Detection of Bovine Milk in Ovine Milk by a Sandwich Enzyme-Linked Immunosorbent Assay (ELISA)

1991 ◽  
Vol 54 (5) ◽  
pp. 366-371 ◽  
Author(s):  
TERESA GARCÍA ◽  
ROSARIO MARTÍN ◽  
ELENA RODRÍGUEZ ◽  
JUAN I. AZCONA ◽  
BERNABÉ SANZ ◽  
...  
Keyword(s):  
Optical Density ◽  
Immunosorbent Assay ◽  
Polyclonal Antibodies ◽  
Sandwich Elisa ◽  
Whey Proteins ◽  
Bovine Milk ◽  
Microtiter Plate ◽  
Enzyme Linked Immunosorbent Assay ◽  
Specific Antibodies ◽  
Specific Antigens

A sandwich ELISA (enzyme-linked immunosorbent assay) was developed for the detection of defined amounts of bovine milk (1–30%) in ovine milk. Polyclonal antibodies were raised in rabbits against bovine whey proteins (BWP). Resultant antibodies were affinity purified by immunoadsorption of the crude antiserum onto columns containing immobilized ovine, caprine, and BWP, followed by elution of the bovine milk specific antibodies (anti-BWP) from the column containing the bovine proteins. The specific anti-BWP antibodies bound to the wells of a microtiter plate were used to capture the BWP from milk mixtures. Further immunorecognition of the captured proteins was attained with the same specific antibodies conjugated to biotin. ExtrAvidin-peroxidase was used to detect the biotinylated antibodies bound to their specific antigens. Subsequent enzymic conversion of substrate gave clear optical density differences when assaying mixtures containing variable amounts of bovine milk.

scholarly journals Production of Monoclonal Antibodies Specific for the i and 1,2 Flagellar Antigens of Salmonella typhimurium and Characterization of Their Respective Epitopes

1998 ◽  
Vol 64 (12) ◽  
pp. 5033-5038 ◽  
Author(s):  
N. de Vries ◽  
K. A. Zwaagstra ◽  
J. H. J. Huis in’t Veld ◽  
F. van Knapen ◽  
F. G. van Zijderveld ◽  
...  
Keyword(s):  
Amino Acids ◽  
Monoclonal Antibodies ◽  
Salmonella Typhimurium ◽  
Immunosorbent Assay ◽  
Specific Antibody ◽  
Polyclonal Antibodies ◽  
Enzyme Linked Immunosorbent Assay ◽  
Specific Antibodies ◽  
Minimal Area

ABSTRACT Salmonella typhimurium expresses two antigenically distinct flagellins, each containing a different H antigen (i and 1,2), the combination of which is highly specific for this serotype. In this study, overlapping recombinant flagellin fragments were constructed from the fliC(H:i) and fljB (H:1,2) flagellin genes, and the expression products were tested for binding to H antigen-specific monoclonal and polyclonal antibodies. A minimal area, 86 amino acids for H:i and 102 amino acids for H:1,2, located in the central variable domain of each flagellin was required for the binding of serotype-specific antibodies, providing further evidence for the presence of a discontinuous H epitope. Two peptides comprising these areas were shown to be highly suitable for application as antigens in an enzyme-linked immunosorbent assay detecting S. typhimurium-specific antibody.

Sandwich ELISA for Detection of Pig Meat in Raw Beef Using Antisera to Muscle Soluble Proteins

1988 ◽  
Vol 51 (10) ◽  
pp. 790-798 ◽  
Author(s):  
ROSARIO MARTÍN ◽  
JUAN I. AZCONA ◽  
CARMEN CASAS ◽  
PABLO E. HERNÁNDEZ ◽  
BERNABÉ SANZ
Keyword(s):  
Horseradish Peroxidase ◽  
Immunosorbent Assay ◽  
Peroxidase Activity ◽  
Sandwich Elisa ◽  
Soluble Proteins ◽  
Enzyme Linked Immunosorbent Assay ◽  
Specific Antibodies ◽  
Double Antibody ◽  
Colored Product ◽  
Pig Meat

A double-antibody sandwich ELISA (enzyme-linked immunosorbent assay) has been successfully developed for the detection of defined amounts of pig meat (1–50%) in raw beef. Antibodies against pig sarcoplasmic extracts were produced in rabbits. Pig-specific antibodies were affinity purified by removing antibodies which crossreacted with horse, chicken or beef extracts followed by immunoadsorption and elution from a pig-extract column. The ELISA involved capturing antigens in sarcoplasmic extracts with pig specific antibodies immobilized on 96-well plates, detecting bound antigen with pig specific, horseradish peroxidase-labeling antibody, and measuring peroxidase activity by the conversion of a clear substrate to a colored product.

