Download - Rapid enzyme immunoassay for measurement of bovine progesterone

Biosensors & Bioelectronics 13 (1998) 1165–1171

Rapid enzyme immunoassay for measurement of bovineprogesterone

Rodney W. Claycomba, Michael J. Delwicheb,*, Coralie J. Munroc,Robert H. BonDurantc

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, USA

Received 16 December 1997; received in revised form 9 June 1998; accepted 9 June 1998

Abstract

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

1. Introduction

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: S0956-5663 (98)00081-5

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-side”test 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, the“cow-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 thebinding time, the unbound antigen and

1166 R.W. Claycomb et al. /Biosensors & Bioelectronics 13 (1998) 1165–1171

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 Al15 logSI0l1

Itl1

D (1)

whereA is the absorbency,I0 is the incident light inten-sity, It is the transmitted light intensity, andl1 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 I0l1

KItl1,no well

D 5 logS I0l1

Itl1,no well

D 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.

2. Objectives

The long-term goal of this research is an on-line sys-tem to automatically monitor luteal function by assay ofeach cow’s 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 enzyme–substrate 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 1014

and cross-reacted 1.5% with 17-a-OH-progesterone. Themonoclonal antibody had an affinity constant of 93 109

and cross-reacted 15% with 17-a-OH-progesterone, 5%

1167R.W. Claycomb et al. /Biosensors & Bioelectronics 13 (1998) 1165–1171

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 (100ml, 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 (100ml, 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 (100ml, 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 (100ml, 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

development

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 enzyme–substrate 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 (10mg/ml) and polyclonal (1mg/ml and 10mg/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 100ml 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 (100ml, 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

development

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.


Table 3Protocol for comparison of TMB and ABTS as substrates

Immobilization of antibody(1) Immobilize polyclonal antibody on untreated polystyrene test wells (100ml, 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 100ml 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 (100ml, 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 (100mlb, 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 50ml 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 (100ml, 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

development

aFurther research showed 2 h with 5mg/ml gave no change.bFurther research showed 150ml 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

There is a critical amount of active antibody neededon the test well surface. If the amount of active antibody

is in excess relative to the antigen and labeled antigen,essentially all the progesterone in the test wells can bind,regardless of the unlabeled progesterone concentration,and no competition occurs (antigen-limited). If the anti-gen and labeled antigen are in excess, though, they willcompete more vigorously for binding sites (antibody-limited). However, as the amount of active antibody con-tinues to decrease, there is a lower detection limit set bythe minimum amount of enzyme that can be measured.For the assay to be effective, it must be operated in theantibody-limited region. In this region, the amount ofactive antibody on the surface must be very repeatable.The particular antibody concentration depends on therequirements of the assay. Higher antibody concen-trations shift the standard curve to the right, raising thelower detection limit and the absolute ODs and shifting


the linear portion of the standard curve. Price and avail-ability of the antibody may also be a factor in favor ofusing lower antibody concentrations.

4.1. Solid support

Antibody immobilization on untreated polystyreneresulted in poor precision with regard to the number ofactive antibody sites. Adsorption probably resulted inrandom orientation of the antibody on the test well sur-face, rendering a fraction of the antibody inactive. Thiscan occur when the antibody adheres to the surface withits variable binding ends covered. Also, untreated poly-styrene is a hydrophobic surface. This hydrophobicitycan denature the antibody over time (Lin et al., 1991).

Fig. 1 shows the development curves for themaximum attainable signal for the four combinations ofantibody treatment and surface type. Covalent couplingof oxidized antibody resulted in the highest rate of devel-opment (1.661 mOD/s) and the lowest CV (8.3%). Thisindicated that more active antibody was immobilized andthat the amount of immobilized active antibody wasmore precise. For adsorption, a higher rate of develop-ment (1.026 mOD/s) and a higher precision (13.9%) wasobtained for the non-oxidized antibody. Since therecould be no covalent coupling on the untreated poly-styrene, all immobilized antibody must have beenadsorbed. Oxidation of the antibody resulted in chargedcarbohydrate moieties, probably reducing the adsorptionon untreated polystyrene. Finally, the signal obtained

Fig. 1. Comparison of antibody immobilization by adsorption and bycovalent coupling for oxidized and non-oxidized antibody (I, adsorp-tion, non-oxidized;j, adsorption, oxidized;G, covalent coupling, oxi-dized; H, covalent coupling, non-oxidized; one standard deviationerror bars,n 5 48).

from the non-oxidized antibody on the hydrazine-acti-vated surface was likely due to some adsorbing proper-ties of the polystyrene and to some “self-oxidized”carbohydrate groups on the antibodies.

4.2. Antibody type

Although the specificity for MAbs is usually higherthan that of PAbs, the affinity is usually several ordersof magnitude lower. This lower affinity is probably anadvantage when addressing the issue of reusing the anti-body surface in repeated tests. MAbs also have anadvantage in their commercial availability and essen-tially unlimited supply. Since the EIA was operating inthe linear region of the binding kinetic profile, affinitywas not the best parameter to examine, since it is relatedto the concentrations of the reagents at equilibrium, anda binding time of 5 min was not close to equilibrium(Claycomb et al., 1994).

