Rapid enzyme immunoassay for measurement of bovine progesterone
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Biosensors & Bioelectronics 13 (1998) 11651171
Rapid enzyme immunoassay for measurement of bovineprogesterone
Rodney W. Claycomb a, Michael J. Delwiche b,*, Coralie J. Munro c,Robert H. BonDurant c
a DDx, Incorporated, 7000 North Broadway, Building 3, Suite 305, Denver, CO 80221, USAb Biological and Agricultural Engineering, University of California, Davis, Davis, CA 95616, USA
c Population Health and Reproduction, School of Veterinary Medicine, University of California, Davis, Davis, CA 95616, USAReceived 16 December 1997; received in revised form 9 June 1998; accepted 9 June 1998
Reproductive management is a primary financial concern of the dairy industry with missed estrus detection one of the majorcauses of lost income. A rapid enzyme immunoassay (EIA) was developed for on-line measurement of progesterone in bovine milkwith a biosensor for detection of estrus. The EIA was developed using covalent binding microtiter wells, monoclonal antibody,horseradish peroxidase, and 3,39,5,59-tetramethylbenzidine (TMB). The EIA took 8 min and had a dynamic response for progesteronein buffer and milk between 0.2 and 20 ng/ml. 1998 Elsevier Science S.A. All rights reserved.
Keywords: Cow; Estrus; Antibody; Hormone
Profitability of the dairy industry is heavily dependentupon satisfactory reproductive performance. In 1982 itwas estimated that total annual losses in the USA causedby inadequate reproductive performance exceededUS$1.2 billion (Strandberg and Oltenacu, 1989; Levinsand Varner, 1987; Ruiz et al., 1992; Britt, 1985). Repro-ductive performance has improved little since that esti-mate. An underlying reason for these extended intervalsis poor estrus detection (Britt, 1985). Estrus is definedas the period of time in the reproductive cycle just priorto ovulation. Present methods of estrus detection suchas visual observation or pedometry are not adequate.Measurements of hormone concentrations in the bloodor milk are more accurate indicators of estrus (Laing andHeap, 1971; Nebel et al., 1987; Nebel, 1988). Serumprogesterone levels decrease from a luteal phase concen-tration of more than 6 ng/ml to less than 0.1 ng/ml duringestrus. These changes are reflected in the milk at some-
* Corresponding author. Tel.: 1 1-530-752-7023; Fax: 1 1-530-752-2640.
0956-5663/98/$ - see front matter 1998 Elsevier Science S.A. All rights reserved.PII: S0956- 56 63 (98)00 08 1- 5
what higher concentrations due to the fat solubility ofthe steroid hormone (Koelsch et al., 1990, 1994).
An enzyme immunoassay (EIA) for progesterone inblood plasma was developed using polyclonal antibodiesand horseradish peroxidase as the enzyme label, with alower sensitivity limit of 25 pg per test well on amicrotiter plate (Munro and Stabenfeldt, 1984). In themid-1980s, on-farm disposable EIA kits (i.e., cow-sidetest kits) became available for detection of progesteronein milk. Currently, these test are manual, requiring 30min to several hours for the result, and are qualitative(Nebel, 1988). Although an elegant technology, thecow-side EIA for milk progesterone has not foundwide use for several reasons, including the difficulty ofusing a single progesterone measurement to detect estrusand the time involved in collecting, storing, and testingmilk samples.
In a typical EIA, the antibody is immobilized on a testcell surface and the test solution with unknown antigenconcentration is added. In a competitive EIA, the antigenand a solution with known concentration of enzyme-lab-eled antigen (i.e., the conjugate) are added simul-taneously. The labeled and unlabeled antigens competefor antibody binding sites. After sufficient time for bind-ing, called the binding time, the unbound antigen and
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conjugate are washed from the test cell and a substratesolution catalyzed by the bound enzyme is added. Thisreaction proceeds for a given amount of time, called thedevelopment time. The extent of the reaction is read asan optical change in the test cell. The degree of opticalchange is inversely proportional to the antigen concen-tration in the sample. Many variations to the general EIAprocedure have been developed, including sandwich-type assays or labeling the antibody rather than the anti-gen (Blum and Coulet, 1991).
Color intensity of the substrate solution is read in amicroplate reader as optical density (OD), given by
OD 5 Al1 5 logSI0l1Itl1D (1)where A is the absorbency, I0 is the incident light inten-sity, It is the transmitted light intensity, and l1 is themeasurement wavelength (ASAE, 1991; Segel, 1991).Using the assumption that the transmitted intensitythrough the solution and the test well is a constant timesthe transmitted intensity through only the solution, theOD becomes
OD 5 logS I0l1KItl1,no wellD 5 logSI0l1
Itl1,no wellD 2 log K (2)
Therefore, using only a single wavelength to measureOD results in a dependence on the absorbance propertiesof the test well. Given this result, it is important to usetest wells having consistent optical properties.
