Solid-Phase Fluorescence Immunoassay for the ... - ACS Publications

Chapter 36

Downloaded by NORTH CAROLINA STATE UNIV on September 20, 2012 | http://pubs.acs.org Publication Date: May 5, 1996 | doi: 10.1021/bk-1996-0621.ch036

Solid-Phase Fluorescence Immunoassay for the Detection of Antibiotic Residues in Milk Amit Kumar, Richard M . Rocco, Danton Κ. Leung, Larry S. Jang, Shanta Kharadia, Caroline Yu, Krista K. Hara-Mikami, Gwendolyn M . Jang, and Marcos Piani IDETEK Inc., 1245 Reamwood Avenue, Sunnyvale, CA 94089

We describe a rapid, inhibition immunoassay for several antibiotics found as residues in milk. The fluorophore is the recently developed compound, Cy-5™, and the excitation source is a semiconductor-diode laser. The solid-phase is the borosilicate glass surface (SiO ) of a capillary tube that has been treated with a silanizing reagent. β-Lactam antibiotics ( penicillin-G, ampicillin, cloxacillin, amoxicillin, ceftiofur and cephapirin) at the parts per billion level can be detected in a total assay time of less than three minutes. These assays were optimized to provide the greatest sensitivity at or near the regulatory safe/tolerance levels for the appropriate antibiotics. The potential for the use of this system for other types of assays will also be discussed. 2

Antibiotics are adrriinistered to cows for the prevention and treatment of infections, such as mastitis, and for the enhancement of animal growth and milk production ( i ) . Antibiotics are also abused through off-label, illegal adrninistration in an attempt to quickly bring a sick animal back into the producing herd. For the purpose of maintaining a safe and healthy food supply, considerable attention has been focused on identifying, monitoring and minimizing the existence of these antibiotic residues in milk and milk products. Since milk and milk products are widely consumed, antibiotic residues should be avoided for several reasons: (i) Some residues can cause allergic reactions in sensitive consumers. Approximately 5 to 10% of the population is hypersensitive to rjemcillin or other antibiotics (2). (ii) Small concentrations of antibiotic residues can aid in the selection of resistant strains of pathogens that are harmful to humans (2). (iii) Residues are often capable of interfering with starter cultures used in the production of processed milk products such as cheese and yogurt (2). Consequently, the United States Food and Drug Adrninistration (US FDA) has established safe/tolerance levels for antibiotic residues in milk and milk products. The antibiotics that are most commonly administered to lactating cows, and consequently those antibiotics that are usually found as contaminants in milk and rnilk-

0097-6156/96/0621-0450$15.00/0 © 1996 American Chemical Society In Immunoassays for Residue Analysis; Beier, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1996.


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products, fall into the β-lactam group (penicillin G, ampicillin, cloxacillin, cephapirin, ceftiofur, and amoxicillin) (3). Several types of assays have been developed and marketed for the detection of β-lactam antibiotics in milk. These assays are based on one of a variety of biochemical phenomena. The most common are inhibition of bacterial growth by antibiotic residues (4\ binding of antibiotic residues to appropriate cel-membrane receptors (5) and binding of the antibiotic residues to appropriately designed polyclonal and monoclonal antibodies (6). These assays have been developed to test milk samples in bulk tanks that contain the commingled milk from many cows or trucks which contain the milk from several farms. Although they are generally useful, these assays involve many manipulations and/or long incubations. Most of these assays require a significant degree of sophistication and training of the operator. In many cases, the assays require visual inspection of a reaction that is transient. Another major problem is that virtually all commercialy available assays produce an unacceptable number of false positives (samples with no or below the safe/tolerance level of antibiotics, that are incorrectly identified as having antibiotic residues above safe/tolerance levels) when used for individual cowside tests (7). Studies have shown that for individual cowside tests, for tests in samples of milk with high somatic cell counts, or samples of milk with high levels of bacteria, bacterial inhibition tests producedfrom10 to 50% false positives. With receptor based assays, 48% of negative milk samples were perceived to be positive (7). Consequently, no commercialy available test has been approved for use in testing samples of milk from individual cows, although studies sponsored by the US FDA are in progress. The high number of false positives is a concern for the dairy industry since they can lead to unwarranted waste of milk, can lead to the untimely slaughter of a potentially valuable dairy animal, and can result in a poor domestic and international perception of the US dairy industry by general and industrial consumers. No diagnostic procedure is infallible. Each procedure entails a probability of incorrectly identifying a negative sample as positive, however, the procedures that perform to specifications under the most stressful conditions are the most useful. A rapid, inexpensive, single-assay method for testing milk samples, for residues of all six β-lactam antibiotics, that requires minimal manipulation, produces semi quantitative and permanent results, and does not produce false positives would be very useful to the dairy industry. This paper describes preliminary studies aimed at developing an assay that fulfills these requirements and addresses the problems mentioned above. The assay, known as solid-phase fluorescence immunoassay (SPFIA) takes advantage of the specificity of antibody-antigen recognition, is conducted in a glass capillary tube and incorporates the use of a fluorescent label, Cy5™, for detection. Figure 1 diagrams the principle of the SPFIA. A milk sample is mixed with a known amount of a fluorescently-labeled antibody which is specific to a β-lactam antibiotic. An instantaneous binding reaction will occur between the antibody and the antibiotic in the milk sample. A solid support, the inside wall of a glass capillary tube in our format, with an antigen coating will then be exposed to the sample, the sample is removed, the capillary tube is washed, dried and examined with a fluorometer. Surfaces incubated with milk samples with high concentrations of an antibiotic will have fewerfreeantibody molecules to react with, thus yielding lower fluorescence signals, and surfaces exposed to milk samples with low concentrations of antibiotic

