Development of a monoclonal antibody-based immunoassay for specific quantification of bovine milk alkaline phosphatase

2007 ◽  
Vol 74 (3) ◽  
pp. 290-295 ◽  
Author(s):  
Nathalie Geneix ◽  
Eric Dufour ◽  
Annie Venien ◽  
Didier Levieux
Keyword(s):  
Monoclonal Antibody ◽  
Alkaline Phosphatase ◽  
Monoclonal Antibodies ◽  
Dairy Products ◽  
Indirect Elisa ◽  
Bovine Milk ◽  
Raw Milk ◽  
Heat Stable ◽  
Nitrophenyl Phosphate ◽  
Enzymatic Methods

The detection of alkaline phosphatase (ALP) activity is used as a legal test to determine whether milk has been adequately pasteurized or recontaminated with raw milk. However, a wide variety of microorganisms produce both heat labile and heat stable ALPs which cannot be differentiated from the milk ALP by current enzymatic methods. Monoclonal antibodies specific of the bovine milk ALP were obtained in mice from a raw bovine milk ALP preparation. Coated in microtitre plates, these antibodies specifically capture the bovine milk ALP from dairy products. After washing, the enzymatic activity of the captured ALP is revealed by adding p-nitrophenyl-phosphate as a substrate. This simple immunoassay does not react with ALPs of intestinal or bacterial origin and, once optimized, was found to be the first immunoassay suitable to detect raw milk in boiled milk down to a 0·02% dilution. Moreover, in contrast with competitive indirect ELISA formats, the capture immunoassay does not require purified ALP.

Bovine milk acid phosphatase: II. Binding to casein substrates and heat-inactivation studies

1974 ◽  
Vol 41 (2) ◽  
pp. 229-237 ◽  
Author(s):  
A. T. Andrews
Keyword(s):  
Acid Phosphatase ◽  
Protein Denaturation ◽  
Bovine Milk ◽  
Raw Milk ◽  
Heat Inactivation ◽  
Heat Stable ◽  
First Order Kinetics ◽  
Nitrophenyl Phosphate ◽  
Substrate Protection ◽  
Hydrolysis Of

SummaryThe acid phosphatase of bovine milk was further purified to yield enzyme with an activity of about 2 units/mg. This was almost 105 times the activity present in milk and enabled a detailed study of heat inactivation to be made, together with further measurements on binding to casein substrates.The effectiveness of caseins as inhibitors of the hydrolysis of p-nitrophenyl phosphate by acid phosphatase paralleled the phosphate content of the casein molecules, so that αs1-casein A was a more potent inhibitor with a K1 of 1·7 mM than β-casein A1A2 (K1 = 4·3 mM), which in turn was more inhibitory than κ-casein A (K1 = 5·9 mM).The heat inactivation of acid phosphatase followed first-order kinetics at pH 4·9, 5·2 and 6·7 and values of E, the activation energy, were between 2·4×105 and 3·0×105 J mole −1 in all cases, consistent with simple protein denaturation. The presence of 1% αs1-casein A, 1% β-casein A1A2, 1% κ-casein A, 1% isoelectrically precipitated ‘whole’ casein and 1% fresh raw milk provided no substrate protection at pH 5·2 or 6·7. Acid phosphatase was somewhat less heat stable at pH 6·7 than at pH 4·9, but may be expected to survive typical milk pasteurization conditions almost completely. However, conventional milk sterilization or ultra-high-temperature (UHT) processes would be expected to give total inactivation.

Proteinase and phospholipase activities and development at different temperatures of yeasts isolated from bovine milk

2011 ◽  
Vol 78 (4) ◽  
pp. 385-390 ◽  
Author(s):  
Priscilla A Melville ◽  
Nilson R Benites ◽  
Monica Ruz-Peres ◽  
Eugenio Yokoya
Keyword(s):  
Dairy Products ◽  
Room Temperature ◽  
Bovine Milk ◽  
Raw Milk ◽  
Pathogenic Potential ◽  
Proteinase Production ◽  
Different Temperatures ◽  
Quality Of Milk ◽  
Means Of Transmission

