Microbiological Assay Procedures for Antibiotic Residues - ACS

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Microbiological Assay Procedures for Antibiotic Residues

Downloaded by UNIV OF CALIFORNIA SAN DIEGO on March 7, 2016 | http://pubs.acs.org Publication Date: September 18, 1986 | doi: 10.1021/bk-1986-0320.ch013

Stanley E. Katz Department of Biochemistry and Microbiology, Rutgers University—The State University of New Jersey, New Brunswick, NJ 08903

The c l a s s i c a l microbial assay approaches to measuring a n t i b i o t i c residues, d i f f u s i o n , turbidimetric and acid production were described and the advantages and l i m i tations reviewed. Other systems so discussed and r e viewed were the affinity or receptor methods and the immunological approach using ELISA or EMIT assay techniques. The c l a s s i c a l systems, i n general, could measure a n t i b i o t i c residues at the f r a c t i o n a l ppm to the ppb l e v e l s . The potentials of the receptor and immunological assay system were discussed.

The appearance o f a n t i b i o t i c r e s i d u e s i n f o o d p r o d u c t s o f a n i m a l o r i g i n a r e f o r t h e most p a r t t h e r e s u l t o f improper and c a r e l e s s usage, from d e l i b e r a t e and i n t e n t i o n a l misusage, from t h e improper f o r m u l a t i o n o f a n i m a l f e e d i n g m a t e r i a l s and from t h e i g n o r i n g o f proper withdrawal times. The paths o f misuse o f a n t i b i o t i c s i n a n i m a l a g r i c u l t u r e c a n be a s v a r i e d as t h e i m a g i n a t i o n o f man can d e v i s e ; a l l however, a r e based upon economic needs o r p e r c e i v e d needs t o p r e v e n t d i s e a s e and/or t o t r e a t r e c a l c i t r a n t i n f e c t i o n s . The a n a l y s i s f o r a n t i b i o t i c r e s i d u e s i n e d i b l e a n i m a l t i s s u e , eggs, m i l k , t r a d i t i o n a l l y , h a s been performed u s i n g m i c r o b i o l o g i c a l a s s a y t e c h n i q u e s . P r i m a r i l y , t h e s e a s s a y p r o c e d u r e s were i n h i b i t i o n a s s a y s u t i l i z i n g t h e a g a r d i f f u s i o n systems (1_). M i c r o b i o l o g i c a l a s s a y p r o c e d u r e s measure a c t i v e s p e c i e s , i . e . , t h o s e t h a t c a n i n h i b i t t h e growth o f m i c r o o r g a n i s m s . Metabolic products that are n o t i n h i b i t o r y , a r e n o t measured; c o n j u g a t e d a n t i b i o t i c s , t y p i c a l l y Phase I I m e t a b o l i t e s , u s u a l l y w i l l n o t be d e t e c t e d . D e t e c t i o n o f such m e t a b o l i c p r o d u c t s r e q u i r e s h y d r o l y s i s o f t h e c o n j u g a t e p r i o r to the assay. O v e r a l l , m i c r o b i o l o g i c a l a s s a y methods have been t h e most s e n s i t i v e o f a l l a s s a y systems and t h e a b i l i t y t o measure r e s i d u e s i n t h e ppb t o ppm range i s common and h a s been f o r over 20 y e a r s ( 1 ) . Howe v e r , most o f t h e r e s i d u e a s s a y systems l a c k s p e c i f i c i t y and r e q u i r e c o n f i r m a t i o n by s p e c t r a l systems f o r a p r o p e r i d e n t i f i c a t i o n o f t h e i n d i v i d u a l a n t i b i o t i c or the a n t i b i o t i c family. There a r e many advantages t o t h e u s e o f m i c r o b i a l a s s a y 0097-6156/ 86/ 0320-0142$06.00/ 0 © 1986 American Chemical Society

Moats; Agricultural Uses of Antibiotics ACS Symposium Series; American Chemical Society: Washington, DC, 1986.


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methods. With rare exception, these procedures are simple to perform, possess a f a i r degree of precision and accuracy f o r the species they measure and require simple equipment to perform. Unfortunately, microbial systems are usually slow labor-intensive and require overnight incubations and multiple platings and measurements to achieve the ± 25% to ± 35% precision at the ppm to ppb concentration. There are several ways to subdivide the a n a l y t i c a l systems encompassing the area defined as microbial assay procedures. These are: Diffusion Systems cylinder-plate well-plate pad-plate Turbidimetric Systems Competitive Receptor Assays Immunological Systems Diffusion Systems Diffusion systems are based upon the a b i l i t y of the a n t i b i o t i c to d i f f u s e through agar and cause the i n h i b i t i o n of the sensitive assay s t r a i n s . Since the substrate to be assayed i s applied i n a "point source," d i f f u s i o n occurs r a d i a l l y . A c i r c u l a r zone of i n h i b i t i o n forms and the size of the zone i s a function of the concentration. This function i s expressed as a linear relationship between the size of the zone of i n h i b i t i o n and the logarithm of the concentration. By comparing the measurable zone with a standard response l i n e , the concentration of the d i l u t i o n can be determined and the potency of the sample may be calculated. For a complete discussion of the mechanics of d i f f u s i o n , the formation of the zone edge, and the relationships between concentration and zone size, the reader should r e f e r to Kavanagh s c l a s s i c text 02). f