In Immunoassays for Residue Analysis; Beier, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1996.

452

IMMUNOASSAYS FOR RESIDUE ANALYSIS


Solid-Phase Fluorescence Immunoassay (SPFIA)

Concentration

Figure 1. General Diagram of the Inhibition Assay. When the labeled antibody is added to the sample of milk, the free β-lactam drug in the sample will bind to a fractional number of antibody molecules. The concentration of unbound antibody will be inversely proportional to the amount of drug in the milk. When the solid-support is added to the sample, the remaining unbound antibody molecules are free to bind to the surface. The support is removed, washed, dried and examined by fluorimetry. The fluorescence intensity is inversely proportional to the initial amount of drug in the milk. Samples with low concentrations of drug will produce high fluorescence signals from the surface, and samples with high concentrations of drugs will produce low fluorescence signals from the surface. The plot at the bottom depicts and ideal, optimized dose-response. Compare this to the actual dose-responses observed in Figures 3 and 4.


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will yield higherfluorescencesignals. Mathematicaly, the measured fluorescence signal is inversely proportional to the concentration of analyte in the sample.


Experimental

Capillary-Tube Surface Preparation. The surface of borosilicate, glass capillary tubes (Drummond Scientific, Broomall, PA) was treated with a silanizing reagent using a proprietary technique. The lengths of individual tubes used in the assay were typically 2.5 cm to 6 cm. The inner diameter was 0.65 mm and the oute diameter was 1 mm. The use of capillary tubes for the assay, instead of the wells of microtiter-plates or test tubes, was implemented to achieve a high surface area to volume ratio and to rninirnize the use of reagents. The high surface area to volume ratio alowed for a short two-minute incubation. In addition, our proprietary surface chemistry is designed for use on silicon dioxide (glass) surfaces. Tubes having other inner diameter dimensions were also examined, however, the 0.65 mm inner diameter tubing was found to be most useful with fresh, raw milk samples, which often contain fat globules that can be as large as several hundred micrometers (8). Such fat particles could potentially clog the capillary channels if tubes of smaler inner diameter were used. One additional point should be noted regarding assays in smal diameter tubes. The transport of reactants to the surface, where the measurable binding reaction occurs, is strictly through diffusion. Therefore, potential problems due to irreproducible agitation are eliminated. Synthesis

of Antigen Conjugates for Coating Capillaries.