Fig. 2 shows standard curves using MAb and PAb asthe molecular recognition elements in the EIA. The rateswere calculated from simple linear regression of OD ondevelopment time (n 5 4). The CVs for all data wereless than 3%. The standard curve for the MAb offeredseveral advantages. First, the standard curve was shiftedto the left compared with those for the PAb. This meantthat the MAb offered a lower limit of detection than thePAb. Also, the maximum rate for the MAb (0 ng/mlprogesterone; not shown on the plot) was 1.6 mOD/scompared with the corresponding maximum signal forthe PAb of 1.1 mOD/s (0 ng/ml progesterone, 10mg/mlPAb). Finally, the slope of the standard curve within the

Fig. 2. Comparison of polyclonal and monoclonal antibody as themolecular recognition element in the modified EIA (I, MAb, 10mg/ml; j, PAb, 1,mg/ml; G, PAb, 10mg/ml).


region of interest was highest for the MAb. Therefore,using a MAb allowed for a larger resolution in determin-ing progesterone concentration. No cross-reactivity stud-ies were performed.

4.3. Substrate

Increasing the rate of color change decreases theamount of enzyme needed to achieve a given opticaldensity in a given development time. Lowering theamount of conjugate (enzyme-labeled progesterone) inthe test well enhances competition, thus improving sen-sitivity.

Different substrates are used with HRP, includingo-phenylenediamine (OPD),o-dianisidine, 5-aminosal-icylic acid (5AS), and TMB. The important parametersfor the enzyme–substrate combinations are the Michaelisconstant,Km, and the maximum initial rate orvelocityof the reaction,Vmax (Lehninger et al., 1993).Km of thereaction is an inverse measure of the affinity of theenzyme for its substrate(s).Vmax is the initial velocity ofsubstrate catalysis under saturating substrate concen-trations. From enzyme theory,Km is defined as the sub-strate concentration that provides a velocity of 1/2Vmax.TMB is a substrate providing a lowKm and a highVmax.Fig. 3 shows a comparison between using TMB andABTS as the enzyme substrate. The rates were calcu-lated from simple linear regression on the OD versusdevelopment time data (n 5 16). The maximum rate forthe TMB (0 ng/ml progesterone; not shown on the plot)was 1.5 mOD/s compared with the correspondingmaximum signal for the ABTS of 0.8 mOD/s (0 ng/mlprogesterone). Since TMB has a lowerKm and higher

Fig. 3. Comparison of TMB and ABTS as the substrate in the modi-fied EIA (I, TMB; j, ABTS).

Vmax, an order of magnitude less conjugate was used inthe assay to achieve similar development rates. Thisshifted the standard curve to the left, thus improving thelower limit of detection of the assay. A second advantagewas in the precision of color development with lowerconjugate concentrations. The mean CV for TMB as thesubstrate was 5.6%, compared to 13.2% for ABTS.

4.4. New assay

Fig. 4 shows a comparison of standard curves inbuffer and estrus milk for the real-time progesterone EIAusing hydrazine-activated polystyrene test wells, oxid-ized monoclonal antibody, and TMB as the enzyme sub-strate. The binding time was 5 min, and the developmenttime was 3 min. Again, the rates were calculated fromsimple linear regression of OD on time (n 5 2). Thestandard curve was linear over the range of physiologicalprogesterone concentrations, and the development rateswere sufficient to measure progesterone concentrationswithin that range. The slope of the standard curve (20.86 mOD/s per decade) was sufficient to resolve pro-gesterone concentrations between 0.2 and 20 ng/ml. Themaximum rate for milk (0 ng/ml progesterone; notshown on the plot) was 1.1 mOD/s and the maximumrate for buffer was 1.1 mOD/s.

The effect of milk as a medium was to shift the stan-dard curve to the right. This increased the lower limitof detection of the assay. We think that progesteroneadded to the estrus milk dissolved in the fat and/or pro-tein globules due to its hydrophobicity. This inhibitedthe progesterone from competing with the conjugate forantibody binding sites, therefore allowing more conju-gate to bind and, thus, increasing the signal. The stan-

Fig. 4. Standard curve for real-time progesterone EIA for standardsin different media (I, estrus milk;j, PBS).


dard curve still had sufficient slope within the physio-logical range of interest, but milk as a medium decreasedthe resolution of the assay.

Further experiments were performed in which themilk was diluted with distilled water to reduce this so-calledmilk effect. A 1:2 dilution of the milk reduced themilk effect such that the standard curve shifted back tothe left, but not back to the buffer standard curve. In anautomated sensor, the milk samples would be diluted 1:2with a conjugate solution that was twice the workingconcentration. Using this procedure, the unlabeled pro-gesterone concentration in the test well during bindingwas one half that of the raw sample. A further modifi-cation was to reduce the conjugate concentration to 28.6ng/ml from 40 ng/ml, which also shifted the standardcurve to the left, allowing smaller concentrations of pro-gesterone to be measured, but lowering the absoluteODs. Although no variation in the source of milk withrespect to breed of animal was studied, we believe thatthe fat variation between breeds will have an effect.

5. Conclusions

A rapid enzyme immunoassay (EIA) was developedfor use in a biosensor to measure progesterone in bovinemilk during the milking operation. To achieve thedesired sensitivity, the EIA used covalent carbohydratebinding microtiter wells, monoclonal antibodies, HRP asthe enzyme label, and TMB as the substrate. Covalentcarbohydrate binding microtiter wells and TMBincreased the maximum rate and reduced the variationof color development. The use of monoclonal rather thanpolyclonal antibodies improved the lower limit of detec-tion, increased the maximum attainable signal, andincreased the slope of the standard curve. The assay wasadjusted so that the dynamic range of the standard curvewas from 0.2 ng/ml to 20 ng/ml for a 5 min bindingtime and a 3 min development time.

Acknowledgements

We express our appreciation to the USDA for fellow-ship support during this research, and to the Universityof California, Davis, Animal Science Department for theuse of the Experimental Dairy Herd and facilities.

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