The EIA for progesterone developed by Munro andStabenfeldt (1984) was used as the starting point. Thereaction kinetics of this assay were studied and evalu-ated, the standard EIA was modified to operate in real-time, and a modified EIA was designed for measurementof progesterone in buffer (Claycomb et al., 1994). Assaytime was reduced from 150 min to as short as 2 min, butthe ODs and slope in the dynamic range of progesteroneconcentration were too low to provide a measurableresponse, and a new assay had to be developed.
The long-term goal of this research is an on-line sys-tem to automatically monitor luteal function by assay ofeach cows milk for progesterone every time the milkingmachine is attached. The first step in attaining such asystem was the development of an assay capable ofincorporation into a biosensor. Our objective was todevelop an enzyme immunoassay with sufficient sensi-tivity for real-time measurement of progesterone in milk.
3. Assay development
To function in an on-line sensor, the assay had to takeno more than 15 min, the normal turn-over time for agroup of cows in a milking parlor. In the biosensing sys-tem, this included not only the binding time and develop-ment time, but also the time required for transferring sol-utions, data collection and analysis, and preparation forthe subsequent test cycle. To satisfy this real-time con-straint, binding time and development time had to be lessthan 10 min.
Although the modified EIA we reported previouslymet the real-time requirements, the slope of the standardcurve between the concentrations of 0.2 and 20 ng/mlwas still not sufficient to resolve progesterone concen-trations within this range (Claycomb et al., 1994).Increasing the slope of the standard curve requireddevelopment of a new EIA. Three methods to achievethis result were modification of the solid support, modi-fication of the antibody, and modification of the enzymesubstrate reaction.
Covalent coupling of antibody to the test well surfaceresults in two advantages. Since the carbohydrates on anantibody molecule are in the hinge region, coupling thecarbohydrate groups to the polystyrene orients the anti-body in a more active configuration, thus optimizing theuse of the antibody, a relatively expensive reagent. Also,covalent coupling is not a random attachment. Rather,it is a specific chemical reaction occurring between thecarbohydrate groups on the antibody and the hydrazidegroups on the polystyrene. Given a repeatable numberof hydrazide groups, the precision in the amount of anti-body coupled to the surface is increased. Immobilizationprotocols using untreated polystyrene (Immulon I, Dyna-tech, Chantilly, VA) and hydrazine-activated polystyrene(CHO-Binding, Corning Costar, Cambridge, MA) wereevaluated using the experimental protocol shown inTable 1. To be covalently coupled to hydrazide groups,the carbohydrate groups on the antibodies needed to beoxidized. To evaluate how much antibody immobiliz-ation was due to adsorption and how much was due tocovalent coupling, both oxidized and non-oxidized anti-body were used on both untreated and hydrazine-acti-vated polystyrene.
Another way of improving sensitivity used the speci-ficity advantages a monoclonal antibody (MAb) offersover a polyclonal antibody (PAb). Experiments wereperformed to compare the performance of a polyclonalantibody (R4861, Department of Population Health andReproduction, School of Veterinary Medicine, Univer-sity of California, Davis) and a purified monoclonal anti-body (ASM0126, The Binding Site, Birmingham, UK).The polyclonal antibody had an affinity constant of 1014and cross-reacted 1.5% with 17-a-OH-progesterone. Themonoclonal antibody had an affinity constant of 9 3 109and cross-reacted 15% with 17-a-OH-progesterone, 5%
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Table 1Protocol for comparison of adsorption (non-primed steps) and covalent coupling (primed steps) for the modified progesterone EIA
Immobilization of antibody for adsorption(1) Immobilize oxidized and non-oxidized polyclonal antibody on untreated polystyrene test wells (100 ml, 1 mg/ml, 0.05 M
carbonate buffer, pH 9.6, 1 h, ambient temperature)(2) Wash test wells (0.05% Tween 20, 0.15 M NaCl)
Immobilization of antibody for covalent coupling(19) Immobilize oxidized and non-oxidized polyclonal antibody on hydrazine-activated test wells (100 ml, 1 mg/ml, 0.1 M acetate,
pH 5.3, 1 h, ambient temperature)(29) Wash test wells (0.05% Tween 20, 0.15 M NaCl)(39) Block test wells (100 ml, 2% BSA, 50 mM Tris, pH 8.2, 30 min, ambient temperature)(49) Wash test wells (0.05% Tween 20, 0.15 M NaCl)
Binding(3) (59) Add 100 ml of 400 ng/ml conjugate combined with varying progesterone concentrations (0, 0.2, 2, 10, 20 ng/ml) in PBS to test
wells(4) (69) Incubate for 5 min at ambient temperature with agitation(5) (79) Wash test wells (0.05% Tween 20, 0.15 M NaCl)
Development(6) (89) Add substrate solution to all test wells (100 ml, 0.4 mM ABTS, 1.6 mM H2O2, 0.05 M citrate, pH 4.0)(7) (99) Read optical density (l 5 405 nm) on microplate reader at 20 s intervals for 3 min with agitation and calculate rates of
with pregnenolone, and 3% with androstenedione. Theexperimental protocol shown in Table 2 was used. Thesubstrate was changed from 2,29-azino-bis(3-ethyl-benzthiazoline-6-sulfonic acid) (ABTS) to 3,39,5,59-tetramethylbenzidine-dihydrochloride (TMB) (34015,Pierce, Rockford, IL), as explained below.