All of the

antigen conjugates, for coating onto the surface of the capillary tubes, were prepared by binding the appropriate β-lactam drug to either bovine serum albumin (BSA) or a polypeptide copolymer consisting of lysine and alanine subunits (Sigma Chemical Co., St. Louis, MO). The covalent linking of antigen to carrier protein was accomplished through the use of conventional homobifunctional or heterobifunctional linkers, such as Succinimidyl 4-(7V-maleimidomethyl)cyclohexane-l-carboxylate (SMCC), Z?/s(sulfosuccinimidyl) suberate, and l-Ethyl-3-(3-Dmiethylaminopropyl)carbodimide Hydrochloride (Pierce Chemical Co., Rockford, IL) (9). Procedure for Coating Antigen-Conjugate on Surface of Tubes. After the silanizing surface treatment the capillary tubes

Capillary

were incubated for 30 min to 24 h in a buffered solution of the antigen-conjugate (20-40 pg/mL). The incubation temperature was usually Φ-7 °C, although occasionally room temperature incubation was used. The capillary tubes were removed, washed with distilled water, dried in a stream of compressed air, and then incubated in a solution of bovine-serum albumin (0.1% BSA in PBS-phosphate buffered saline-pH = 7.2, 0.05% Proclin 300a biocide) for 1 h at room temperature. The purpose of this second incubation was to block any regions of the surface that were bare. The tubes were again removed, washed with distilled water and dried in a stream of compressed air (20-30 psi). They were stored in the dark, at room temperature in a sealed, foil pouch with an indicating desiccant package. Maintaining dry and dark conditions were important to maintain the stability of the coated tubes.



454


Antibody Production. Antibodies to the antibiotics in the penicillin family were produced by irrimunizing goats with a conjugate of keyhole limpet hemocyanin (KLH) and ampicillin. Ampicillin was used since it has an amino-group available for conjugation. The resulting bleeds were screened for cross-reactivity with r^nicillin G , ampicillin, cloxacillin and amoxicillin. The cross-reactivity of this antibody with penicillin G and ampicillin was comparable, while the cross-reactivity for cloxacillin and amoxicillin was approximately 50% less. This antibody also exhibited less than 1% cross-reactivity with ceftiofur and cephapirin. Antibodies to ceftiofur were developed by immunizing goats with KLH-ceftiofur. A monoclonal to cephapirin was developed using conventional techniques with KLH-cephapirin as the conjugate. Enzyme immunoassay studies indicated that the cross-reactivity of the ceftiofur antibody to the other five β-lactam drugs of interest was less than 1%. The crossreactivity of the cephapirin antibody to the other five β-lactam antibiotics was less than 0.1%. Antibody Conjugates. Monoclonal and polyclonal antibodies were prepared using conventional purification techniques. Cy-5-antibody conjugates were prepared using the protocols previously published and provided by the manufacturer (Biological Detection Systems, Pittsburgh, PA) (70). The Cy-5 fluorophore, available with an NHS ester functionality, was linked to amino groups of the antibody. Purification and isolation of the conjugated dye was performed through chromatography. Spectroscopic examination indicated that the number of Cy-5 dye molecules that were bound to each antibody molecule was usually between two and four. The ratio of antibody molecule to Cy-5 molecule could be controlled by modifying reaction conditions such as time of conjugation and/or ratio of Cy-5 concentration to antibody concentration in the reaction mixture. Batch to batch studies indicated that there was no significant increase in fluorescence intensity with increase in the number of Cy-5 molecules per antibody molecule. In fact, the fluorescence intensity often decreased when the fluorophore/antibody ratio was greater than four. This phenomenon was especially apparent when using monoclonal antibodies. It is unclear at this time whether this decrease was due to increased quenching with high loading of the fluorophore or due to decreased antibody binding affinity resulting from inactivation of the antibodies' recognition sites. Fluorescence Measurements. Fluorescence signal intensities from individual capillary tubes were measured using a fluorometer that was designed and built inhouse. The excitation source was a 3 mW semiconductor-diode laser (A^ax = 635 nm, TOLD 9521 (s), Toshiba, Japan). The fluorescence signal from the capillary tubes was collected and filtered through appropriate optics and measured using the current response of a silicon p-i-n junction diode. The resulting photocurrent was amplified, converted from an analog to a digital signal and processed on a 386-PC compatible. Assay Protocol. The assay was begun by combining a measured amount of antibody-Cy-5 conjugate with the sample in a small container such as the well of a microtiter-plate. The antibody reagent was typically lyophilized and used as such in the assays. The raw milk sample and antibody reagent were mixed on a vibratory