A Competitive Enzyme-Linked Immunosorbent Assay for Detection of Bovine Milk in Ovine and Caprine Milk and Cheese Using a Monoclonal Antibody against Bovine β-Casein

1997 ◽  
Vol 60 (1) ◽  
pp. 64-66 ◽  
Author(s):  
G. ANGUITA ◽  
R. MARTÍN ◽  
T. GARCÍA ◽  
P. MORALES ◽  
A. I. HAZA ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Immunosorbent Assay ◽  
Binding Sites ◽  
Bovine Milk ◽  
Microtiter Plate ◽  
Competitive Elisa ◽  
Quantitative Detection ◽  
Enzyme Linked Immunosorbent Assay ◽  
Caprine Milk ◽  
Mouse Immunoglobulin

A competitive ELISA (enzyme-linked immunosorbent assay) was performed to detect and quantify bovine milk in ovine and caprine milk and cheese using a monoclonal antibody (AH4 MAb) against bovine β-casein. Ovine or caprine milk and cheese containing bovine milk were added simultaneously with the AH4 MAb to the wells of a microtiter plate that had been previously sensitized with commercial bovine β-casein. The bovine caseins in milk or cheese samples compete with the bovine β-casein bound to the plate for the AH4 MAb binding sites. Further immunorecognition of AH4 MAb bound to the bovine β-casein immobilized onto the plate was attained with rabbit anti-mouse immunoglobulin conjugated to peroxidase. Subsequent enzymic conversion of the substrate showed clear differences in absorbance values during assay of mixtures of ovine and caprine milk and cheese containing various amounts of bovine milk. The competitive ELISA developed in this work allows the quantitative detection of bovine milk in ovine and caprine milk and cheese samples in the range of 0.5 to 25% of substitution.

scholarly journals Rubella-specific IgG subclass avidity ELISA and its role in the differentiation between primary rubella and rubella reinfection

1988 ◽  
Vol 101 (3) ◽  
pp. 591-598 ◽  
Author(s):  
H. I. J. Thomas ◽  
P. Morgan-Capner
Keyword(s):  
Optical Density ◽  
Immunosorbent Assay ◽  
Primary Infection ◽  
Enzyme Linked Immunosorbent Assay ◽  
Igg Subclass ◽  
Antibody Avidity ◽  
Specific Antibodies ◽  
Specific Igg ◽  
Protein Denaturant

SUMMARYAn antiglobulin enzyme-linked immunosorbent assay for rubella-specific IgG1and IgG3was adapted to measure antibody avidity by incorporating a mild protein denaturant, diethylamine (DEA), into the serum diluent. Sera were tested at varying dilutions, both with and without DEA, if they contained sufficient specific IgG1or IgG3. The optical density (OD) was measured and curves were plotted. The highest OD (V) was noted and halved (V/2). The distance between the OD curves at V/2 was measured as the DEA shift value.Sera were examined from people whose sera contained rubella-specific antibodies as a consequence of infection or vaccination in the distant past (24 sera), recent primary rubella (66 sera), symptomatic reinfection (11 sera) or asymptomatic reinfection (64 sera). For specific IgG1the DEA shift value was <O·6 for cases of rubella in the distant past, compared with >0·8 for the first month after primary infection. The maximum DEA shift value for the sera from cases of reinfection was 0·65.No serum from cases of rubella in the distant past contained sufficient specific IgG3to estimate avidity. The sera collected within 1 month of onset of primary rubella gave DEA shift values >O·7 compared with sera from reinfections, which gave DEA shift values <O·6, except for two sera from a case of symptomatic reinfection.Thus the assessment of specific IgG subclass avidity is of value in differentiating serologically primary rubella from reinfection.

Enzyme-linked immunosorbent assay for serum amyloid A apolipoprotein with use of specific antibodies against synthetic peptides.

