Detection of genetic variants of alpha 1-antitrypsin with site-specific monoclonal antibodies

1991 ◽  
Vol 37 (9) ◽  
pp. 1606-1611 ◽  
Author(s):  
N D Zegers ◽  
E Claassen ◽  
K Gerritse ◽  
C Deen ◽  
W J Boersma
Keyword(s):  
Monoclonal Antibodies ◽  
Electrophoretic Pattern ◽  
Base Substitution ◽  
Severe Deficiency ◽  
Normal Individuals ◽  
Increased Risk ◽  
Peptide Carrier ◽  
Flanking Sequences ◽  
At Deficiency ◽  
Alpha 1 Antitrypsin

Abstract The serum protein alpha 1-antitrypsin (alpha 1-AT) serves as the major inhibitor of neutrophil elastase. The most common allele of the alpha 1-AT gene is designated as PiM. The Z mutation is a single-base substitution of the normal M allele, causing a Glu----Lys change at position 342 in the molecule. The ZZ phenotype is associated with a severe deficiency of alpha 1-AT, serum concentrations of the protein being 10% of normal. Individuals with an alpha 1-AT deficiency are at an increased risk of developing emphysema. To generate antibodies that specifically detect the 342 position in the context of the flanking sequences, we synthesized several peptides that included the 342 position for both the M and the Z variant. Immunization with variant-specific peptide-carrier conjugates elicited alpha 1-AT variant-specific responses, as determined in a direct enzyme-linked immunoassay. Monoclonal antibodies (MAbs) were selected with different specificity for the 342 region: MAbs F43 recognize only the alpha 1-AT sequence with 342Glu, i.e., all variant proteins that are non-Z, either from hetero- or homozygous individuals; MAbs F50 recognize only the sequence with 342Lys, i.e., all Z-variant proteins in ZZ or heterozygous individuals; MAbs F46 recognize alpha 1-AT with either 342Lys or 342Glu, all variant proteins with sequences as in the peptides used. Z homo- and heterozygotes were detected with our MAbs in a rapid and simple immunoblot assay. Other variants (M, S, and F) can also be assigned on the basis of the electrophoretic pattern. This sensitive detection method is very easy, rapid, and straightforward and provides a powerful tool for diagnosis of the alpha 1-AT deficiencies, allowing early treatment (augmentation of alpha 1-AT) and proper advice on lifestyle practices.

The Interaction of Botrocetin with Normal or Variant von Willebrand Factor (Types IIA and IIB) and Its Inhibition by Monoclonal Antibodies that Block Receptor Binding

1992 ◽  
Vol 68 (04) ◽  
pp. 464-469 ◽  
Author(s):  
Y Fujimura ◽  
S Miyata ◽  
S Nishida ◽  
S Miura ◽  
M Kaneda ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Von Willebrand Factor ◽  
Binding Activity ◽  
Von Willebrand Disease ◽  
Platelet Glycoprotein ◽  
Normal Individuals ◽  
Rag 1 ◽  
The One ◽  
Von Willebrand ◽  
Willebrand Factor

SummaryWe have recently shown the existence of two distinct forms of botrocetin (one-chain and two-chain), and demonstrated that the two-chain species is approximately 30 times more active than the one-chain in promoting von Willebrand factor (vWF) binding to platelet glycoprotein (GP) Ib. The N-terminal sequence of two-chain botrocetin is highly homologous to sea-urchin Echinoidin and other Ca2+-dependent lectins (Fujimura et al., Biochemistry 1991; 30: 1957–64).Present data indicate that purified two-chain botrocetin binds to vWF from plasmas of patients with type IIA or IIB von Willebrand disease and its interaction is indistinguishable from that with vWF from normal individuals. However, an “activated complex” formed between botrocetin and IIB vWF expresses an enhanced biological activity for binding to GP Ib whereas the complex with IIA vWF has a decreased binding activity. Among several anti-vWF monoclonal antibodies (MoAbs) which inhibit ristocetin-induced platelet aggregation and/or vWF binding to GPIb, only two MoAbs (NMC-4 and RFF-VIII RAG:1) abolished direct binding between purified botrocetin and vWF. This suggests that they recognize an epitope(s) on the vWF molecule in close proximity to the botrocetin binding site.