The presence of yeasts in milk may cause physical and chemical changes limiting the durability and compromising the quality of the product. Moreover, milk and dairy products contaminated by yeasts may be a potential means of transmission of these microorganisms to man and animals causing several kinds of infections. This study aimed to determine whether different species of yeasts isolated from bovine raw milk had the ability to develop at 37°C and/or under refrigeration temperature. Proteinase and phospholipase activities resulting from these yeasts were also monitored at different temperatures. Five genera of yeasts (Aureobasidium sp., Candida spp., Geotrichum spp., Trichosporon spp. and Rhodotorula spp.) isolated from bovine raw milk samples were evaluated. All strains showed one or a combination of characteristics: growth at 37°C (99·09% of the strains), psychrotrophic behaviour (50·9%), proteinase production (16·81% of the strains at 37°C and 4·09% under refrigeration) and phospholipase production (36·36% of the isolates at 37°C and 10·9% under refrigeration), and all these factors may compromise the quality of the product. Proteinase production was similar for strains incubated at 37°C (16·81% of the isolates) and room temperature (17·27%) but there was less amount of phospholipase-producing strains at room temperature (15·45% of the isolates were positive) when compared with incubation at 37°C (36·36%). Enzymes production at 37°C by yeasts isolated from milk confirmed their pathogenic potential. The refrigeration temperature was found to be most efficient to inhibit enzymes production and consequently ensure better quality of milk. The viability of yeasts and the activity of their enzymes at different temperatures are worrying because this can compromise the quality of dairy products at all stages of production and/or storage, and represent a risk to the consumer.

Use of monoclonal antibodies to detect human placental alkaline phosphatase.

1983 ◽  
Vol 29 (1) ◽  
pp. 115-119 ◽  
Author(s):  
G De Groote ◽  
P De Waele ◽  
A Van de Voorde ◽  
M De Broe ◽  
W Fiers
Keyword(s):  
Monoclonal Antibody ◽  
Alkaline Phosphatase ◽  
Monoclonal Antibodies ◽  
Solid Phase ◽  
Enzymatic Reactions ◽  
Placental Alkaline Phosphatase ◽  
Human Placental Alkaline Phosphatase ◽  
Human Sera ◽  
Starch Gel ◽  
Alkaline Phosphatase Isoenzyme

Abstract Convenient, sensitive, and specific solid-phase immunoassays involving monoclonal antibody are described for the determination of human placental alkaline phosphatase (hPLAP). An endogenous enzyme immunoassay combined the specificity of the immunological and the enzymatic reactions. Alternatively, a solid-phase "sandwich" radioimmunoassay involving immobilized polyclonal rabbit anti-hPLAP in combination with iodinated monoclonal antibody provided some additional advantages. Both tests can be used to detect hPLAP from various sources, e.g., in human sera during pregnancy or as a tumor marker. The radioimmunoassay detected an increase in hPLAP at nine weeks of gestation. We discuss the use of monoclonal antibodies for the differentiation of different alkaline phosphatase isoenzyme types by electrophoresis on starch gel.

Fluorometric Analysis of Alkaline Phosphatase in Fluid Dairy Products

1990 ◽  
Vol 53 (7) ◽  
pp. 588-591 ◽  
Author(s):  
RICHARD M. ROCCO
Keyword(s):  
Alkaline Phosphatase ◽  
Dairy Products ◽  
Raw Milk ◽  
Test Sample ◽  
Reaction Rates ◽  
Product Formation ◽  
Test Time ◽  
Whole Milk ◽  
Repeated Analysis ◽  
Total Test Time

A new quantitative assay has been developed for measuring residual alkaline phosphatase (ALP) activity in a wide variety of dairy products including whole milk, low fat and skim milks, chocolate milk, and creams. ALP in the test sample hydrolyzes a nonfluorescent substrate, FluorophosR, to a highly fluorescent product. Product formation is monitored continuously during a short incubation period and enzyme activity is calculated from the rate of fluorescence increase. Total test time is 3 min. Reaction rates are linear up to 0.5% raw milk (equivalent to 5 μg phenol/ml/15 min) with a detection limit of 0.006% raw milk. Within and between run precision of the fluorometric method was assessed by repeated analysis of a pasteurized milk sample containing added mixed herd raw milk. The within run (N=10) mean was 190.4 mU/L, standard deviation (SD) 3.2, and a coefficient of variance (CV) of 1.7%. The procedure provides a rapid, sensitive, precise, and easy-to-use ALP assay, applicable to a wide variety of dairy products.