1988 ◽  
Vol 34 (9) ◽  
pp. 1767-1771 ◽  
Author(s):  
R Saïle ◽  
C Delpierre ◽  
P Puchois ◽  
G Hocke ◽  
C Cachera ◽  
...  
Keyword(s):  
Immunosorbent Assay ◽  
Synthetic Peptides ◽  
Solid Phase ◽  
Polyclonal Antibodies ◽  
Enzyme Linked Immunosorbent Assay ◽  
Specific Antibodies ◽  
Amyloid A ◽  
Microtiter Plates ◽  
Sensitivity Specificity ◽  
As Solid Phase

Abstract We describe a noncompetitive enzyme-linked immunosorbent assay for amyloid A apolipoprotein in human serum (apo SAA) in which specific antibodies against synthetic peptides are used. Microtiter plates were used as solid phase and coated with affinity-purified antibodies raised against SAA1-(95-104) peptide. After incubation with delipidated plasmas, the bound apo SAA was revealed by labeled antibodies raised against SAA1-(58-69) peptide. The assay offers several advantages over existing techniques: sensitivity, specificity, simplicity, and non-use of radioisotopes. Results correlate well with those by a nephelometric method in which polyclonal antibodies are used.

Lactate Dehydrogenase as Safe Endpoint Cooking Indicator in Poultry Breast Rolls: Development of Monoclonal Antibodies and Application to Sandwich Enzyme-Linked Immunosorbent Assay (ELISA)

1993 ◽  
Vol 56 (2) ◽  
pp. 120-124 ◽  
Author(s):  
MOHAMED M. ABOUZIED ◽  
CHENG HSING WANG ◽  
JAMES J. PESTKA ◽  
DENISE M. SMITH
Keyword(s):  
Monoclonal Antibodies ◽  
Lactate Dehydrogenase ◽  
Immunosorbent Assay ◽  
Polyclonal Antibodies ◽  
Sandwich Elisa ◽  
Enzyme Linked Immunosorbent Assay ◽  
Department Of Agriculture ◽  
Poultry Products ◽  
Chicken Muscle ◽  
Assay Detection

A sandwich enzyme-linked immunosorbent assay (ELISA) was developed to detect lactate dehydrogenase (LDH) as a marker protein for verifying endpoint cooking of uncured poultry products. Monoclonal antibodies were prepared against chicken muscle LDH and used with rabbit polyclonal antibodies developed against turkey or chicken muscle LDH for capture and detection in the assay, respectively. Minimum assay detection limits for turkey and chicken muscle LDH were 1 ng/ml. Turkey and chicken muscle LDH, but not LDH from other species cross reacted in the ELISA. The ELISA was further verified using extracts of turkey breast rolls processed to internal temperatures between 68.3 and 72.1°C. The LDH content of extracts diluted 3- to 6-fold was below 15 ng/ml for turkey rolls processed to 70.9 and 72.1°C. At a 6-fold dilution, LDH content of extracts from rolls processed to 69.7°C was approximately 10 times greater than those processed to 70.9°C. A survey of market precooked poultry products indicated assay validity with precooked turkey roast, but not turkey hams with maximum internal temperature requirements of 68.3°C. Results suggested the sandwich ELISA should be applicable for determining whether turkey breast rolls are processed to the required U.S. Department of Agriculture endpoint temperature of 71.1°C.

scholarly journals Rapid and Sensitive Detection of Immunoglobulin M (IgM) and IgG Antibodies against Canine Distemper Virus by a New Recombinant Nucleocapsid Protein-Based Enzyme-Linked Immunosorbent Assay

1999 ◽  
Vol 37 (4) ◽  
pp. 1049-1056 ◽  
Author(s):  
Veronika von Messling ◽  
Timm C. Harder ◽  
Volker Moennig ◽  
Peter Rautenberg ◽  
Ingo Nolte ◽  
...  
Keyword(s):  
Immunosorbent Assay ◽  
Sandwich Elisa ◽  
Immunoglobulin M ◽  
Enzyme Linked Immunosorbent Assay ◽  
Rater Agreement ◽  
Canine Distemper ◽  
Wild Type ◽  
Rt Pcr ◽  
Specific Antibodies ◽  
Type Isolate