The Capacity of a Specific Anti-Streptococcus Mutans Monoclonal Antibodies Test to Identify the Diabetic Patients with Increased Risk of Periodontal Disease

2017 ◽  
Vol 68 (11) ◽  
pp. 2556-2559
Author(s):  
Mona Ionas ◽  
Sebastian Ioan Cernusca Mitariu ◽  
Adela Dancila ◽  
Tiberiu Horatiu Ionas ◽  
Raluca Monica Comaneanu ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Periodontal Disease ◽  
Streptococcus Mutans ◽  
Mutans Streptococci ◽  
Diabetic Patients ◽  
A Value ◽  
Salivary Level ◽  
Increased Risk ◽  
The Status

By means of a specific anti-Streptococcus mutans monoclonal antibodies test we want to identify the diabetic patients which have an increased risk to develop the periodontal disease. The highest percentage, of 88.1% of all patients included in this study represents the subjects with a level greater than 500,000 cfu / mL of streptococcus mutans. The Kruskal-Wallis test reveals a value of p = 0.283 resulted from the status of diabetes in patients and the level of streptococcus mutans in saliva. In conclusion, the status of diabetes in patients seems not to influence the salivary level of mutans streptococci determined with the method used in our study.

Alpha-1 antitrypsin deficiency: clarifying the role of the putative protective threshold

2021 ◽  
pp. 2101410
Author(s):  
Alessandro N. Franciosi ◽  
Daniel Fraughen ◽  
Tomás P. Carroll ◽  
Noel G. McElvaney
Keyword(s):  
Risk Estimation ◽  
Specific Treatment ◽  
Weekly Administration ◽  
Risk Assessment Models ◽  
Increased Risk ◽  
Antitrypsin Deficiency ◽  
Dosing Regimens ◽  
Alpha 1 Antitrypsin Deficiency ◽  
Alpha 1 Antitrypsin ◽  
Protective Threshold

AATD is the only readily identifiable monogenic cause of COPD. To date the only condition-specific treatment for AATD-associated COPD is weekly administration of intravenous purified pooled human AAT (IV-AAT). Uncertainties regarding which AATD genotypes should benefit from IV-AAT persist. IV-AAT is costly and involves weekly administration of a plasma product. Much of the risk stratification has been centred around the long-accepted hypothesis of a “putative protective threshold” of 11 µM (0.57 g·L−1) in serum. This hypothesis has become central to the paradigm of AATD care, though its derivation and accuracy for defining risk of disease remain unclear.We review the literature and examine the association between the 11 µM threshold and clinical outcomes to provide context and insight into the issues surrounding this topic.We found no data which demonstrates an increased risk of COPD dependent on the 11 µM threshold. Moreover, an abundance of recent clinical data examining this threshold refutes the hypothesis. Conversely, the use of 11 µM as a treatment target in appropriate ZZ individuals is supported by clinical evidence, although more refined dosing regimens are being explored.Continued use of the 11 µM threshold as a determinant of clinical risk is questionable, perpetuates inappropriate AAT-augmentation practices, may drive increased healthcare expenditure and should not be used as an indicator for commencing treatment.Genotype represents a more proven indicator of risk, with ZZ and rare ZZ-equivalent genotypes independently associated with COPD. New and better risk assessment models are needed to provide individuals diagnosed with AATD with reliable risk estimation and optimised treatment goals.

scholarly journals Chronic Obstructive Lung Disease in young: Alpha 1 anti trypsin deficiency

2015 ◽  
Vol 3 (2) ◽  
pp. 65-67
Author(s):  
S.S. Dhakal ◽  
K.K. Agrawaal ◽  
N.K. Bhatta
Keyword(s):  
Bronchial Asthma ◽  
Clinical Manifestations ◽  
Lower Lobe ◽  
Chronic Obstructive Lung Disease ◽  
Chronic Obstructive ◽  
Obstructive Pulmonary Disease ◽  
Severe Deficiency ◽  
History Of ◽  
Pulmonary Arterial ◽  
Alpha 1 Antitrypsin

Alpha-1 antitrypsin (AAT) deficiency is a clinically under recognized inherited disorder. The main clinical manifestations relate to three separate organs: the lung, the liver, and the skin. In the lung, severe deficiency of AAT predisposes to chronic obstructive pulmonary disease. We present a case of 34 years male with a history of recurrent chest infections in past and treated in the line of bronchial asthma but not relieved. He was admitted on 22nd May 2011 at BPKIHS. He presented with type 2 respiratory failure and had features of severe pulmonary arterial hypertension and left lower lobe pneumonia. The patient got improved with the treatment and is doing well on follow up. The diagnosis should be strongly suspected in patients with history suggestive of bronchial asthma and with obstructive features.Journal of Advances in Internal Medicine 2014;3(2):65-67