Fluorometric Determination of Alkaline Phosphatase in Fluid Dairy Products: Collaborative Study

1990 ◽  
Vol 73 (6) ◽  
pp. 842-849 ◽  
Author(s):  
Richard M Rocco
Keyword(s):  
Alkaline Phosphatase ◽  
Dairy Products ◽  
Collaborative Study ◽  
Skim Milk ◽  
Raw Milk ◽  
Chocolate Milk ◽  
Phenyl Phosphate ◽  
Whole Milk ◽  
Standard Deviations

Abstract Official methods for the measurement of alkaline phosphatase (ALP) in dairy products use either phenyl phosphate or phenolphthaleln monophosphate as substrate. Quantitation of results requires butanol extraction of the Indophenol (Scharer) or 3-h dialysis of the liberated phenolphthaleln (Rutgers). The Advanced Fluorophos® assay Is based on a self-indicating substrate which, when acted upon by ALP, loses a phosphate radical and becomes a highly fluorescent compound. The rate of fluorophore formation Is monitored for 3 mln In a fluorometer and the enzyme activity In mU/L Is calculated. Eight laboratories participated in a collaborative study to evaluate the Fluorophos® assay for determining ALP activity In whole milk, skim milk, chocolate milk, and cream (half and half). The comparative method was the AOAC quantitative phenyl phosphate method, 16.121-16.122 (14th Ed.). Mixed herd raw milk was added to pasteurized samples at 0.05, 0.1, and 0.2% (v/v). Method performance at 0.1% (v/v) added raw milk as measured by repeatability and reproducibility standard deviations (sr and sR) and relative standard deviations (RSDr and RSDR), respectively, were: whole milk, sr = 21.7%, sR = 34.6%, RSDr = 4.4%, RSDR = 7.0%; skim milk, sr = 19.2%, sR = 31.4%, RSDr = 3.8%, RSDR = 6.2%; chocolate milk, sr = 27.6%, sR = 45.8%, RSDr = 5.3%, RSDR = 8.8%. The method has been adopted official first action by AOAC for determination of alkaline phosphatase in whole milk, skim milk, and chocolate milk.

Monoclonal Antibody Detection of Porcine Meat

1994 ◽  
Vol 57 (2) ◽  
pp. 146-149 ◽  
Author(s):  
P. MORALES ◽  
T. GARCÍA ◽  
I. GONZÁLEZ ◽  
R. MARTÍN ◽  
B. SANZ ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Monoclonal Antibodies ◽  
Bovine Serum Albumin ◽  
Horseradish Peroxidase ◽  
Indirect Elisa ◽  
Cross Reactivity ◽  
Antibody Detection ◽  
Muscle Proteins ◽  
Elisa Format ◽  
Soya Proteins

A stable hybridoma cell line (DD9) has been produced secreting a monoclonal antibody specific for porcine muscle proteins. The DD9 monoclonal antibody (mAb) failed to show a significant cross-reactivity when tested against beef, horse, chicken, and soya proteins, as well as bovine caseins, gelatin, and bovine serum albumin. The DD9 mAb was further used in an indirect ELISA format for detection of defined amounts of porcine meat (1–100%) in beef and chicken meat mixtures immobilized on 96-well plates. Immunorecognition of monoclonal antibodies adsorbed to porcine meat was made with rabbit anti-mouse immunoglobulins conjugated to the enzyme horseradish peroxidase.

Evaluation of Alkaline Phosphatase Detection in Dairy Products Using a Modified Rapid Chemiluminescent Method and Official Methods

2011 ◽  
Vol 74 (7) ◽  
pp. 1144-1154 ◽  
Author(s):  
S. M. ALBILLOS ◽  
R. REDDY ◽  
R. SALTER
Keyword(s):  
Public Health ◽  
Alkaline Phosphatase ◽  
Dairy Products ◽  
Limit Of Detection ◽  
Raw Milk ◽  
Modified Method ◽  
European Public ◽  
Single Laboratory ◽  
Chemiluminescent Method ◽  
The U.S