The Cylinder-Plate Procedure. In this procedure the substance being assayed diffuses from cylinders placed upon a uniform thickness of seeded agar, f i l l e d or charged with a fixed volume of the analyte, or reference standards or a series of standard solutions. The p e t r i dishes are incubated at a predetermined temperature and the zones of i n h i b i t i o n measured to the nearest 0.1 mm. The Cup-Plate or Well Procedure. This procedure i s similar to the cylinder-plate system except that wells are cut into the agar with cutters capable of cutting uniform, completely c i r c u l a r wells. As with the cylinder-plate assays, the wells are f i l l e d . Zones are measured after incubation and the concentration determined u t i l i z i n g a comparison with a standard response l i n e . The Pad-Plate Procedure. The pad-plate approach u t i l i z e s f i l t e r paper discs saturated with solution of the analyte as the reservoir. In a l l other respects, the system i s i d e n t i c a l to the other d i f f u s i o n systems. The advantages of the d i f f u s i o n system are: ( i ) variations are adaptable to provide reasonably sensitive assays, ( i i ) the approach i s adaptable to assay most i f not a l l a n t i b i o t i c s , and ( i i i ) the



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analyte solution need not be s t e r i l e or treated s p e c i a l l y . The disadvantages are: ( i ) f i l l i n g cylinder or wells or saturating and placing pads on agar pads i s labor intensive, slow and tedious, ( i i ) most assays require overnight incubation and hence any assay covers a two-day period, and ( i i i ) the pad-plate v a r i a t i o n i s the least sensitive usually capable of measuring ug/mL quantities; i n comparison, the cylinder or well v a r i a t i o n can measure ng/mL l e v e l s , suspended materials interfere i n the cylinder-plate system by plugging the bottoms of the cylinder and l i m i t i n g d i f f u s i o n ; i n contrast the well system i s unaffected since the analyte solution diffuses only horizontally rather than v e r t i c a l l y and horizontally. Several factors affect the d i f f u s i o n assay and must be cont r o l l e d c a r e f u l l y . The depth of the agar i n the cylinder-plate system must be minimal, as thin as possible and as uniform as possible to maximize d i f f u s i o n of the analyte. The temperature of incubators must be uniform throughout and should not vary more than ± 0.2°C. The vegetative assay organism must be sensitive to the analyte, be stable (resistant to spontaneous change), be i n the logarithmic growth phase (for uniformity of response), and be e a s i l y cultured, maintained and standardized. Spores suspension have similar c r i t e r i a except that the spores must be capable of germinating with reasonable synchrony. U t i l i z i n g the d i f f u s i o n assay systems, primarily the cylinderplate procedure, the following l i m i t s of detection and measurement are r e a l i s t i c . Table I. Detection and Measurement Levels of A n t i b i o t i c Residues i n Products of Animal Origin Using Diffusion Assays ReferAnimal Dairy Muscle ences Eggs Products or units/g or mL Penicillins 0.005-0.01 0.01-0.02 0.01-0.02 0.025-0.03 (1) (313) Streptomycins 0.06 -0.10 0.20-0.40 0.20-0.40 0.30 -0.50 (1) (14) (15) Chlortetracycline 0.005-0.10 0.02-0.03 0.02-0.03 0.02 -0.04(1X16-20) Oxytetracycline 0.025-0.03 0.08-0.10 0.08-0.10 0.08 -0.10 U ) (2122) Chloramphenicol 0.025-0.05 0.10-0.20 0.025-0.05 (1) (23) Neomycin 0.05-0.10 0.25-0.50 0.20 -0.30 (1) (2425) Erythromycin 0.025-0.05 0.10-0.20 0.10-0.20 0.10 -0.20 (1) (26Antibiotic

Milk

-17) Some other a n t i b i o t i c s commonly used i n animal production such as the b a c i t r a c i n s , bambermycins and virginiamycins as well as the streptomycins are poorly absorbed from the i n t e s t i n a l tract and residues usually do not occur from feeding. Chloramphenicol i s used i l l e g a l l y i n the United States i n many species; i t i s used l e g a l l y i n Europe, Canada and other parts of the world. The maximum s e n s i t i v i t y (the lower l i m i t of detection and measurement) that can be achieved for any d i f f u s i o n procedure i s a function of the response of the test organism to the a n t i b i o t i c being assayed. In order to increase the s e n s i t i v i t y of such