Enzyme immunoassay for intact human insulin in serum or plasma

1993 ◽  
Vol 39 (4) ◽  
pp. 578-582 ◽  
Author(s):  
L Andersen ◽  
B Dinesen ◽  
P N Jørgensen ◽  
F Poulsen ◽  
M E Røder
Keyword(s):  
Monoclonal Antibodies ◽  
Detection Limit ◽  
Human Serum ◽  
Clinical Importance ◽  
Porcine Insulin ◽  
Microtiter Plates ◽  
Relative Response ◽  
Normal Individuals ◽  
Insulin Dependent ◽  
Murine Monoclonal Antibodies

Abstract We describe an enzyme-linked two-site immunoassay for quantitation of intact insulin in human serum and plasma. The method uses two murine monoclonal antibodies that bind to two different epitopes on the insulin molecule. The immunoassay is specific. Human proinsulin is not bound by the antibodies, and the binding of partially processed proinsulin intermediates is believed to be of minor clinical importance. The relative response of human, bovine, and porcine insulin is 1, 1, and 3, respectively. The assay is sensitive (detection limit 5 pmol/L), accurate (101% recovery with 50 pmol/L insulin added to samples, 95% with 100 pmol/L, and 89% with 300 pmol/L), and fast (results within 3 h), and has a high analytical capacity (done in microtiter plates). The working assay range selected is 5-600 pmol/L, corresponding to a clinically useful range. Because of its specificity, this two-site immunoassay gives results that are lower than those obtained by using a competitive radioimmunoassay, both in normal individuals and in non-insulin-dependent diabetics.

scholarly journals Protocol for the EARCO Registry: a pan-European observational study in patients with α1-antitrypsin deficiency

2020 ◽  
Vol 6 (1) ◽  
pp. 00181-2019 ◽  
Author(s):  
Timm Greulich ◽  
Alan Altraja ◽  
Miriam Barrecheguren ◽  
Robert Bals ◽  
Jan Chlumsky ◽  
...  
Keyword(s):  
Natural History ◽  
Research Collaboration ◽  
Medical Decision ◽  
Case Report Form ◽  
Increased Risk ◽  
Antitrypsin Deficiency ◽  
Study Team ◽  
Alpha 1 Antitrypsin Deficiency ◽  
Alpha 1 Antitrypsin

Rationale and objectivesAlpha-1 antitrypsin deficiency (AATD) is a genetic condition that leads to an increased risk of emphysema and liver disease. Despite extensive investigation, there remain unanswered questions concerning the natural history, pathophysiology, genetics and the prognosis of the lung disease in association with AATD. The European Alpha-1 Clinical Research Collaboration (EARCO) is designed to bring together researchers from European countries and to create a standardised database for the follow-up of patients with AATD.Study design and populationThe EARCO Registry is a non-interventional, multicentre, pan-European, longitudinal observational cohort study enrolling patients with AATD. Data will be collected prospectively without interference/modification of patient's management by the study team. The major inclusion criterion is diagnosed severe AATD, defined by an AAT serum level <11 µM (50 mg·dL−1) and/or a proteinase inhibitor genotype ZZ, SZ or compound heterozygotes or homozygotes of other rare deficient variants. Assessments at baseline and during the yearly follow-up visits include lung function testing (spirometry, body plethysmography and diffusing capacity of the lung), exercise capacity, blood tests and questionnaires (symptoms, quality of life and physical activity). To ensure correct data collection, there will be designated investigator staff to document the data in the case report form. All data will be reviewed by the EARCO database manager.SummaryThe EARCO Registry aims to understand the natural history and prognosis of AATD better with the goal to create and validate prognostic tools to support medical decision-making.

scholarly journals Studies of T-lymphocytes in preleukemic disorders and acute nonlymphocytic leukemia: in vitro radiosensitivity, mitogenic responsiveness, colony formation, and enumeration of lymphocytic subpopulations

