US 4,347,312 AGranted

Detection of antibiotics in milk

AssigneeResearch Triangle Institute

InventorsRodney J. Brown, Harold E. Swaisgood

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Abstract

A method for detecting the presence of antibiotics in milk which comprises the steps of: (a) contacting a solid matrix having attached thereto a purified immobilized antibody with a volume of milk and an enzyme-labeled antibiotic, said antibody being specific to said antibiotic; (b) separating the matrix from the milk and rinsing the matrix with water to remove excess milk and enzyme-labeled antibiotic; (c) contacting the rinsed matrix with a substrate, said substrate in the presence of said enzyme-labeled antibiotic exhibiting a color change the amount of which is quantitatively related to the amount of enzyme-labeled antibiotic; and (d) measuring the amount of antibiotic present in said milk by comparing the color change in said substrate with a standard. Also provided is a method for producing purified antibodies for use in the foregoing detection method by: (a) convalently conjugating an antibiotic having a lactam-ring in the molecule to a protein capable of binding thereto through said lactam-ring; (b) injecting into a host animal capable of raising antibodies specific to said antibiotic the conjugate obtained in step (a) so as to raise said specific antibodies; (c) covalently conjugating the same antibiotic in step (a) to a second protein capable of binding thereto through said lactam-ring and different than the protein used in step (a) to form a second conjugate; (d) covalently binding said second conjugate to a solid matrix to form an affinity matrix for purifying the antibodies; (e) isolating and purifying the specific antibodies raised in step (b) by contacting the host animal serum with the affinity matrix; and (f) recovering the specific antibodies in pure form.

Claims14

Dependent claims

A method for detecting the presence of antibiotics having a lactam ring in milk which comprises the steps of:

(a) contacting a solid matrix having attached thereto a purified immobilized antibody with a volume of milk and an enzyme-labeled antibiotic having a lactam ring, said antibody being specific to said antibiotic and said enzyme-labeled antibiotic being covalently conjugated to the antibiotic through a carboxy group in the antibiotic molecule;
(b) separating the matrix from the milk and rinsing the matrix with water to remove excess milk and enzyme-labeled antibiotic;
(c) contacting the rinsed matrix with a substrate, said substrate in the presence of said enzyme-labeled antibiotic exhibiting a color change the amount of which is quantitatively related to the amount of enzyme-labeled antibiotic;
and
(d) measuring the amount of antibiotic present in said milk by comparing the color change in said substrate with a standard, said purified immobilized antibody having been prepared according to the steps which comprise:
(i) covalently conjugating an antibiotic having a lactam-ring to a protein through said lactam-ring;
(ii) injecting into a host animal capable of raising antibodies specific to said antibiotic the conjugate obtained in step (i) so as to raise said specific antibodies;
(iii) covalently conjugating the sme antibiotic as used in step (i) to a second protein capable of binding thereto through said lactam-ring and different than the protein used in step (i) to form a second conjugate;
(iv) covalently binding said second conjugate to a solid matrix to form an affinity matrix for purifying the antibodies;
(v) isolating and purifying the specific antibiodies raised in step (ii) by contacting the host animal serum with the affinity matrix;
and
(vi) recovering the specific antibodies in a pure form and immobilizing the purified antibodies on a solid matrix.

9 more elements

Dependencies are inferred from the claim text.