Lateral flow immunoassay (LFIA) system for measuring reombinant bovine somatotrophin (rBST) in whole milk and other dairy products

Abstract

The present invention discloses a competitive lateral flow immunoassay for detection of recombinant bovine somatotropin (rbSt) in bovine milk or a milk product or extract thereof from lactating dairy cows treated/injected with rbSt. The rbSt (analyte) in milk or its derivatives, is added to a sample pad. The rbSt mouse or rat monoclonal antibody (primary antibody) is complexed to gold nano-particles and dried down on the conjugate pad. This complex (gold conjugated to monoclonal anti-rbSt) then flows to the test and control lines by capillary action through their respective channels, as a result of the osmolarity of the wicking pad. A solution of rbSt-BSA (Bovine Serum Albumin) is striped/bound to the test line. As the conjugate is rehydrated by the sample and flows down the strip, rbSt, if present in the test sample (milk), competes with rbS-BSA at the test line for binding to the gold labeled rbSt monoclonal primary antibody. If rbSt is present in the sample, the test line signal will be lighter than if there was no rbSt. The test line signal is inversely proportional to the rbSt concentration in the milk sample. The control line contains a second antibody (IgG) directed against the primary monoclonal antibody bound to the gold particle. The test line and control lines are analyzed by a reader containing a photodiode (leelu reader.)

Claims

1. An immunological method of determining whether a lactating cow is being treated with recombinant bovine somatotropin (rbSt). a. Providing the production of an antigen to produce specific monoclonal mouse/rat antibody(s) against rbSt. b. Providing a specific monoclonal antibody(s) specific for the analyte rbSt. c. Providing a process whereby milk can be handled and processed, so that it can be tested for the presence of rbSt in milk. d. Providing a lateral-flow immunodiffusion platform for detection of rbSt in milk. e. Providing a process for milk sample analysis. The milk sample is either undiluted or diluted before exposing it to the immunoassay. The cream may be removed from the top of the milk before exposing the sample to the immunoassay. In one embodiment, the milk sample is treated to remove at least 50% of the fat and/or casein. The treatment may be by adding detergent to remove fat globules and/or precipitating the casein with acid. The monoclonal antibody (s) to be used is specific to the n-terminal methionine of the rbSt and therefore does not cross react with the native pituitary (pb St).

2. An immunological method of determining whether a thawed milk sample, dairy product, i.e. cheese, milk powder or dairy derived extract contains recombinant bovine somatotropin (rbSt) comprising the steps of: a. Providing a sample of frozen then thawed milk or its derivatives from a lactating animal treated with rbSt to be tested. b. The proposed assay will detect the presence of rbSt in whole milk, pasteurized milk, whole milk and pasteurized milk in solution/diluents, whole milk, pasteurized milk extracts and extracts of processed dairy products. c. An immune-analytical device for the rapid and simultaneous detection of recombinant bovine somatotropin (rbSt). The method is based on a lateral flow immunoassay technique performed to detect the antigen specific for rbSt in milk or derivatives of milk containing products (dairy products). d. The method comprising of: Exposing said milk sample to a test membrane having monoclonal antibody that is specific for the antigenic determinant that is the n-terminal amino acid, methionine, of rbSt.

3. The method of claim 1 wherein the cream is removed from the top of the milk before exposing.

4. The method of claim 1 wherein said milk sample is treated to remove at least 50% of the fat and/or the casein.

5. The method of claim 1 wherein said treatment comprises adding a detergent to remove milk fat globules and precipitating the casein with acid.

6. A method whereby exposed milk sample or milk component from said dairy product is exposed to a lateral flow immune diffusion platform described herein for the detection of rb St.

Description

DESCRIPTION OF FIGURES

[0078] FIG. 1 describes the purification procedure of rbSt from Posilac.

[0079] FIG. 2 describes the Leelu readings for average test line peak intensity of various concentrations of rbSt from Posilac.

[0080] FIG. 3 describes the Leelu readings for average test line/control like peak intensity of various concentrations of rbSt from Posilac.

[0081] FIG. 4 describes the average test line peak intensity Leelu readings for rbSt spiked milk.

[0082] FIG. 5 describes t describes the Leelu readings for test line/control line peak intensity Leelu spiked milk.

[0083] FIG. 6 describes the rbSt lateral flow immunoassay test strip for detecting rbSt in milk.

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