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shaker. After mixing the antibody reagent and the sample for 10 s, the solution was sipped into and incubated in the appropriate capillary tube for 2 min. Sipping was performed through the use of a manifold device such as a modified pipettor or a cartridge (vide infra). All reagents were then washed out of the tubes with flowing distilled water, and they were dried with a stream of compressed air (20-30 psi). The fluorescence intensity of the tubes was then measured. Homemade cartridges were prepared to conduct multiple assays at once. These cartridges consisted of a common manifold and support structure for capillary tubes. The cartridge facilitated the removal of used tubes and insertion of new tubes. All of these procedures were conducted manualy. An automated system to perform the assay has been developed and will be discussed in another publication (Kumar, Α., Idetek Corp. Sunnyvale, CA, in progress). Results and Discussion

Figure 2 shows the chemical structures of the six β-lactam antibiotics of interest (77). The structures of those in the penicillin family (penicillin-G, ampicillin, cloxacillin, amoxicillin) are quite similar. Consequently, one polyclonal antibody cross-reacts with all four drugs in that family. The structures of cephapirin and ceftiofur are sufficiently different from each other and from the antibiotics of the rjemciUin family that individual antibodies are required for each. For the studies discussed in this report, a monoclonal antibody was used for cephapirin and a polyclonal antibody for ceftiofur. Table I lists the US FDA mandated safe/tolerance levels for the six β-lactam drugs (72). Table I.

Safe/tolerance levels for β-lactam drugs in milk. (Data from ref. 12) 1

Drug Safe/Tolerance Level (ppb) Penicillin G 5 Amoxicillin 10 Ampicillin 10 Cloxacillin 10 Cephapirin 20 Ceftiofur 50 ^pb = parts per billion, 1 ppb is equal to 1 ng/mL. A tremendous analytical challenge is posed by the these requirements. Since all of the safe/tolerance levels are different, the most efficient method for analysis is to conduct independent assays specific for each analyte. Consequently, the use of individual antibodies for each antibiotic or antibiotic family is necessary. The use of individual antibodies for each drug or drug-family also allows for the independent optimization of each assay.



F a m i l y

Figure 2. Structures of β-Lactam Antibiotics. The structures of the penicillin family, are so similar that a single polyclonal antibody cross-reacts with each. The epitope is the β-lactam structure that is common to each. The affinity of the antibody to each is similar. Although, ceftiofur and cephapirin are in the βlactam class, their structures are sufficiently different from the r^mcillin family and from each other to warrant the use of additional antibodies. A n additional polyclonal antibody was used for ceftiofur and a monoclonal antibody was used for cephapirin.

Penicillin



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Figures 3 and 4 display dose-response data for all six drugs of the β-lactam family that are regulated. Each point on the curve is an average of measurements made with between 30 and 120 individual samples of fresh, raw milk that were spiked with the appropriate drug concentration. These data were obtained after complete optimization of the assay protocol. The data depicted were also obtained in a blind study: one individual prepared the spiked samples and coded them randomly, while others ran the tests on the unknown samples. The data were collated after completion of the full study. A few notable points can be made from the graphs of Figure 3 and 4. The goal was to optimize the assay so that the greatest response would be observed at or near the safe/tolerance level for the particular drug. This goal was achieved for each analyte: note that both axes are linear, not logarithmic. Normalized fluorescence values were calculated as the signal of interest divided by the signal for the sample containing no analyte, multiplied by 100%. The depicted error bars reflect one standard deviation, + (σ/2). The dose-response curves for all of the drugs in the pemcillin family (Figure 3ad) are similar, since the same antibody, which cross-reacts somewhat equally with each drug in the family, was used for all four drugs. The dose-response curve for ceftiofur begins to plateau for concentrations greater than 35 ppb (Figure 4b). The ceftiofur curve begins to lose sensitivity due to the nature of the antibody. However, this performance is still better than any other commercial assay system. The data indicate that coefficients of variation (CV = (signal/standard deviation) χ 100) for each of the systems was less than 10% with few exceptions. In fact, the coefficients of variation of the cephapirin assay (Figure 4a), which uses a high affinity monoclonal antibody, was typically in the 1-2% range. The data of Figures 3 and 4 are especially notable since all of the assays were conducted in fresh, raw milk. Milk is a complex mixture of fat, protein and numerous other constituents (7,8). Raw, unpasteurized and non-homogenized milk is especially complex(7,&), however, no dilution of the samples was required for excellent performance of these assays. Figures 3 and 4 demonstrate the exceptional performance of this high sensitivity SPFIA. Although fluorescence assays are ubiquitous in the clinical diagnostics industry (13), solid-phase fluorescence formats have traditionally been difficult to develop (14). This difficulty is primarily due to a number of factors such as quenching of the fluorescent reagent when bound to the surface, background fluorescence from non-specifically adsorbed compounds from the sample matrix, background interference caused by scattering of the excitation light from a rough or porous surface, and others (14). We feel that through the combination of Cy-5, our proprietary surface chemistry on smooth glass surfaces, and high intensity laser excitation, many of the usual problems have been alleviated if not eliminated. Cy-5 is a cyanine dye, that is provided by the manufacturer as a Nhydroxysucciriirnide (NHS) ester (10). It has an absorbance maximum at 650 nm (extinction coefficient = 2 χ 10 M^cnr), with an emission maximum at 667 nm (10). Such a small Stokes shift, 17 nm, requires a high degree of optimization of the filtering components on the measurement system. The quantum yield for Cy-5 in solution is 0.28 (10). Absorbance in the red region of the spectrum allows excitation using a helium-neon laser (He-Ne, λ = 632.8 nm) or some recently developed 5