Alkaline phosphatase is a ubiquitous milk enzyme that historically has been used to verify adequate pasteurization of milk for public health purposes. Current approved methods for detection of alkaline phosphatase in milk include the use of enzyme photoactivated substrates to give readings in milliunits per liter. The U.S. and European public health limit for alkaline phosphatase in pasteurized drinks is 350 mU/liter. A modified chemiluminescent method, fast alkaline phosphatase, was compared with the approved fluorometric and chemiluminescent alkaline phosphatase methods to determine whether the modified method was equivalent to the approved methods and suitable for detecting alkaline phosphatase in milk. Alkaline phosphatase concentrations in cow's, goat's, and sheep's milk and in flavored drinks and cream were determined by three methods. Evaluations in each matrix were conducted with pasteurized samples spiked with raw milk to produce alkaline phosphatase concentrations of 2 to 5,000 mU/liter. The tests were performed by the method developer and then reproduced at a laboratory at the National Center for Food Safety and Technology following the criteria for a single laboratory validation. The results indicated that the fast alkaline phosphatase method was not significantly different from the approved chemiluminescent method, with a limit of detection of 20 to 50 mU/liter in all the studied matrices. This modified chemiluminescent method detects alkaline phosphatase in the 350 mU/liter range with absolute differences from triplicate data that are lower and within the range of the allowed intralaboratory repeatability values published for the approved chemiluminescent method.

Antibiotic Residue Detection in Milk - A Review

1984 ◽  
Vol 47 (8) ◽  
pp. 647-652 ◽  
Author(s):  
J. R. BISHOP ◽  
C. H. WHITE
Keyword(s):  
Monoclonal Antibody ◽  
Dairy Products ◽  
Dairy Industry ◽  
Raw Milk ◽  
Residue Detection ◽  
The Past ◽  
Antibiotic Residue ◽  
Antibody Technology

Methods for antibiotic residue detection in dairy products, especially raw milk, have greatly improved as to their rapidity, accuracy and sensitivity over the past 30 years. An assay requiring overnight coagulation was available in the mid-1950's, whereas now there is an immunologically-based test using monoclonal antibody technology requiring only 6 min. These advances have not come about without extensive research efforts. The following is an overview of the developments and their significance to the dairy industry.

Heat-Stable Proteases from Psychrotrophs in Milk

1980 ◽  
Vol 43 (3) ◽  
pp. 197-200 ◽  
Author(s):  
A. GEBRE-EGZIABHER ◽  
E. S. HUMBERT ◽  
G. BLANKENAGEL
Keyword(s):  
Molecular Weight ◽  
Gel Electrophoresis ◽  
Dairy Products ◽  
Raw Milk ◽  
Gram Negative ◽  
Psychrotrophic Bacteria ◽  
Heat Resistant ◽  
Heat Stable ◽  
Single Enzyme

Twelve gram-negative psychrotrophic bacteria producing heat-resistant proteases that hydrolyzed casein were isolated from refrigerated raw milk. All were pseudomonads and the enzymes of the six most proteolytic cultures were examined further. The proteases were partially purified, and gel electrophoresis indicated that only a single enzyme was present in the preparation. The molecular weight of most of the proteases was approximately 45,000. All six enzymes retained some activity after being heated at 121 C for 10 min and casein was hydrolyzed at pH levels found in normal milk and many cultured dairy products. Although proteolysis was highest at about 40 C, considerable activity was evident at refrigeration temperatures.

scholarly journals Preparation and use of monoclonal antibodies  to detect Phytophthora nicotianae  var. nicotianae

1999 ◽  
Vol 35 (No. 2) ◽  
pp. 41-46
Author(s):  
B. Pekárová-Kyněrová ◽  
M. Kutíková
Keyword(s):  
Monoclonal Antibody ◽  
Monoclonal Antibodies ◽  
Fusarium Oxysporum ◽  
Indirect Elisa ◽  
Phytophthora Nicotianae ◽  
Clavibacter Michiganensis ◽  
Tomato Plants ◽  
Clavibacter Michiganensis Subsp

A monoclonal antibody (MAb 18) was prepared against purified mycelial proteins from Phytophthora nicotianae var. nicotianae. The specificity of MAb 18 (lgG class) was tested using indirect ELISA (PTA-ELISA).It cross-reacted with Phytophthora cacto­ rum, P. cinrzamomi, P. cryptogea, P. fragariae) but not with other fungi (Fusarium  oxysporum, Pythium ultimwn and P. oligan­ drwn) and bacteria (Clavibacter michiganensis subsp. michiganensis) isolated from tomato. Phytophthora nicotianae var. nicotianae was detected in roots and basal stems of artificially infected young tomato plants using indirect ELISA and immunoprinting.

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