Differential titration of plasmin and plasminogen in milk using sandwich ELISA with monoclonal antibodies

1997 ◽  
Vol 64 (1) ◽  
pp. 77-86 ◽  
Author(s):  
DIDIER DUPONT ◽  
CHRISTELLE BAILLY ◽  
JEANNE GROSCLAUDE ◽  
JEAN-CLAUDE COLLIN
Keyword(s):  
Monoclonal Antibodies ◽  
Proteinase Inhibitors ◽  
Sandwich Elisa ◽  
Bovine Milk ◽  
Milk Proteins ◽  
Cross Reactivity ◽  
The Other ◽  
Milk Samples ◽  
Skimmed Milk ◽  
Mastitic Milk

Two sandwich enzyme-linked immunosorbent assays (ELISA) have been developed for quantitation of bovine milk plasminogen and plasmin. The assays used two monoclonal antibodies, one specific for plasminogen and the other specific for plasminogen plus plasmin. Plasmin concentration was obtained by subtracting the first concentration from the second. The assays were sensitive (linear range, 5–75 ng/ml), repeatable (CV, 8 and 5% for plasmin and plasminogen titration respectively), specific (no cross reactivity with the major milk proteins) and directly applicable to skimmed milk with no particular pretreatment of the sample. However, ELISA did not permit complete titration of milk plasminogen and plasmin (only 60–90% could be quantified). This lack of accuracy was due to casein interfering with the plasmin–plasminogen titration by ELISA. Results obtained with ELISA or an enzymic technique on 20 milk samples collected from individual cows milked throughout pregnancy showed that the ELISA was particularly suitable for analysis of late lactation or mastitic milk where proteinase inhibitors interfered with enzymic quantitation.

scholarly journals Distribution of Antibiotic Resistance Genes in Enterococcus spp. Isolated from Mastitis Bovine Milk

2016 ◽  
Vol 66 (3) ◽  
pp. 336-346 ◽  
Author(s):  
Goksel Erbas ◽  
Ugur Parin ◽  
Suheyla Turkyilmaz ◽  
Nese Ucan ◽  
Mehmet Ozturk ◽  
...  
Keyword(s):  
Resistance Genes ◽  
Bovine Milk ◽  
Antibiotic Resistance Genes ◽  
High Rate ◽  
Resistant Isolate ◽  
Chloramphenicol Resistance ◽  
Milk Samples ◽  
Enterococcus Spp ◽  
Pcr Method ◽  
Mastitic Milk

AbstractIn this study, determination of enterococcus species that were isolated from mastitic milk samples, investigation of their susceptibilities to antibiotics and identification of the existence of resistance genes in resistant strains were conducted. The specimens consist of 600 mastitic milk samples that were collected from 242 cows. Isolation of enterococcus was carried out in selective media and 94 (15.6%)Enterococcusspp. were isolated. A total of 94 species of Enterococci were identified using both sequencing and polymerase chain reaction (PCR).Enterococcusspp. isolates belong to 5 different species (E. faecalis, E. faecium, E. durans, E. hirae, E. mundtii) in sequence analysis and 4 different species (E. faecalis, E. faecium, E. durans, E. hirae) were identify by PCR method with specific primers. Analyzing 94 enterococcus strains by antibiotic sensitiveness test a high rate of resistance to tetracycline in 77 (81.9%) isolates was shown. Thetetresistance genes were identified as follows: 54 weretetM positive, 23 weretetK positive and 17 were positive ontetM andtetK. Resistance to erythromycin was established in 27 (28.7%) isolates (25ermB) while the chloramphenicol resistance gene was found in 10 (10.7%) of isolates and thecatgene was identified in nine samples and one isolate was resistant to vancomycin (1.06%) with theVanA gene confirmed. In conclusion, it was shown thatE. faecalishas the biggest role inenterococcusoriginated mastitis and these strains were found to be mostly resistant to tetracycline. One vancomycine resistant isolate that had theVanA gene was also determined.