1


458



Penicillin G

Ml' ιι 1

2

ι Ϊ j

3

Concentration (ppb)

Concentration (ppb)

1 4

0

1

2

3

4

5

6

7

8

9

Concentration (ppb)

Concentration (ppb)

Figure 3. Dose-response data for rjerricillin family drugs. Note that all axes are linear. The error bars reflect one standard deviation. The normalized fluor escence was calculated by dividing the signal of interest by the signal for rnilk with no drug and multiplying by 100%. The response of all of the drugs is similar. Note that the assay for penicillin G was performed only to 5 ppb, since the USFDA safe/tolerance level is 5 ppb.


10


a)

I 4 I 12

I 14

Concentration (ppb)

I 10

I 16

I 18

I 20

I 22

b)

10

20

25

30

40

45

• D 35

- τ 5

50

ι

Ceftiofur

π

Concentration (ppb)

15

π

Figure 4. Dose-response data for Cephapirin and Ceftiofur. Note that all axes are linear. The error bars reflect one standard deviation. The normalized fluorescence was calculated by dividing the signal of interest by the signal for milk with no drug and multiplying by 100%. A monoclonal antibody was used for cephapirin and a polyclonal was used for ceftiofur.

1 2

Cephapirin


.u


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IMMUNOASSAYS F O RRESIDUE ANALYSIS

semiconductor-diode lasers (630-650 nm). The optical properties of the dye make it useful for immunoassays, since very little background fluorescence is observed from biological fluids, such as milk, blood, and others in the spectral regions of interest (10). Although the quantum yield for the Cy-5-antibody complex bound to the surface is probably much less than 0.28, it is sufficient to provide the detection sensitivity required for the antibiotic assays. In addition to providing assays for the β-lactam antibiotics, other potential assays can be conducted in the SPFIA format. To deterrnine the applicability of this format to other systems, we conducted proof-of concept experiments for a number of analytes in addition to the β-lactam antibiotics, as shown in Table Π.

Table II.

Solid-phase fluorescence immunoassays (SPFIAs).

Analyte

Range of Detection

Total Assay Time (min.)

β-L Antibiotics Amoxicillin Ampicillin Cloxacillin Penicillin G Cephapirin Ceftiofur

1-10 1-10 1-10 1-5 1-20 1-50

ppb ppb ppb ppb ppb ppb

3 3 3 3 3 3

Other Antibiotics Tetracycline Sulfamethazine

1-30 1-10

ppb ppb

3 3

1-103 ppb

3

Clinical Drugs Digoxin in Buffer

1-2

3

Pesticides/Herbicides

in progress

Hydrocarbons ΒΤΈΧ (benzene, toluene, ethyl-benzene, and xylenes)

ppb

For milk screening applications, the same format can be used to test for other antibiotics and contaminants. In addition, assays for hydrocarbon and pesticide wastes in water and soil, and assays for drugs of clinical significance should be possible. In principle, it should be possible to develop assays that can be used to screen any and all potential analytes for which an antibody of appropriate affinity exist. Certain matrices such as soil and solid food samples will require some sort of extraction protocol before conducting the assay.



