Development and validation of a simple, sensitive, second antibody format enzyme immunoassay for LH determination in plasma

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<ul><li><p>Protocol</p><p>Development and validation of a simple, sensitive, second antibody</p><p>format enzyme immunoassay for LH determination in plasma</p><p>B.S. Prakash*, Vijay Paul, N. Anandlaxmi</p><p>Division of Dairy Cattle Physiology, National Dairy Research Institute, Karnal, Haryana 132001, India</p><p>Received 25 July 2002; accepted 25 July 2002</p><p>Abstract</p><p>The objective of this study was to develop and validate a direct simple and highly sensitive enzyme immunoassay (EIA) for</p><p>luteinizing hormone (LH) determination in buffalo plasma on microtiter plates using the biotinstreptavidin amplification</p><p>system and the second antibody coating. Biotin was coupled to LH and used to bridge between streptavidin-peroxidase and</p><p>immobilized antiserum in competitive assay. The EIA was carried out directly in 20 Al buffalo plasma. The LH standardsranging from 6.25 to 200 pg/well/20 Al were prepared in hormone free plasma collected from a buffalo on day 4 post-calving.The sensitivity of EIA procedure was 6.25 pg/well LH, which corresponds to 0.31 ng/ml plasma; the 50% relative binding</p><p>sensitivity was seen at 50 pg/well/20 Al. Plasma volumes for the EIA, viz. 10 and 20 Al, did not influence the shape of standardcurve even though a slight drop in the OD450 was seen with higher plasma volumes. A parallelism was carried out to compare</p><p>the endogenous buffalo plasma LH with bovine LH standards. For the biological validation of assay, 10 Murrah buffaloes were</p><p>used. These were administered (10 Ag im) with a synthetic analogue of gonadotropin-releasing hormone (GnRH) and bloodsamples were collected at 15-min interval using indwelling jugular catheter beginning just prior to GnRH injection till 6 h and</p><p>thereafter 2-h interval for another 18 h. In all animals, sharp increases in LH concentrations were recorded post-GnRH</p><p>administration, which confirms the biological validation of the EIA. To record the LH peak during periestrus in a cycling</p><p>buffalo, the blood samples were collected at 2-h intervals from onset of behavioral estrus signs till ovulation. The LH peak was</p><p>observed after the initial behavioural estrus signs followed by the gradual decline in the levels towards the ovulation.</p><p>D 2002 Elsevier Science B.V. All rights reserved.</p><p>Keywords: Buffalo endocrinology; LH; EIA; Plasma</p><p>1. Background</p><p>The hypophysial hormone LH plays an important</p><p>role in ovulation and luteinization in females. The</p><p>control of ovulation is brought about by the interactions</p><p>between the pituitary gonadotropins, FSH and luteiniz-</p><p>ing hormone (LH) and intraovarian factors such as</p><p>steroids, cytokines and other growth factors (Findley et</p><p>al., 1996).Measurement of LH in peripheral circulation</p><p>of buffaloes is important for understanding the phe-</p><p>0022-1759/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved.</p><p>PII: S0022 -1759 (02 )00301 -0</p><p>Abbreviations: LH, luteinizing hormone; EIA, enzyme immuno-</p><p>assay; PBS, phosphate-buffered saline; BSA, bovine serum</p><p>albumin; GnRH, gonadotropin-releasing hormone.</p><p>* Corresponding author. Fax: +91-184-250042.</p><p>E-mail address: (B.S. Prakash).</p><p></p><p>Journal of Immunological Methods 270 (2002) 281290</p></li><li><p>nomena limiting its fertility. LH measurements in</p><p>buffalo plasma are currently being carried out by</p><p>sensitive radioimmunoassay (RIA) procedures which</p><p>were established several years ago using 125I as the</p><p>label (Heranjal et al., 1976; Kaker et al., 1980; Galhotra</p><p>et al., 1981; Arora and Pandey, 1982; Rao and Pandey,</p><p>1983; Kanai and Shimizu, 1984; Avenell et al., 1985;</p><p>Singh, 1998). Although these methods are reliable and</p><p>accurate, they suffer from the problems associated with</p><p>the use of radioisotopes, which restricts their use to</p><p>specialized laboratories. The RIA procedure also suf-</p><p>fers from the disadvantage of using 125I as the label,</p><p>which has a short half-life. While enzyme immuno-</p><p>assay (EIA) procedures have been developed for</p><p>bovine LH (Mutayoba et al., 1990), GH (Hennies and</p><p>Holtz, 1993) and FSH (Prakash et al., 1999), no EIA</p><p>has so been established for buffalo LH. Hence, we</p><p>decided to develop a sensitive and convenient second</p><p>antibody EIA for LH determination in buffalo plasma</p><p>using the biotinstreptavidin-peroxidase amplification</p><p>system.