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procedures, an extraction system must be devised to concentrate the a n t i b i o t i c . Solvent extraction and concentration, and subsequent p a r t i t i o n i n g into a suitable buffer has not achieved any large degree of success simply because of complications from co-extraction of interferences. Use of column concentration/clean-up techniques also has not been exploited since there appears to be l i t t l e advantage to i t , at present. Interferences have been handled, t r a d i t i o n a l l y , by the use of a matrix compensation response curve. B a s i c a l l y , the system i s a series of standard additions to samples of a matrix and the use of these supplementations as the standards i n a response curve. Thus, the recoveries of a n t i b i o t i c s , affected p o s i t i v e l y or negatively, can be corrected for matrix e f f e c t s over a wide range of concentrations. Absolute recoveries are, of course, determined against standards i n buffer. Extractions t r a d i t i o n a l l y have been performed using buffers (jL); the same used to obtain the maximum response i n standard curves. Unfortunately t h i s has been a major f a i l i n g of the plate d i f f u s i o n assay systems. I t i s rare that the pH can be adjusted to the optimum necessary for greatest response simply by blending a matrix with buffer. As much as a 30 to 40% loss of a c t i v i t y can occur by not adjusting the pH properly; analysis f o r residues of the streptomycins and erythromycin, f o r example, can y i e l d results 20% lower by having the pH of the analyte 0.2 units below 8.0; i f the pH i s 0.5 units below 8.0, the loss of potency approaches 50% (14-15). The conventional assay systems (1) include d i l u t i o n s of 1:2 to 1:5, as part of the extraction. Hence, the levels of detection are limited. The use of minimum amounts of extractant coupled with the physical removal of s o l i d s can improve the l i m i t s of detection and measurement. Again, i t i s important to r e i t e r a t e one important fact, only free a n t i b i o t i c i s measured. Bound residues are rarely measured d i r e c t l y using these assays. Another problem with a l l such assays i s the supplementation system. The assumption that the simple addition of drug to a matrix followed by analysis was r e f l e c t i v e of the problems of assaying for a n t i b i o t i c residues i s s i m p l i s t i c and does not address the o v e r a l l problem of assaying f o r a n t i b i o t i c residues. Turbidimetric Systems The methodology i s based upon the relationship between the increasing concentrations of an a n t i b i o t i c and the resulting i n h i b i t i o n of the growth of a microorganism as measured by the development of t u r b i d i t y . The presence of increasing amounts of a n t i b i o t i c i n the assay medium result i n an increasing i n h i b i t i o n of growth. By comparing the response of the assay organism exposed to an unknown quantity of a n t i b i o t i c with the response found from known concent r a t i o n s , the potency of the a n t i b i o t i c i n the sample can be determined (absorbance vs concentration). The procedure requires that the standards and the samples be assayed under exactly the same cond i t i o n s . The most general method u t i l i z e d i s based upon the growth rate. This involves a short (usually 3-4 h) incubation period after which the incubation i s terminated and the absorbance (turbidity)




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measured i n a suitable spectrophotometer, using a flow-through c e l l system. Required for this assay are medium control, uniform test organism seeding, incubation temperature control, and a quenching system to cause the cessation of growth 02). This approach has certain advantages over the d i f f u s i o n system; i t i s more sensitive to low concentrations and the assay i s rapid. However, the l i m i t a t i o n s precluded t h i s approach from widespread a p p l i c a t i o n . Extracts of tissue or body f l u i d s are often turbid or have i n t e r f e r i n g colors and can cause errors. Solvents can i n t e r fere much more i n the turbidimetric systems than i n d i f f u s i o n systems. Surprisingly, s t e r i l i t y i s not a s i g n i f i c a n t problem unless the samples contain very large numbers of organisms. If one inoculates the assay medium to y i e l d a density of 1 x 10 organisms/ mL, i n 4 h assuming no lag phase; the organism concentration would be 4 x 10® organisms/mL (assuming no i n h i b i t o r y material and an organism generation time of 20 min). At 10 organisms/mL there i s minimal measurable t u r b i d i t y . Only i f a rapid growing organism i s present i n large numbers i n the sample extract would an interference be noted. 5