Blood ◽  
1983 ◽  
Vol 61 (3) ◽  
pp. 449-455 ◽  
Author(s):  
SJ Knox ◽  
BR Greenberg ◽  
RW Anderson ◽  
LS Rosenblatt
Keyword(s):  
Whole Blood ◽  
Colony Formation ◽  
Tritiated Thymidine ◽  
Lymphocyte Stimulation Test ◽  
Acute Nonlymphocytic Leukemia ◽  
Clinically Normal ◽  
Normal Individuals ◽  
Increased Risk ◽  
X Irradiation

Abstract Tritiated thymidine incorporation in a whole blood lymphocyte stimulation test (LST) and lymphocyte colony formation (CFU-L) from whole blood were measured following in vitro x-irradiation. Lymphocytes from patients with myelodysplastic disorders, acute nonlymphocytic leukemia, and patients at increased risk for leukemia because of their primary disease and/or cytotoxic therapy were found to be significantly more sensitive to in vitro x-irradiation than lymphocytes from clinically normal individuals. Cloning efficiencies and mitogenic responsiveness of patient lymphocytes were significantly depressed as compared to normal values. Using monoclonal antibodies to specific surface markers, quantitative abnormalities in lymphocytic subpopulations from myelodysplastic patients also were observed. These findings are suggestive of a defect at the T-cell level that may directly or indirectly affect hematopoiesis.

Rapid screening for deficiency of alpha 1-proteinase inhibitor.

1989 ◽  
Vol 35 (9) ◽  
pp. 1971-1975 ◽  
Author(s):  
C Lloyd ◽  
J Travis
Keyword(s):  
Human Plasma ◽  
Inhibitory Activity ◽  
Proteinase Inhibitor ◽  
Rapid Screening ◽  
Early Screening ◽  
Pancreatic Elastase ◽  
Low Concentrations ◽  
Normal Individuals ◽  
Porcine Pancreatic Elastase ◽  
Alpha 1 Antitrypsin

Abstract This rapid screening procedure for detection of low but functional elastase-inhibitory activity in human plasma is based on the fact that incubation of excess porcine pancreatic elastase (EC 3.4.21.36) with plasma results in formation of a complex with active alpha 1-proteinase inhibitor (alpha 1PI, also called alpha 1-antitrypsin). In normal individuals all of the elastase is complexed, leaving no free enzyme to hydrolyze the elastase substrate, and the reaction mixture remains clear. Because individuals homozygous for the Z allele have relatively low concentrations of alpha 1PI, their plasma cannot complex all of the elastase in the assay. The uncomplexed enzyme hydrolyzes the elastase-specific p-nitroanilide substrate, producing a yellow reaction mixture. Use of this simple assay for early screening of individuals for alpha 1PI deficiency may substantially decrease the number of untreated cases of familial emphysema, a disorder that develops as a result of a genetically derived proteinase-proteinase inhibitor imbalance.

Creation of an Additional Glycosylation Site as a Mechanism for Type I Antithrombin Deficiency

2001 ◽  
Vol 86 (10) ◽  
pp. 1023-1027 ◽  
Author(s):  
Krzysztof Lewandowski ◽  
Robin Olds ◽  
Alison Fitches
Keyword(s):  
Molecular Weight ◽  
Glycosylation Site ◽  
Base Substitution ◽  
Type I ◽  
Antithrombin Deficiency ◽  
Expression Studies ◽  
Glycan Chain ◽  
Reticulocyte Lysate ◽  
History Of ◽  
At Deficiency

SummaryWe report the identification of a new mutation resulting in type I antithrombin (AT) deficiency and the mechanism by which the deficiency arose. The single base substitution of G to A at nucleotide 2709 was identified in a proband with a family history of venous thrombosis. The mutation results in a substitution of 82 Ser by Asn, creating a new glycosylation site. Expression studies were then carried out, to confirm Asn-linked glycosylation occurred at this consensus site and that this resulted in the AT deficient phenotype. Cell-free translations using rabbit reticulocyte lysate in the presence of microsomes demonstrated that the 82 Asn variant was post-translationally processed efficiently. The 82 Asn variant protein was of a higher molecular weight than normal AT, consistent with the addition of a fifth glycan chain. Incubation of translation product with endoglycosidase H, confirmed that the higher molecular weight product had resulted from additional carbohydrate. Expression of the 82 Asn variant in COS-7 cells resulted in intracellular accumulation, with a low level of secretion of the protein into culture supernatant, consistent with type I AT deficiency. The addition of an extra carbohydrate side chain to residue 82 of antithrombin may block post-translational folding, trapping the variant intracellulary.

Sign in / Sign up

Export Citation Format

Share Document