Characterisation ofStaphylococcus aureusstrains isolated from mastitis bovine milk in Argentina

2018 ◽  
Vol 85 (1) ◽  
pp. 57-63 ◽  
Author(s):  
Mariela E Srednik ◽  
Valentine Usongo ◽  
Sarah Lépine ◽  
Xavier Janvier ◽  
Marie Archambault ◽  
...  
Keyword(s):  
Large Scale ◽  
Susceptibility Testing ◽  
Restriction Analysis ◽  
Bovine Milk ◽  
Dairy Herds ◽  
Antibiotic Susceptibility Testing ◽  
Chromosomal Dna ◽  
Isolation Rate ◽  
Milk Samples ◽  
Mastitic Milk

The study reported in this Research Communication was conducted to characteriseStaphylococcus aureusisolates recovered from mastitic bovine milk from dairy herds in Argentina. A total of 829 mastitic milk samples, both clinical and subclinical, were collected from 21 farms by veterinarians and submitted to the laboratory for testing from which 229S. aureusisolates were recovered, an isolation rate of 28·1%. These isolates were tested for susceptibility to the antibiotics penicillin, erythromycin and clindamycin. Of the 229 isolates, 53 (23·1%) were resistant to penicillin, 31 (13·5%) to erythromycin and 28 (12·2%) to clindamycin. All isolates were negative for themecA,mecC andpvlgenes by PCR. Southernblot hybridisation revealed that theermC gene was located on plasmid bands. Eighty isolates were randomly selected from the 229 for further characterisation. Restriction analysis of chromosomal DNA with Cf9I followed by PFGE of the 80 isolates revealed 23 distinct pulsotypes at 80% similarity. Seven major types (A, B, N, P, S, T, U and V) accounted for 68·7% of these isolates and 12 pulsotypes (A, B, F, G, J, K, M, N, P, S, T and U) occurred on more than one farm indicating genetic diversity within the farms. MLST of a representative isolate from dominant types identified the STs 97 705, 746, 2102 and 2187 with ST97 being the most predominant. Antibiotic susceptibility testing showed that 53·7% of the 80 randomly selected isolates were resistant to at least one of the three antibiotics tested. To our knowledge, this study represents the first large scale molecular studies onS. aureusisolates from dairy farms in Argentina.

scholarly journals The effect of pre-enrichment on recovery ofStreptococcus agalactiae, Staphylococcus aureusand mycoplasma from bovine milk

1989 ◽  
Vol 103 (3) ◽  
pp. 465-474 ◽  
Author(s):  
M. C. Thurmond ◽  
J. W. Tyler ◽  
D. M. Luiz ◽  
C. A. Holmberg ◽  
J. P. Picanso
Keyword(s):  
Dairy Cows ◽  
Storage Temperature ◽  
Bovine Milk ◽  
The Other ◽  
Bovine Blood ◽  
Sample Storage ◽  
Milk Samples ◽  
California Mastitis Test ◽  
Direct Inoculation ◽  
Culture Procedure

SUMMARYThe study was conducted to determine whether pre-enrichment would increase sensitivity of detectingStreptococcus (Str.) aqalactiae, Staphylococcus (S.) aureus, and mycoplasma in bovine milk. Two procedures were followed, one involving direct inoculation of milk on bovine blood agar, and the other involving preenrichment in broth followed by inoculation on agar. Logistic regression was used to predict the probability of isolation as a function of culture procedure and two additional covariates. the California Mastitis Test (CMT) score of the milk and the type of sample (indicating sample storage temperature and herd mastitis status). A total of 13778 milk samples was cultured for each of the three bacteria. By using results of both direct inoculation and pre-enrichment, the probability of isolation compared to use of direct inoculation only and adjusted for effects of other variables was increased 3·6-fold forStr. agalactiae, 1·6-fold forS. aureusand 1·7- fold for mycoplasma. The probability of isolation for all three bacteria increased as the CMT score increased. ForStr. agalactiae, there was a statistical interaction predicting that enrichment improved the odds of isolation more from milk with high CMT scores than from milk with low scores. Results indicate that preenrichment can substantially increase the sensitivity of bacteriological screening of dairy cows for mastitis caused byStr. agalactiae,S. aureus, and mycoplasma.

scholarly journals Development of Vasoinhibin-Specific Monoclonal Antibodies

2021 ◽  
Vol 12 ◽  
Author(s):  
Nils Müller ◽  
Juan Pablo Robles ◽  
Magdalena Zamora ◽  
Johannes Ebnet ◽  
Hülya Markl-Hahn ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Serum Proteins ◽  
Limit Of Detection ◽  
Sandwich Elisa ◽  
Vascular Diseases ◽  
High Specificity ◽  
Cross Reactivity ◽  
Dimensional Structure ◽  
Serum Samples ◽  
Limit Of Quantitation