5

Competitive Receptor Assays This assay, commonly referred to as the Charm Test, i s based upon the a f f i n i t y of a n t i b i o t i c s for s p e c i f i c s i t e s on the c e l l wall of microorganisms and the i r r e v e r s i b l e binding of the a n t i b i o t i c to these s i t e s . By adding C - l a b e l l e d or H - l a b e l l e d a n t i b i o t i c to a sample of milk, urine or the aqueous extract of tissues together followed by microbial binding s i t e s and measuring the quantity of the l a b e l l e d a n t i b i o t i c that binds to the microbial s i t e s , the a n t i b i o t i c residue can be measured. The competition f o r receptor s i t e s prevents the radiolabelled a n t i b i o t i c from binding. Thus the more radiolabelled a n t i b i o t i c bound, the less a n t i b i o t i c i n the sample. The Charm Test was i n i t i a l l y applied to the analysis of 3lactam residues i n milk although i t s application to the analysis of body, f l u i d s , meat extracts, and fermentation broths was indicated. There appears to be no rationale why t h i s basic procedure cannot be applied to a l l types of matrices (water, s o i l , animal feeds, premixes). The primary application of the procedure i s the determination of the presence or absence of 3-lactam (7) residues i n milk and secondarily to measure the levels quantitatively. The receptor assay system has now been expanded to q u a l i t a t i v e l y detect residues of t e t r a c y c l i n e , erythromycin, streptomycin, chloramphenicol, novob i o c i n , and sulfamethazine i n milk, serum and urine (Table II) (30). B a s i c a l l y the procedure to detect 3-lactam residues i n milk i s remarkably simple. A 5 mL sample of milk i s used. To this i s added the C - l a b e l l e d 3-lactam and the b a c t e r i a l receptor s i t e s . The mixture i s incubated f o r 4 min at 85°C to complete the competition f o r receptor s i t e s and centrifuged. The supernatant i s discarded, the p e l l e t i s washed gently so as not to disturb the p e l l e t . The p e l l e t i s resuspended i n water and s c i n t i l l a t i o n f l u i d i s added. For quantitative work, the sample i s counted for 5 min, for screening purposes 1 min. The l a b e l l e d a n t i b i o t i c s contain either a H- or C - l a b e l . ll+

3

1I+

3

ll+


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An entire a n t i b i o t i c screen can be carried out using 4 samples with the assay time being 15 min to 1 h depending upon whether the q u a l i tative or quantitative mode i s desired. Table I I . Limits of Detection and Measurement of A n t i b i o t i c s


Milk A n t i b i o t i c Family Penicillins Tetracyclines Macrolides Aminoglycosides Chloramphenicol Sulfonamides Novobiocin

0.0025 0.25 0.05 0.025 0.020 0.025 0.010

Serum ugs or units/mL 0.0025 0.25 0.05 0.10 0.50 0.25 —

Urine 0.0025 — 0.05 0.10 — 0.25 —

From these data the p o t e n t i a l of the receptor assay i s evident. Comparison with the microbial d i f f u s i o n assay system, Table I, i n d i cates that the levels of detection and measurement are reasonably s i m i l a r . The receptor assay has the added v i r t u e of allowing for the completion by the analysis within an hour, generally, rather than several hours or the next day. Miscellaneous Assays f o r Residues of A n t i b i o t i c s i n Milk In conjunction with the discussion of the receptor assay system, i t i s l o g i c a l to discuss the variations of the plate assay systems and/or growth systems using colorimetric indicators of i n h i b i t i o n of metabolism or growth. Disc Assay - This i s the simplist of the procedures and involves the placing of a standard 1/2" disc saturated with milk onto the surface of IS. stearothermophilus seeded agar plate and co-incubating with suitable control discs at 55° or 64°C u n t i l w e l l defined zones of i n h i b i t i o n are obtained, usually 3-4 h. Confirmation using p e n i c i l l i n a s e - t r e a t e d milk i s required. Zones 14.0 mm are p o s i t i v e . The lower l i m i t of detection i s 0.008 units penicillin/mL. This type of assay i s simple, reasonably rapid and reasonably sensitive. Quantitation i s possible by using graded concentrations of p e n i c i l l i n i n the control milk. The technique i s limited, however, to 3-lactam a n t i b i o t i c s , primarily p e n i c i l l i n (8). A v a r i a t i o n of the disc assay i s the quantitative estimate using a central point. Each p e t r i dish contains three reference discs which contain 0.016 units penicillin/mL and three discs saturated with the unknown milk. A p e n i c i l l i n a s e disc i s placed i n the center of each plate to help confirm the presence of p e n i c i l l i n . Three plates are used for each milk sample; 13. stearothermophilus i s the assay organism. After incubation for 2-4 h, zones are measured and compared to the diameters of the reference concentration. V a l i d i t y of the difference between zone size of the reference and sample i s determined s t a t i s t i c a l l y . This procedure i s less sensitive and attempts to set the qualitative presence of 3-lactams at 0.016 units/mL rather than at lower levels (3).

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Moats; Agricultural Uses Antibiotics Washington, D.C.of 20036 ACS Symposium Series; American Chemical Society: Washington, DC, 1986.