</p><p>2. Type of research</p><p>1. Standardization and determination of LH in buffalo</p><p>plasma by EIA.</p><p>2. Biological validation of enzyme immunoassay by</p><p>measuring LH level after administration of gona-</p><p>dotropin-releasing hormone (GnRH) analogue.</p><p>3. Measurement of plasma LH during estrus in buffalo.</p><p>3. Time required</p><p>(1) Preparation of biotinylLH conjugate: 2 days.</p><p>(2) First coating of microtiter plates with goat</p><p>antirabbit IgG overnight.</p><p>(3) Second coating with 1% BSA in PBS 40 to 50 min.</p><p>(4) Immune reaction between antigen and antibody</p><p>overnight.</p><p>(5) Addition of biotinylLH conjugate and streptavi-</p><p>din-peroxidase 30 min for each step.</p><p>(6) Substrate reaction: 40 min.</p><p>(7) Addition of 4 N H2SO4 and reading of optical</p><p>density in Microtiter plate reader: 5 min.</p><p>(8) Microtiter plate washing (Four times each for 15</p><p>min).</p><p>4. Materials</p><p>4.1. Preparation of biotinylLH conjugate</p><p>Special equipment:</p><p> Dialysis sack (250-7U, Sigma, USA). Dialysis assembly (2 l beaker filled with PBS and</p><p>having a magnetic stirrer).</p><p>Chemicals and reagents:</p><p> Bovine LH (USDA-bLH-B-6, Beltsville, USA). PBS pH 7.4 (50 mM NaPO4, 0.15 M NaCl, pH</p><p>adjusted with 5 N HCl). Biotinamidocaproate-N-hydroxysuccinimideester</p><p>(Biotin; Sigma, Germany). 1 M NH4Cl solution in distilled water. 1% bovine serum albumin solution in PBS (BSA;</p><p>Sigma, Germany). Glycerol (Hi Media, India).</p><p>4.2. Preparation of affinity purified goat IgG anti-</p><p>rabbit IgG</p><p> See Anandlaxmi and Prakash (2001).</p><p>4.3. EIA procedure: first coating with goat IgG anti-</p><p>rabbit IgG and second coating with 1% BSA</p><p>Special equipments:</p><p> Microtiterplate shaker (Titertek, Flow Laboratories,Germany).</p><p> Microtiter plates (Greiner, Labortechnik, Ger-many).</p><p> Digital multichannel pipette (Flow TitertekR, Fin-land).</p><p>Chemical and reagents:</p><p> Goat IgG antirabbit IgG (Anandlaxmi and Prakash,2001).</p><p> Coating buffer pH 9.6 (15 mM Na2 CO3, 35 mMNaHCO3).</p><p> 1% bovine serum albumin in PBS (BSA, Sigma,Germany).</p><p>B.S. Prakash et al. / Journal of Immunological Methods 270 (2002) 281290282</p></li><li><p>4.4. Washing of coated microtiter plates</p><p>Special equipments:</p><p> Automated microtiter plate washer (Model: EL50x8MS, USA).</p><p>Chemical and reagents:</p><p> Washing solution (0.05% polyoxyethylenesorbitanmonolaurate, Tween 20 in distilled water, Sigma,</p><p>Germany).</p><p>4.5. Assay protocol</p><p>Special equipments:</p><p> Dilutor dispenser (Hamilton, MicrolabR 500series, Switzerland).</p><p> Digital multichannel pipette (Flow TitertekR). Automated microtiter plate washer (Model: EL50x</p><p>8MS).</p><p>Chemical and reagents:</p><p> Bovine LH standards (USDA-bLH-B-6). Hormone free buffalo plasma having LH concen-</p><p>trations lower than the measurable limit collected</p><p>on day 4 of the parturition. Rabbit polyclonal anti bovineLHantiserum(USDA-</p><p>309-684P, Beltsville, USA); very specific for LH</p><p>(USDA-bLH-B-6), as provided by the USDA the</p><p>cross-reactivity of the bLH antisera (USDA-309-</p><p>684P) with USDA-bFSH-B-1, USDA-bTSH-I-1,</p><p>USDA-bGH-B-1, and USDA-bPRL-B-1 was less</p><p>than 0.7%. Assay buffer pH 7.4 (50 mM NaPO4, 0.15 M</p><p>NaCl, 0.02% thimerosal; pH7.4 adjusted with 5 N</p><p>HCl). BiotinylLH conjugate. Streptavidin-peroxidase (Sigma, Germany).</p><p>4.6. Substrate reaction</p><p>Special equipments:</p><p> Microtiter plate reader (Model: ECIL, Microscan,India).</p><p> Graph pad PRISMR 2.01, software package.