The third possibility for increasing the sensitivity ofthe EIA was modifying the enzymesubstrate reaction.Experiments were performed to compare TMB withABTS as the enzyme substrates in the assay. The experi-mental protocol shown in Table 3 was used. The TMBused for this experiment was 3,39,5,59-tetramethylbenzi-dine (T2885, Sigma, St. Louis, MO). TMB is not water-
Table 2Protocol for comparison of monoclonal and polyclonal antibodies as the molecular recognition element
Immobilization of antibody(1) Immobilize monoclonal (10 mg/ml) and polyclonal (1 mg/ml and 10 mg/ml) antibody on untreated polystyrene test wells (100
ml, 0.05 M carbonate buffer, pH 9.6, 1 h, ambient temperature)(2) Wash test wells (0.05% Tween 20, 0.15 M NaCl)
Binding(3) Add 100 ml of 400 ng/ml conjugate combined with varying progesterone concentrations (0, 0.2, 2, 10, 20 ng/ml) in PBS to test
wells(4) Incubate for 5 min at ambient temperature with agitation(5) Wash test wells (0.05% Tween 20, 0.15 M NaCl)
Development(6) Add substrate solution to all test wells (100 ml, 0.32 mM TMB, 0.16 mM H2O2, 51.4 mM phosphate/24.3 mM citrate, pH 5.0)(7) Read optical density (l 5 650 nm) on microplate reader at 20 s intervals for 3 min with agitation and calculate rates of
soluble and, therefore, an organic solvent must be usedbefore making the substrate solution. Dimethylsulfoxide(D 8779, Sigma, St. Louis, MO) was used as the organicsolvent. In subsequent experiments, the dihydrochloridederivative of TMB (34015, Pierce, Rockford, IL) wasused to make the substrate water-soluble and, thus, sim-plify the preparation.
Based on these experiments, a new EIA was designedusing hydrazine-activated polystyrene as the solid sup-port, monoclonal antibodies as the molecular recognitionelement, and TMB as the substrate. The binding timewas 5 min, and the development time was 3 min. Theprotocol for the new EIA is shown in Table 4.
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Table 3Protocol for comparison of TMB and ABTS as substrates
Immobilization of antibody(1) Immobilize polyclonal antibody on untreated polystyrene test wells (100 ml, 1 mg/ml, 0.05 M carbonate buffer, pH 9.6, 1 h,
ambient temperature)(2) Wash test wells (0.05% Tween 20, 0.15 M NaCl)
Binding(3) Add 100 ml of conjugate (40 ng/ml for TMB; 400 ng/ml for ABTS) combined with varying progesterone concentrations (0, 0.2,
2, 10, 20 ng/ml) in PBS to test wells(4) Incubate for 5 min at ambient temperature with agitation(5) Wash test wells (0.05% Tween 20, 0.15 M NaCl)
Development(6) Add substrate solution to all test wells
TMB (100 ml, 0.83 mM TMB, 1.6 mM H2O2, 0.05 M acetate, pH 4.8)ABTS (100 ml, 0.40 mM ABTS, 1.6 mM H2O2, 0.05 M citrate, pH 4.0)
(7) Read optical density (l 5 650 nm for TMB, 405 nm for ABTS) on microplate reader at 20 s intervals for 3 min with agitationand calculate rates of development
Table 4Protocol for real-time progesterone EIA
Antibody immobilization(1) Immobilize monoclonal antibody on hydrazine-activated plates (100 ml, 10 mg/ml, 0.1 M acetate, pH 5.3, 1 ha, ambient
temperature)(2) Wash test wells (0.05% Tween 20, 0.15 M NaCl)(3) Block test wells (100 mlb, 2% BSA, 50 mM Tris, pH 8.2, 30 min, ambient temperature)(4) Wash test wells (0.05% Tween 20, 0.15 M NaCl)
Binding(6) Add 50 ml of 80 ng/ml conjugate combined with 50 ml of varying progesterone concentrations in PBS to test wells(7) Incubate for 5 min at ambient temperature with agitation(8) Wash plate (0.05% Tween 20, 0.15 M NaCl)
Development(9) Add substrate solution to all test wells (100 ml, 0.32 mM TMB, 0.16 mM H2O2, 51.4 mM phosphate/24.3 mM citrate, pH 5.0)(10) Read optical density (l 5 650 nm) on microplate reader at 10 s intervals for 3 min with agitation and calculate rates of
aFurther research showed 2 h with 5 mg/ml gave no change.bFurther research showed 150 ml reduced non-specific binding.
Another issue was the sensing of progesterone in milkrather than in buffer. Many results have shown that milkdoes not adversely affect the assay sensitivity (Sauer etal., 1986; Bulman, 1979). The new EIA protocol shownin Table 4 was used with the standards made in milkrather than phosphate-buffered saline (PBS). This milkwas taken from cows thought to be in estrus (i.e., lowprogesterone) based on observation. This experimentwas replicated three times using milk from three differ-ent cows.
4. Results and discussion