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Colorimetric Assay f o r 3-Lactams The system discussed, commercially known as the Delvotest (9-11), u t i l i z e s a s t r a i n of B^. stearothermophilus which grows at a very rapid rate and produces acid from the nutrients i n the medium i n the absence of i n h i b i t o r y substances. When i n h i b i t o r y substances such as the 3-lactam a n t i b i o t i c s are present, acid production i s i n h i b i t e d By incorporating bromcresol purple i n the medium, i t i s easy to observe acid production, a change from purple to yellow. Incubation i s performed at 65°C f o r approximately 3 h. Table I I I shows the actual combination of colors that can be obtained and t h e i r interpretation. Table I I I . Interpretation of Delvotest Results

Sample Color Yellow Purple or Purple Yellow Purple Purple/Yellow Purple

Heated Confirm —

Penicillinase Treated —

Interpretation -

Yellow

Yellow

+

Purple Purple/Yellow Purple

Purple Purple/Yellow Yellow

4-

The levels of detection are quite good and are shown f o r a number of dairy products i n Table IV. Table IV. Limits of Detection of 3-Lactams i n Milk Using the Delvotest System Milk Type Raw Skim Low fat 1-2% Homogenized Half and Half

Units Penicillin/mL 0.004-0.005 0.006 0.004-0.005 0.004-0.006 0.007

Other miscellaneous assays f o r p e n i c i l l i n or other 3-lactams in milk i s the Penzyme Test which uses c e l l wall enzymes inhibited by 3-lactam drugs i n a k i n e t i c assay. This test system i s purported to be able to detect 0.005 units penicillin/mL and requires approximately 30 min to complete. I t , l i k e many other assays, detects 3-lactam a n t i b i o t i c s only. Application of Delvotest or the disc assay systems to detecting other a n t i b i o t i c s i n milk has not been successful. Only the receptor assay system appears to be v e r s a t i l e and p o t e n t i a l l y applicable to determine the presence of d i f f e r e n t a n t i b i o t i c residues i n d i f f e r e n t matrices. Immunological Systems M i c r o b i o l o g i c a l l y based assay systems invariably measure the active a n t i b i o t i c ( s ) or forms of the a n t i b i o t i c that can be i n h i b i t o r y to microorganisms. Immunological assays can measure both the active a n t i b i o t i c as well as m i c r o b i o l o g i c a l l y inactive species.


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Immunological assays measure those moieties that can cause an a n t i genic response. For the most part, immunological assays should not be interfered with by a n t i b i o t i c s from the other a n t i b i o t i c families, the s p e c i f i c i t y of the antibodies being vaguely similar to the s p e c i f i c i t y of enzyme systems. The basic p r i n c i p l e governing immunoassays for a n t i b i o t i c s i s indicated i n the following reaction:


Ag Antigen

+

Ab Antibody

• *c

=*» Ag:Ab » Antigen:Antibody Complex

This reaction i s an equilibrium reaction and w i l l continue u n t i l the concentration of antigen i n both the free and complexed form becomes a constant (30). Applications of the immunological systems are limited by the a b i l i t y to develop suitable antibodies. Most a n t i b i o t i c s are r e l a t i v e l y small molecules having molecular weights under 500. Hence these molecules must be complexed with some c a r r i e r protein to create a molecule that can evoke the immune response and the development of antibodies. An antibody i s produced when the antigen carrying a number of antigenic determinants i s introduced into an animal s body. Lines of B c e l l s mature into plasma c e l l s and each produces an immunoglobulin molecule that f i t s a single determinant or a segment of the determinant. In a conventional sense, antibodies are polyclonal proteins because they can be directed against several other components rather than against the antigen alone. Separation of the d i f f e r e n t antibodies i n a polyclonal mixture i s extremely d i f f i c u l t i f not impossible. In contrast, monoclonal antibodies are directed against a s p e c i f i c antigen or a s p e c i f i c segment of the antigenic molecule. Kohler and M i l s t e i n (31) revolutionized immunology by demons t r a t i n g that antibody producing c e l l s (spleen c e l l s ) when fused with malignant mouse myeloma c e l l s produced hybrid c e l l lines whose c e l l s produced only a single antibody. These hybrid c e l l s , known as hybridomas,were e s s e n t i a l l y immortal meaning that they could be grown i n c e l l culture. A complete discussion of the production of antibody by hybridoma i s given by Goding (32). T

Immunological Techniques f o r Analyzing A n t i b i o t i c s Over the l a s t 8-10 years, monoclonal as well as conventional a n t i body techniques have gained popularity f o r the analysis of hormones, various drugs, proteins, bacteria, viruses and parasites. Application of immunological systems f o r the analysis of drug and a n t i b i o t i c residues has lagged because of the general lack of f a m i l i a r i t y with the p r i n c i p l e s of immunology, the d i f f i c u l t i e s i n producing stable reagents, and the d i f f i c u l t i e s i n developing methods using the crude material i n i t i a l l y available. Agglutination. The agglutination assay or the passive hemagglutination i n h i b i t i o n assay i s based upon the least amount of soluble antigen necessary to i n h i b i t agglutination or the clumping of c e l l s that occurs following the union of antigen and antibody.