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The purpose of these demonstrations was not to develop optimized assays at this time, but to show the versatility of the system. In fact, most of the assays in Table II, except those for the β-lactam drugs, were not optimized at all. One very important point to note has to do with the ability of this SPFIA to be formatted to operate multiple assays at once. Since each assay is conducted in its own capillary tube, a cartridge that allows the performance of several assays at once is possible. Such a system provides tremendousflexibilityto the assay operator. Once specific assays have been optimized, they can be included in the cartridge in any combination desired. This type of commercial system will be discussed in another publication (Kumar, Α., Idetek Inc. Sunnyvale, CA, in progress). Conclusion In addition to the versatility demonstrated by the number of analytes shown in Table II, there are numerous other factors that can make the SPFIA in capillary tubes even more versatile and superior. For example, the incubation time for all of the β-lactam assays was 2-min. This number was chosen due to market requirements. One of the requirements for an assay for antibiotics in milk is speed. The fastest current assay requires 7 min of time, therefore, our goal was to cut this time in half. With longer incubation times, several minutes or even hours, the assay would become correspondingly more sensitive and reproducible. Also, the inner diameter of the capillary tube can control assay sensitivity. Since this assay was being developed for testing raw milk which can contain fat particles that can be several tenths of a millimeter in diameter, a relatively large inner diameter capillary tube was used (ID = 0.65 mm). For assays in matrices that do not have particulates that may plug the tube, smaller diameter tubes can be used. Smaller diameter tubes will produce tremendous increases in surface area to volume ratios resulting in faster and more sensitive assays. In conclusion, the SPFIA discussed in this paper should provide for better and more efficient screening for β-lactam antibiotics in milk. It also is apparent that this assay format will support additional tests in many different areas, and the potential exists to improve the performance of this assay by controlling factors such as the geometries of tubes and incubation times. Acknowledgments Funding from the Small Business Innovation Research (SBIR) is acknowledged. Phase I grant # 1 R43 HD32266-01. Literature Cited 1. 2. 3. 4.

Mitchell, M.J.;Yee, A. J. Dairy, Food, Env. Sanit. 1995, 15, 484-487. Jones, G. M.; Seymour, Ε. H. J. Dairy Sci. 1988, 71, 1691-1699. Brady, M. S.; Katz, S. E. J. Food Prot. 1988, 51, 8-11. Gilbertson, T.J.;Mejeur, R. L.; Yein, F. S.; Jagian, P. S. J. Dairy Sci. 1995, 78, 1032-1038.



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5. Tyler, J. W.; Cullor, J. S.; Erskine, R. J.; Smith, W. L.; Dellinger, J.; McClure, K. J. Am. Vet. Med. Assoc. 1992, 201, 1378-1384. 6. Van Eenennaam, Α.; Cullor, J. S.; Perani, L. Gardner, I. Α.; Smith, W.L.; Dellinger,J.;Guterbock, W. M.; Jensen, L. J. Dairy Sci. 1993, 76, 30413053. 7. Cullor, J. S. Vet. Med. 1992, 87, 1235-1242. 8. Protein and Fat Globule Modifications by Heat Treatment, Homogenization and Other Technological Means for High Quality Dairy Products; Intemational Dairy Federation: Brussels, Belgium, 1993. 9. Immunotechnology Handbook; Pierce Chemical Co., Rockford, IL, 1992/3. 10. Mujumdar, R. B.; Ernst, L. Α.; Mujumdar, R. S.; Lewis, C. J.; Waggoner, A. S. Bioconjugate Chem. 1994, 4, 105-111. 11. The Merck Index, An Encyclopedia of Chemicals, Drugs and Biologicals; Budavari, S., ed.; Merck & Co.: Rahway, NJ, 1989. 12. United States Code of Federal Regulations (CFR); Title 21, Part 556, April 1993; Grade "A" Pasteurized Milk Ordinance, 1993 Revision; U.S. Department of Health and Human Services, Public Health Service, Food and Drag Administration. 13. Hemmila, I. A. Applications of Fluorescence in Immunoassays; John Wiley and Sons: New York, NY, 1991. 14. Glad, C. Appl. Biochem. Biotechnol. 1982, 7, 75-89. RECEIVED October 11, 1995


Solid-Phase Fluorescence Immunoassay for the ... - ACS Publications

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