Vasoinhibin is a protein hormone with antiangiogenic, antivasodilatatory, and antivasopermeability effects generated by the proteolytic cleavage of prolactin. The discovery of its role in diabetic retinopathy and peripartum cardiomyopathy led to the evaluation of new pharmacological treatments in clinical interventional trials. However, the quantitative evaluation of vasoinhibin in biological samples from patients has not been possible due to the lack of vasoinhibin-specific antibodies. Recently, loop 1 of vasoinhibin was identified to have a different three-dimensional structure compared to PRL, and thus to contain vasoinhibin-specific epitopes. Here, we report the development of two sets of vasoinhibin-specific monoclonal antibodies against two neighboring regions of the vasoinhibin loop 1. An experimental sandwich ELISA with two monoclonal anti-vasoinhibin antibodies was developed, which had no cross-reactivity to recombinant human full-length prolactin. The ELISA had a quantitation limit of 100 ng/ml, and intra-assay- and inter-assay coefficients of variation of 12.5% and 14%, respectively. The evaluation of 15 human serum samples demonstrated concentrations of below limit of detection (n=3), below limit of quantitation (n=1) and between 0.23 µg/ml (230 ng/ml) to 605 µg/ml (n=12) in the quantifiable range. Despite the high specificity of the monoclonal-monoclonal antibody sandwiches which discriminate vasoinhibin from PRL, there might be cross-reactivities by serum proteins other than vasoinhibin. A fully established vasoinhibin ELISA may support diagnostic and therapeutic measures in vascular diseases.

scholarly journals Monoclonal antibodies for the S2 subunit of spike of SARS-CoV-1 cross-react with the newly-emerged SARS-CoV-2

2020 ◽  
Vol 25 (28) ◽  
Author(s):  
Zhiqiang Zheng ◽  
Vanessa Marthe Monteil ◽  
Sebastian Maurer-Stroh ◽  
Chow Wenn Yew ◽  
Carol Leong ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Common Cold ◽  
Sandwich Elisa ◽  
Cell Receptor ◽  
Cross Reactivity ◽  
S Protein ◽  
Mammalian Cell Lines ◽  
Infected Cells ◽  
The Cross ◽  
Novel Coronavirus

Background A novel coronavirus, SARS-CoV-2, which emerged at the end of 2019 and causes COVID-19, has resulted in worldwide human infections. While genetically distinct, SARS-CoV-1, the aetiological agent responsible for an outbreak of severe acute respiratory syndrome (SARS) in 2002–2003, utilises the same host cell receptor as SARS-CoV-2 for entry: angiotensin-converting enzyme 2 (ACE2). Parts of the SARS-CoV-1 spike glycoprotein (S protein), which interacts with ACE2, appear conserved in SARS-CoV-2. Aim The cross-reactivity with SARS-CoV-2 of monoclonal antibodies (mAbs) previously generated against the S protein of SARS-CoV-1 was assessed. Methods The SARS-CoV-2 S protein sequence was aligned to those of SARS-CoV-1, Middle East respiratory syndrome (MERS) and common-cold coronaviruses. Abilities of mAbs generated against SARS-CoV-1 S protein to bind SARS-CoV-2 or its S protein were tested with SARS-CoV-2 infected cells as well as cells expressing either the full length protein or a fragment of its S2 subunit. Quantitative ELISA was also performed to compare binding of mAbs to recombinant S protein. Results An immunogenic domain in the S2 subunit of SARS-CoV-1 S protein is highly conserved in SARS-CoV-2 but not in MERS and human common-cold coronaviruses. Four murine mAbs raised against this immunogenic fragment could recognise SARS-CoV-2 S protein expressed in mammalian cell lines. In particular, mAb 1A9 was demonstrated to detect S protein in SARS-CoV-2-infected cells and is suitable for use in a sandwich ELISA format. Conclusion The cross-reactive mAbs may serve as useful tools for SARS-CoV-2 research and for the development of diagnostic assays for COVID-19.