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A n a l y t i c a l l y t h i s i s the amount of antigen i n the l a s t tube of a d i l u t i o n series that w i l l give a wide ring agglutination pattern. Tubes containing less antigen than this tube allow agglutination to occur. I t i s quite common to use a two-fold d i l u t i o n sequence obviously the greater the i n t e r v a l between concentration, the greater the inaccuracy. The converse i s also evident, the narrower the range, the greater the accuracy. Application of this technique i s very limited for assaying a n t i b i o t i c residues. Steiner (33) developed such a model system u t i l i z i n g gentamicin as the p i l o t a n t i b i o t i c i n matrices such as urine, blood serum, milk and animal feeds. The procedure was r e l a t i v e l y simple. A measured volume of a gentamicin-treated red c e l l suspension was added to the previously mentioned gentamicin supplemented matrices. A fixed volume of gentamicin antibody was added and the mixture incubated at room temperature for 30 to 60 min. The hemagglutination reactions were observed and the concentration of a n t i b i o t i c determined. The l i m i t s of this system for gentamicin were 0.4 ppm f o r chicken serum, burnine, 1.9 ug/mL for milk and 20 g/ton for feeds. Although these l e v e l s are not e s p e c i a l l y s e n s i t i v e , the hemagglutination offers two d i s t i n c t advantages, speed of analysis and s p e c i f i c i t y . The t o t a l assay usually can be completed within 2 h with no observable i n t e r ferences from other a n t i b i o t i c s . Radioimmunoassay. Radioimmunoassay (RIA) was f i r s t described by Berson and Yalow (34) and Luft and Yalow (35). The assay i s based upon the competition for an antibody between a radiolabelled antigen and i t s unlabelled counterpart. The greater the amount of unlabelled antigen i n the test sample, the less radiolabelled antigen bound. The concentration of antigen i n a test sample can be determined from comparisons with standard curves. The primary application of RIA for a n t i b i o t i c s has been i n the medical area, and primarily for a n t i b i o t i c s not used i n agriculture. Assays have been developed f o r gentamicin tobramycin, sisomicin, n e t i l m i c i n and for hygromycin B, an a n t i b i o t i c used primarily i n agriculture (36-37, 22-24). Gentamycin could be measured as low as 80 pg, tobramycin 280 pg, n e t i l m i c i n 300 pg mL. The RIA has d e f i n i t e advantages, small samples, sizes, speed, accuracy, precision, s p e c i f i c i t y . There are s i g n i f i c a n t disadvantages also. The labelled reactant i s unstable ( I ) and costs are r e l a t i v e l y high. The great s e n s i t i v i t y requires considerable d i l u t i o n s ; antibody-bound fractions must be separate from free fractions i n order to obtain accurate counts. In a sense, the receptor assay system i s a RIA-type technique that has been applied to a n t i b i o t i c residue analysis. 1 2 5

Nonisotopic Immunoassays. Nonisotopic immunoassays d i f f e r from the isotopic assays only i n the type of label used, the end-point measurement, and the separation of bound and free fractions (41-43). Fluoroimmunoassays. This assay requires the drug being assayed to be l a b e l l e d with umbelliferyl-B-D-galactoside. The enzyme 3-galactosidase i s added and the fluorescent products are released from the l a b e l l e d a n t i b i o t i c . The antibody i n the bound f r a c t i o n