</p><p>Chemical and reagents:</p><p> Substrate buffer pH 4.0 (0.05 M citric acid, 0.11 MNa2HPO4, 0.05% ureum peroxide; pH4.0 adjusted</p><p>with 5 N HCl). Substrate solution: 17 ml substrate buffer plus 340Al 3,3V,5,5V-tetramethyl benzidene (Sigma, Ger-many); 12.5 mg/ml dimethyl sulfoxide (Sigma,</p><p>Germany). 4 N H2SO4 solution.</p><p>4.7. Biological validation of the buffalo plasma LH</p><p>enzyme immunoassay</p><p>Special equipment:</p><p> Refrigerated centrifuge (IEC, India).</p><p>Chemical and reagents:</p><p> Buserelin-Acetate (ReceptalR, Intervet, India). Local anesthesia (XylocaineR 2%, Astra Zeneca,</p><p>India) and antibiotic (OxytetracyclineR, SarabhaiZydus, India) given during catheterization.</p><p> Indwelling jugular catheter (60 cm surgical tubingNo. 51, Romsons, India; Three way valve with</p><p>stopper and sterilized 14-gauge stainless steel</p><p>needle). 10 and 5 ml disposable syringes (Dispo vanR,</p><p>India). 18-gauge stainless steel needle. Heparin sodium salt (SRL, India) solutions con-</p><p>taining 200 IU/100 Al and 200 IU/ml in normalsaline.</p><p> 15 ml polypropylene tubes (Chemtron, India). 2 ml Storage vials (Tarson, India).</p><p>5. Detailed procedure</p><p>5.1. Preparation of biotinylLH conjugate</p><p>(i) Add 40 Ag bovine LH (USDA-bLH-B-6)dissolved in 200 Al of phosphate buffered salinesolution (PBS: pH7.4), 12 Al biotinamidocap-</p><p>B.S. Prakash et al. / Journal of Immunological Methods 270 (2002) 281290 283</p></li><li><p>roate-N-hydroxysuccinimideester dissolved in</p><p>dimethyl sulfoxide (1 mg/ml) and immediately</p><p>vortex the mixture and incubate further for 3 h at</p><p>room temperature under constant agitation.</p><p>(ii) Stop the coupling reaction by the addition of 20</p><p>Al NH4Cl (1 M) and incubate the reactionmixture further for 30 min before addition of 2</p><p>ml of a solution of 1% BSA in PBS pH7.4.</p><p>(iii) BiotinLH conjugate is isolated by dialysis of</p><p>mixture in dialysis sack overnight at 4 jC withfour changes in PBS.</p><p>(iv) After dialysis, the conjugate is mixed with an</p><p>equal volume of glycerol to prevent freezing and</p><p>preserved at 20 jC in 1 ml aliquots.</p><p>5.2. Preparation of affinity purified goat IgG anti-</p><p>rabbit IgG</p><p>For preparation of affinity purified goat IgG anti-</p><p>rabbit IgG see Anandlaxmi and Prakash (2001). The</p><p>brief procedure is detailed below:</p><p>(i) About 40 ml plasma from a goat immunized</p><p>against rabbit IgG containing 20 IU heparin/ml of</p><p>blood is vortexed with rabbit IgG agarose and</p><p>loaded onto a small column.</p><p>(ii) First non-specific proteins are eluted with PBS</p><p>(10 mM NaPO4, 0.5 M NaCl, pH7.2) buffer.</p><p>(iii) Proteins bound specifically are eluted with 15 ml</p><p>of 0.1 M glycineHCl (pH2.0).</p><p>(iv) All steps are performed at room temperature.</p><p>(v) The eluted fractions (3 ml each) are collected in</p><p>vials containing 0.2 ml of 1 M TrisHCl (pH</p><p>8.0).</p><p>(vi) The eluted IgG is dialyzed overnight against PBS</p><p>and the protein content determined by measuring</p><p>the absorbance spectrophtometrically at 260 nm</p><p>and 280 nm, and extrapolated from a normo-</p><p>graph.</p><p>5.3. EIA procedure</p><p>a. First coating with goat IgG antirabbit IgG:</p><p>(i) The first coating is performed by adding 0.63 Agof goat IgG dissolved in 100 Al of coating buffer(pH 9.6) per well of the microtiterplate. The</p><p>plates are subsequently incubated overnight at 4</p><p>jC.</p><p>b. Second coating with 1% BSA:</p><p>(ii) For blocking the remaining binding sites, add 300</p><p>Al of 1% BSA in PBS to all the wells andincubate for 40 to 50 min at room temperature</p><p>under constant shaking.</p><p>c. Washing of coated microtiter plates:</p><p>(iii) The coated plates are washed twice with 350 Al/well of washing solution (0.05% Tween 20) using</p><p>an automated microtiterplate washer.</p><p>5.4. Assay protocol</p><p>(i) Duplicate of 20 Al of unknown buffalo plasmasample or bovine LH standards (USDA-bLH-B-</p><p>6; prepared in hormone free plasma collected on</p><p>day 4 of parturition) ranging from 6.25 to 400 pg/</p><p>20 Al/well are simultaneously pipetted intorespective wells along with 100 Al of LH anti-body diluted 1:160,000 in assay buffer (pH7.4)</p><p>with the aid of a dilutor dispenser.