Screening for Fibrin Specific Monoclonal Antibodies: The Development of a New Procedure

1992 ◽  
Vol 68 (01) ◽  
pp. 048-053 ◽  
Author(s):  
P M Tymkewycz ◽  
L J Creighton-Kempsford ◽  
D Hockley ◽  
P J Gaffney
Keyword(s):  
Electron Microscopy ◽  
Scanning Electron Microscopy ◽  
Monoclonal Antibodies ◽  
Chemical Composition ◽  
Cross Reactivity ◽  
The Other ◽  
Screening Procedure ◽  
Screening Procedures ◽  
Soluble State ◽  
Scanning Electron

SummaryThe acquisition of monoclonal antibodies specific for human fibrin has been impaired by the similarity in chemical composition between fibrinogen and fibrin and the conformational difference between immobilised and soluble fibrinogen. Five monoclonal antibodies (mabs) with a known affinity for fibrin have been subjected to screening procedures which involved the presentation of different forms of both fibrinogen and fibrin to the test mabs. It was observed by scanning electron microscopy that dried fibrin (denoted fibrin D), immobilised on the wells of PVC plates was morphologically similar to the fibrin found in human clots whereas PVC-immobilised fibrin monolayers (fibrin M) and a homogenised form of fibrin (fibrin FF) presented two very different morphological appearances. It was shown that lack of cross reactivity of a mab with an antigen (e.g. fibrinogen) was validly demonstrated only when both mab and antigen were present in the soluble state. These findings have allowed the generation of a screening procedure which involves the use of fibrin D on PVC plates in conjunction with whole human plasma incubated with the test antibody. This screening procedure has been validated using two mabs, one of which has an exclusive fibrin affinity while the other has a broad spectrum crossreactivity with both fibrinogen and fibrin. This procedure would ensure the acquisition of all the five fibrin-specific mabs used in this study while other less reliable screening procedures might not.

scholarly journals Validation Study of a Receptor-Based Lateral Flow Assay for Detection of Beta-Lactam Antibiotics in Milk

2009 ◽  
Vol 92 (3) ◽  
pp. 959-974 ◽  
Author(s):  
Mohamed Abouzied ◽  
Michael Sarzynski ◽  
Aaron Walsh ◽  
Heather Wood ◽  
Mark Mozola
Keyword(s):  
Bovine Milk ◽  
Operating Parameter ◽  
Raw Milk ◽  
Cross Reactivity ◽  
Penicillin G ◽  
Beta Lactam ◽  
Milk Samples ◽  
Beta Lactam Antibiotics ◽  
Cell Counts ◽  
Lactam Antibiotic

Abstract Avalidation study designed tomeet the requirements of the AOAC Research Institute and the U.S. Food and Drug Administration (FDA), Center for Veterinary Medicine, was conducted for a receptor-based, immunochromatographic method (BetaStar US) for detection of beta-lactam antibiotic residues in raw, commingled bovine milk. The assay was found to detect amoxicillin, ampicillin, cephapirin, cloxacillin, and penicillin G at levels below the FDA tolerance/safe levels but above the maximum sensitivity thresholds established by the National Conference on Interstate Milk Shipments. Results of the Part I (internal) and Part II (independent laboratory) dose-response studies using spiked samples were in very close agreement for all five drugs tested, with differences between the Part I and Part II 90/95 sensitivity values ranging from 0 to 1 ppb. The test was able to detect all five drugs at the approximate 90/95 sensitivity levels when present as incurred residues in milk collected from cows that had been treated with the specific drug. Asixth drug, ceftiofur, was found to be undetectable at levels of 500 ppb (as total ceftiofur metabolites from incurred residues in milk samples). The selectivity of the assay was 100, because no false-positive results were obtained in tests of >1000 control milk samples. The assay was found to be applicable to the testing of frozen raw milk samples. Results of ruggedness experiments established the operating parameter tolerances for the BetaStar US assay. Results of cross-reactivity testing established that the assay detects certain other beta-lactam drugs (dicloxacillin and ticarcillin), but it does not cross-react with any of 30 drugs belonging to other classes. Abnormally high bacterial or somatic cell counts in rawmilk produced no interference with the ability of the test to detect beta-lactams at tolerance/safe levels.