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i n h i b i t s the enzymatic hydrolysis. The d i f f e r e n t i a l i n fluorescence i s proportional to the a n t i b i o t i c concentration. This technique could o f f e r an excellent approach i f endogenous fluorophores can be removed or minimized. Enzyme M u l t i p l i e d Immunoassay Technique (EMIT). This technique employs enzyme-labelled a n t i b i o t i c s which react analogously to the fluroimmunoassay i n that a reduction of enzyme a c t i v i t y i s a t t r i buted to antibody binding. Higher concentrations of unlabelled drug i n the sample result i n less enzyme-labelled drug bound to the antibody. To perform the EMIT assay i s rather simple and straightforward: The sample i s added to a p l a s t i c tube. [Substrate-antibody reagents could be 3-NAD (B-nicotinamidoadenine dinucleotide and 1-malic acid) 3-NAD and glucose-6-phosphate] The enzyme reagent i s added, malic dehydrogenase glucose-6-dehydrogenase The reaction i s stopped a f t e r 10 min with sodium borate. Measure the i n t e n s i t y of the r e s u l t i n g color. The range of a n t i b i o t i c that can be measured i s usually 0.01 to 1.00 ug antibiotic/mL and has been used f o r gentamicin, c a r b e n i c i l l i n , t i c a r c i l l i n and amikacin (44-46). The use of the EMIT system, to-date, has been i n the c l i n i c a l area and unrelated to measuring residues of a n t i b i o t i c s . The procedure has p o t e n t i a l for residue analysis i f interferences by non-specific factors can be overcome. Enzyme-Linked Immunosorbent Assays (ELISA). Three methods are commonly used: d i r e c t competition, double antibody sandwish and antibody i n h i b i t i o n . Direct competition. The s o l i d phase (a m i c r o t i t e r plate) i s coated with an antibody s p e c i f i c for the antigen being assayed. The sample and enzyme-labelled antigen ( a n t i b i o t i c ) are added. There i s a competition for the antibody between the l a b e l l e d and unlabelled antigen ( a n t i b i o t i c ) . Substrate i s added and the color produced by the enzymatic hydrolyse i s inversely proportioned to the concentration of antigen i n the sample (48>Double-antibody sandwich. Antibody i s coated on or adsorbed to the p l a s t i c plate. The sample to be assayed containing the antigen ( a n t i b i o t i c ) i s added followed by a second antibody that i s conjugated to the enzyme (horseradish peroxidase, a l k a l i n e phosphatase, or 3-galactosidase). The substrate i s added and the intensity of the color produced i s d i r e c t l y proportional to the antigen i n the test sample. Antibody i n h i b i t i o n . Antibody i s preincubated with the sample being assayed. I f any antigen i s present i n the sample i t w i l l bind with antibody. When the assay mixture i s added to a microtiter plate coated with antigen, there i s a decrease i n the i n t e n s i t y of the color produced. The aforementioned procedures (techniques) have s i g n i f i c a n t potential and as assay problems are worked out could provide rapid, s e n s i t i v e , s p e c i f i c and precise methods for the analysis of low l e v e l s of a n t i b i o t i c s i n food and feed products.


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Acknowledgment s New Jersey A g r i c u l t u r a l Experiment Station Publication Number F-01112-01-86 supported by State and U. S. Hatch Act Funds. Literature Cited 1.


2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24.

Kramer, J . ; Carter, G. G.; Arret, B.; Wilner, J.; Wright, W. W.; Kirshbaum, A. Methods, Reports, and Protocols. Food and Drug Administration, Washington, D.C., 1968. Kavanagh, F. "Analytical Microbiology"; Academic: New York, 1971; Vol. I I . Ginn, R. E.; Case, R. A.; Packard, V. S.; T a t i n i , S. R. J. Assoc. Off. Anal. Chem. 1982, 1407-12. Katz, S. E.; Fassbender, C. A.; Hackett, A. J . ; M i t c h e l l , R. G. J. Assoc. Off. Anal. Chem. 1974, 57, 819-22. Katz, S. E.; Fassbender, C. A. J . Assoc. Off. Anal. Chem. 1978, 61, 918-22. Kelley, W. N. J . Assoc. Off. Anal. Chem. 1982, 65, 1193-1207. Messer, J. W.; L e s l i e , J . E.; Houghtby, G. A.; Peeler, J . T.; Barnett, J . E. J. Assoc, Off. Anal. Chem. 1982, 65, 1208-14. Ouderkirk, L. A. J . Assoc. Off. Anal. Chem. 1979, 62, 985-88. Packard, V. S.; T a t i n i , S.; Ginn, R. E. J . Milk Food Technol. 1975, 38, 601-03. Van Os, J . L.; Beukers, R. J . Food Protection. 1980, 43, 510-11. Van Os, J . L.; Lameris, S. A.; Doodeward, T.; Oostendorp, J . G. Netherlands Milk Dairy J. 1975, 29, 16-34. Katz, S. E.; Fassbender, C. A.; Dinnerstein, P. S.; Dowling, Jr., J. J . J . Assoc. Off. Anal. Chem. 1974, 57, 522-26. Katz, S. E.; Fassbender, C. A.; DePaolis, A. M.; Rosen, J . D. J. Assoc. Off. Anal. Chem. 1978, 61, 564-68. I n g l i s , J . M.; Katz, S. E. J . Assoc. Off. Anal. Chem. 1978, 61, 1098-1102. I n g l i s , J . M.; Katz, S. E. Appl. Environ. Microbiol. 1978, 35, 517-20. Katz, S. E.; Fassbender, C. A. J . Assoc. Off. Anal. Chem. 1970, 53, 968-72. Katz, S. E.; Fassbender, C. A. J . Agr. Food Chem. 1970, 18, 1164-67. Katz, S. E.; Fassbender, C. A.; Dorfman, D. B u l l . Environ. Contam. Toxicol. 1971, 6, 110-16. Katz, S. E.; Fassbender, C. A.; Dowling, J . J . J . Assoc. Off. Anal. Chem. 1972, 55, 123-33. Katz, S. E.; Fassbender, C. A.; Dorfman, D.; Dowling, J r . , J . J . J. Assoc. Off. Anal. Chem. 1972, 55, 134-38. Katz, S. E.; Fassbender, C. A. B u l l . Environ. Contam. Toxicol. 1972, 7, 229-36. Katz, S. E.; Fassbender, C. A.; Dowling, J r . , J . J . J . Assoc. Off. Anal. Chem. 1973, 56, 77-81. Singer, C. J . ; Katz, S. E. J . Assoc. Off. Anal. Chem. 1985, 1037-41. Katz, S. E.; Levine, P. R. J . Assoc. Off. Anal. Chem. 1978, 61, 1103-06.


