assay had 92.88% (95% confidence interval 89.42–95.48) sensitivity and 100% specificity and in the samples from Europe the Elecsys® Zika IgG assay specificity was ≥99.62%. The Elecsys® Zika IgG assay was highly specific in samples from both prevalent and non-endemic regions.

Radioimmunoassay of lysergic acid diethylamide (LSD) in serum and urine by using antisera of different specificities.

1977 ◽  
Vol 23 (2) ◽  
pp. 169-174 ◽  
Author(s):  
W A Ratcliffe ◽  
S M Fletcher ◽  
A C Moffat ◽  
J G Ratcliffe ◽  
W A Harland ◽  
...  
Keyword(s):  
Biological Fluids ◽  
Cross Reactivity ◽  
High Titre ◽  
Lysergic Acid ◽  
Side Chain ◽  
Satisfactory Method ◽  
Related Compounds ◽  
Amide Side Chain ◽  
Different Parts ◽  
Serum And Urine

Abstract We raised high-titre antisera to two LSD-bovine serum albumin conjugates, one linked via the indole nitrogen, the other via the amide side-chain. The antisera were specific for different parts of the LSD molecule, as demonstrated by cross-reactivity studies with LSD, its metabolites, ergot alkoloids, and closely related compounds. The antisera were used to develop a double-antibody radioimmunoassay with a detection limit of about 0.4 mug of LSD per liter of unextracted urine or serum. We saw no nonspecific interference by urine, serum, or from a series of commonly used drugs. There was good correlation between immunoassay values obtained with the two antisera (r = 0.91). However, the antiserum linked via the indole nitrogen gave consistently higher results for samples from persons who had taken LSD, owing to greater cross-reactivity with LSD metabolites. Radioimmunoassay by use of two such antisera is a more specific screening procedure for LSD abuse than has been available previously. In addition, antisera cross-reacting with LSD metabolites allow measurement of these compounds, for which there is no satisfactory method at the concentrations found in biological fluids in man.

Sign in / Sign up

Export Citation Format

Share Document