Canine distemper morbillivirus (CDV) infection causes a frequently fatal systemic disease in a broad range of carnivore species, including domestic dogs. In CDV infection, classical serology provides data of diagnostic and prognostic values (kinetics of seroconversion) and is also used to predict the optimal vaccination age of pups. Routine CDV serology is still based on time- and cost-intensive virus neutralization assays (V-NA). Here, we describe a new capture-sandwich enzyme-linked immunosorbent assay (ELISA) that uses recombinant baculovirus-expressed nucleocapsid (N) protein of a recent CDV wild-type isolate (2544/Han95) for the detection of CDV-specific antibodies in canine sera. Recombinant antigen was produced with high efficacy in Heliothis virescens larvae. The capture-sandwich ELISA enabled a clear-cut qualitative evaluation of the CDV-specific immunoglobulin G (IgG) and IgM serostatuses of 196 and 35 dog sera, respectively. Inter-rater agreement analysis (κ = 0.988) indicated that the ELISA can be used unrestrictedly as a substitute for the V-NA for the qualitative determination of CDV-specific IgG serostatus. In an attempt to semiquantify N-specific antibodies, a one-step-dilution (alpha method) IgG-specific ELISA was implemented. Alpha values of ≥50% showed very good inter-rater agreement (κ = 0.968) with V-NA titers of ≥1/100 50% neutralizing dose (ND50) as measured against the central European CDV wild-type isolate 2544/Han95 in canine sera originating from northern Germany. An ND50 titer of 1/100 is considered a threshold, and titers of ≥1/100 indicate a resilient, protective immunity. CDV N-specific antibodies of the IgM class were detected by the newly developed ELISA in 9 of 15 sera obtained from dogs with symptoms of acute distemper. In leucocytes of 5 of the 15 dogs (all of which were also IgM positive) CDV RNA was detected by reverse transcription (RT)-PCR. The recombinant capture-sandwich ELISA detecting N-specific antibodies of the IgG class provided superior sensitivity and specificity and thus represents a rapid and cost-effective alternative to classical CDV V-NA. By detection of specific IgM antibodies, the ELISA will be complementary to RT-PCR and V-NA in the diagnosis of acute distemper infections.

Development of an Enzyme-Linked Immunosorbent Assay for the Identification of Smoked Salmon (Salmo salar),Trout (Oncorhynchus mykiss) and Bream (Brama raii)

1996 ◽  
Vol 59 (5) ◽  
pp. 521-524 ◽  
Author(s):  
ESTHER CARRERA ◽  
ROSARIO MARTÍN ◽  
TERESA GARCÍA ◽  
ISABEL GONZÁLEZ ◽  
BERNABE SANZ ◽  
...  
Keyword(s):  
Oncorhynchus Mykiss ◽  
Salmo Salar ◽  
Immunosorbent Assay ◽  
Polyclonal Antibodies ◽  
Microtiter Plate ◽  
Enzyme Linked Immunosorbent Assay ◽  
Smoked Fish ◽  
Smoked Salmon ◽  
Fish Samples ◽  
Species Specific

An indirect enzyme-linked immunosorbent assay (ELISA) has been developed for the identification of smoked meat from salmon (Salmo salar), trout (Oncorhynchus mykiss), and bream (Erama raii). The assay uses polyclonal antibodies raised in rabbits against soluble proteins of muscle from salmon (anti-SSP), trout (anti-TSP), and bream (anti-BSP) which are rendered species-specific by blocking them with the heterologous soluble muscle proteins. The blocked antibodies were used to detect the samples from smoked fish bound to the wells of a microtiter plate. Immunorecognition of polyclonal antibodies adsorbed to fish samples was made with goat anti-rabbit immunoglobulins conjugated to the enzyme horseradish peroxidase. Subsequent enzymic coversion of the substrate allowed clear species identification of smoked meat of salmon, trout, and bream.

Detection of Pseudomonas fluorescens and Related Psychrotrophic Bacteria in Refrigerated Meat by a Sandwich ELISA

1994 ◽  
Vol 57 (8) ◽  
pp. 710-714 ◽  
Author(s):  
I. GONZÁLEZ ◽  
R. MARTÍN ◽  
T. GARCÍA ◽  
P. MORALES ◽  
B. SANZ ◽  
...  
Keyword(s):  
Pseudomonas Fluorescens ◽  
Polyclonal Antibodies ◽  
Cell Envelope ◽  
Sandwich Elisa ◽  
Detection Threshold ◽  
Enzyme Linked Immunosorbent Assay ◽  
Psychrotrophic Bacteria ◽  
Specific Antigens ◽  
Protein F ◽  
Meat Samples

A sandwich enzyme-linked immunosorbent assay (ELISA) was developed for detection of Pseudomonas fluorescens and related psychrotrophic bacteria in refrigerated meat. Polyclonal antibodies were raised in rabbits against protein F from the cell envelope of P. fluorescens AH-70. The ELISA involved capturing antigens from the microorganisms present on meat samples with the anti-protein F antibodies immovilized on 96-well plates, and detecting bound antigens with the same anti-PF antibodies conjugated to biotin. Commercial ExtrAvidin-peroxidase conjugate was used to detect the biotinylated antibodies bound to their specific antigens. Subsequent enzymatic conversion of substrate gave distinct absorbance differences when assaying meat samples containing P. fluorescens strains of different origin as well as related psychrotrophic microorganisms. The detection threshold for the ELISA assay developed in this work is 105 CFU/cm2.

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