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KATZ

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25.

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Levine, P. R. Ph.D. Thesis, Rutgers University, New Brunswick, N. J . , 1980. 26. Harpster, C. P.; Katz, S. E. J . Assoc. Off. Anal. Chem. 1980, 63, 1144-48. 26a. Harpster, C. A. Ph.D. Thesis, Rutgers University, New Brunswick, N. J . , 1979. 27. Charm, S. E. U.S. Patents 5 238 521; 4 239 745; 4 239 852, 1980. 28. Charm, S. E. Cultured Dairy Products J . 1979, 14, 24-26. 29. Charm, S. E.; Chi, R. K. J . Assoc. Off. Anal. Chem. 1982, 65, 1186-92. 30. S e l l , S. "Immunology, Immunopathology and Immunity"; Harper and Row: Hagerstown, 1980; 3rd Ed. 31. Kohler, G.; M i l s t e i n , C. Nature. 1975, 256, 495-97. 32. Goding, J . W. J . Immunol. Meth. 1980, 39, 285-308. 33. Steiner, S. J . Ph.D. Thesis, Rutgers University, New Brunswick, N. J . , 1981. 34. Berson, S. A.; Yalow, R. S. In "Radioimmunoassay: A Status Report i n Immunology"; Good, R. A.; Fisher, D. W., Eds.; Sinauer: Stanford, 1971. 35. Luft, R.; Yallow, R. S. "Radioimmunoassay Methodology and Applications i n Physiology and i n C l i n i c a l Studies"; George Thieme Verlag: Stuttgard, 1974. 36. Broughton, A.; Strong, J . E.; Pickering, L. K.; Bodey, G. P. Antimicrob. Agents Chemother. 1976, 10, 652-56. 37. Lewis, J . E.; Nelson, J . C.; Elder, H. A. Nature New B i o l . 1972, 239, 214-16. 38. Watson, R. A. A.; Wenk, M. In "Current Chemotherapy"; Siegenthaler, W.; Lathy, R., Eds.; American Society for Microbiology: Washington, D.C., 1978; Vol. 2. 39. Watson, R. A. A.; Shaw, E. J . ; Edwards, G. R. W. In "Chemotherapy"; Williams, J . D.; Goeddes, A. M., Eds.; Plenum: New York, 1976; Vol. 2. 40. Foglesong, M. A.; LeFeber, D. S. J . Assoc. Off. Anal. Chem. 1982, 65, 48-51. 41. Shaw, E. J . ; Watson, R. A. A.; Landon, J . ; Smith, D. S. J . C l i n . Pathol. 1977, 30, 526-31. 42. Shaw, E. J . ; Watson, R. A. A.; Smith, D. A. C l i n . Chem. 1979, 25, 322-24. 43. Bund, J . ; Wong, R. C.; Feeney, J . E.; Carrico, R. J . ; Boguslaski, R. C. C l i n . Chem. 1977, 23, 1402-08. 44. O'Leary, T. D.; R a t c l i f f , R. M.; Geary, T. D. Antimicrob. Agents Chemother. 1980, 17, 776-78. 45. Erwin, J . R.; Bullock, W. E.; N a t t a l l , C. E. Antimicrob. Agents Chemother. 1976, 9, 1004-11. 46. Hindler, J . Abstracts, Annual American Society for Microbiology Meeting, 1983. 47. V o l l e r , A.; Bidwell, D. E.; B a r t l e t t , A. "The Enzyme-linked Immunosorbent Assay (ELISA), A Guide with Abstracts of MicroPlate Applications"; Dynatech Publication, Nuffield Laboratories of Comparative Medicine; Zoological Soc. of London: Reagents Park. 48. Campbell, G. S.; Mageau, R. P.; Schwab, B.; Johnston, R. W. Antimicrob. Agents Chemother. 1984, 25, 205-11. RECEIVED May 8, 1986


Microbiological Assay Procedures for Antibiotic Residues - ACS

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