</p><p>(ii) Thereafter, the plates are incubated overnight at</p><p>room temperature after 30 min constant agitation.</p><p>(iii) The next day plates are decanted and washed</p><p>twice with washing solution before addition of</p><p>100 Al of biotinylLH conjugate diluted 1:400 inassay buffer.</p><p>(iv) The plates are further incubated for 30 min with</p><p>constant agitation, decanted and washed four</p><p>times with washing solution.</p><p>(v) Then add 20 ng streptavidin-peroxidase in 100 Alof assay buffer to all the wells and wrap the plates</p><p>in aluminum foils and incubate further for 30 min</p><p>under constant agitation.</p><p>(vi) All steps are performed at room temperature.</p><p>5.5. Substrate reaction</p><p>(i) The plates are then washed five times with</p><p>washing solution and incubated further in the</p><p>dark for 40 min after addition of 150 Al ofsubstrate solution per well.</p><p>(ii) Stop the reaction by the addition of 50 Al 4 NH2SO4 and measure the colour produced at 450</p><p>nm with a 12-channel microtiter plate reader.</p><p>(iii) Calculate LH concentration in buffalo plasma</p><p>samples from the graph; plotted LH concen-</p><p>tration against absorbance at 450 nm by using</p><p>Graph pad PRISMR 2.01, software package.</p><p>B.S. Prakash et al. / Journal of Immunological Methods 270 (2002) 281290284</p></li><li><p>5.6. Biological validation of the buffalo plasma LH</p><p>enzyme immunoassay</p><p>(i) For the biological validation of the assay, 10 non-</p><p>lactating cycling Murrah buffaloes maintained at</p><p>the National Dairy Research Institute farm are</p><p>used.</p><p>(ii) These are administered (10 Ag im) with asynthetic analogue of GnRH (Buserelin-Acetate)</p><p>and blood samples (4 ml) are collected at 15-min</p><p>interval using indwelling jugular catheter begin-</p><p>ning just prior to GnRH injection till 6 h and</p><p>thereafter 2-h interval for another 18 h.</p><p>(iii) All experimental protocols and animal care met</p><p>IACUC regulations. Before catheterization, the</p><p>local anesthesia (XylocaineR) is given and afterremoval of catheter the animal is treated with</p><p>antibiotic (Oxytetracycline) for 3 days.</p><p>(iv) In another experiment designed to measure the</p><p>levels of LH during periestrus, blood samples are</p><p>collected at 2-h intervals by jugular venipuncture</p><p>from the onset of behavioural estrus till 2 h of the</p><p>ovulation.</p><p>(v) The blood samples are collected in heparinized</p><p>polypropylene tubes and immediately kept in ice-</p><p>box (4 jC) and then centrifuged at 3000 rpm for20 min at 4 jC, plasma separated out is stored at 20 jC till assayed for LH.</p><p>6. Results</p><p>6.1. Standardization of enzyme immunoassay for</p><p>buffalo plasma LH determination</p><p>Titration of biotinylLH antiserum: A two dimen-</p><p>sional titer determination for the optimum dilution of</p><p>LH label and the antiserum was carried out. Antibody</p><p>dilutions ranging from 1:5000 to 1:640,000 and the</p><p>biotinylLH dilutions of 1:100 to 1:1600 were tested.</p><p>The antibody titer of 1:160,000 and the biotinylLH</p><p>conjugate titer of 1:400 were found to be the most</p><p>suitable and achieved an OD450 of around 1.5.</p><p>6.2. Assay validation</p><p>6.2.1. Assay interference and sensitivity</p><p>To determine the possible interference of plasma</p><p>with the assay sensitivity, bovine LH standards in va-</p><p>rious amounts of plasma (10, 20 and 40 Al) were run in</p><p>Fig. 1. Influence of different volumes of 10, 20 and 40 Al of buffalo plasma on optical density displacement in LH standard curve. Along withdifferent volumes of plasma, the standards were also prepared in assay buffer. Optical density was measured at 450 nm.</p><p>B.S. Prakash et al. / Journal of Immunological Methods 270 (2002) 281290 285</p></li><li><p>assay. There was no difference in the absolute binding</p><p>sensitivity between 10 and 20 Al plasma volumes,which were similar to that observed in buffer standards;</p><p>however, a slight decrease in sensitivity was seen when</p><p>standar...</p></li></ul>


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