Development and validation of a nephelometric immunoassay for IgG1 in milk

2002 ◽  
Vol 69 (1) ◽  
pp. 27-35 ◽  
Author(s):  
ROGER COLLIN ◽  
COLIN PROSSER ◽  
ROB MCLAREN ◽  
MARY THOMSON ◽  
DAVID MALCOLM
Keyword(s):  
Quality Assurance ◽  
High Throughput ◽  
Bovine Milk ◽  
Cross Reactivity ◽  
Detection Range ◽  
Milk Samples ◽  
Development And Validation ◽  
Β Lactoglobulin ◽  
Good Agreement

A nephelometric immunoassay, with a detection range of 0·3 to 5 g IgG1/l, was developed for the determination of immunoglobulin in bovine milk. The assay exhibited no significant cross-reactivity with αS1-casein, αS2-casein, β-casein, κ-casein or β-lactoglobulin and 39% cross-reactivity with IgG2. The nephelometric assay was compared with ELISA and RID (24 h and 48 h incubations) assays using 105 duplicate milk samples covering IgG1 values ranging from 0·45 to 1·8 g1. The results obtained from all assays showed good agreement with the exception of those obtained by the RID assay (24 h incubation) which gave lower results in samples containing more than 1·2 g IgG1/l. It was concluded that the nephelometric assay is a reliable, rapid and convenient method suitable for the quantification of IgG1 in milk. The assay can be configured for routine high-throughput milk quality assurance for IgG1 in dairy laboratories.

Time-resolved immunofluorometric assay of human pancreatic isoamylase in serum, with use of two monoclonal antibodies.

1989 ◽  
Vol 35 (9) ◽  
pp. 1915-1920 ◽  
Author(s):  
E P Diamandis ◽  
A Papanastasiou-Diamandi ◽  
V Lustig ◽  
M J Khosravi ◽  
A Tan
Keyword(s):  
Enzyme Activity ◽  
Monoclonal Antibodies ◽  
Amylase Activity ◽  
Cross Reactivity ◽  
The Other ◽  
Analytical Performance ◽  
New Method ◽  
Time Resolved ◽  
Pancreatic Isoamylase ◽  
Capture Antibody

Abstract This new method for determining pancreatic isoamylase (EC 3.2.1.1) in serum involves two monoclonal antibodies: one immobilized in a microtitration well (the capture antibody), the other biotinylated. After the sample is incubated with the two antibodies, the captured immunocomplex is quantified by adding streptavidin labeled with a europium chelator and measuring the specific Eu3+ fluorescence in a time-resolved mode. Three assay protocols are proposed, involving incubation times of 90, 45, or 25 min. The assay has low (0.005%) cross-reactivity with the salivary isoenzyme. Analytical performance was satisfactory. Results correlate well with results obtained by measuring total amylase activity or by measuring pancreatic isoamylase activity after immunoinhibition. Unlike numerous current amylase assays, this method measures enzyme mass rather than enzyme activity. Potentially, this is a highly specific assay.

scholarly journals Formulation and Assessment of Cashew Kernel Milk as an Alternative to Cow’s Milk

2020 ◽  
Vol 1 (1) ◽  
pp. 86-92
Author(s):  
Jayeola CO ◽  
◽  
Yahaya LE ◽  
Ogunwolu SO ◽  
Mokwunoye FC ◽  
...  
Keyword(s):  
Nutritional Value ◽  
Mineral Content ◽  
Coconut Milk ◽  
The Other ◽  
Overall Acceptability ◽  
Milk Samples ◽  
Ph Values ◽  
Cashew Nut ◽  
Skimmed Milk ◽  
Dairy Milk

Dairy free milk alternative was produced from tree nuts and legumes with high nutritional value, lactose free, cholesterol free and unsaturated fats unlike the mammalian milk. Dairy free milk alternative was obtained from cashew nut, coconut while skimmed milk from cow was used as reference standard. The prepared milk samples were analyzed for proximate, minerals and sensory attributes. The result revealed a reasonable amount of protein (12.26%) and coconut milk for cashew milk, 11.17% for soymilk, 8.52% for coconut milk while the standard cows skimmed milk, values obtained for cashew milk of 72.67%, soymilk and coconut milk was 64.22% contain the highest of 0.69 for ash while the plant milk also contain appreciable quantity which is directly related to the mineral content of the milk alternative. Cashew nut milk had the lowest pH values of 6. 10 and the lowest specific gravity of 1.010 g/cm3 . Fat content was high for cashew nut milk compared with the other milk samples. Sensory evaluation performed indicated that cashew milk was most preferred for the attributes of colour, mouthfeel while it ranked next to skimmed milk in taste and flavor. Cashew milk was ranked highest with values of 8.33 for overall acceptability compares well with skimmed milk with the value of 8.46 while the other milk alternatives had the values of 6.22 and 6.44 for soy milk and coconut milk respectively. The results therefore indicate that cashew milk can be used as alternative to dairy milk.

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