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

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Development and validation of a simple, sensitive, second antibody

format enzyme immunoassay for LH determination in plasma

B.S. Prakash*, Vijay Paul, N. Anandlaxmi

Division of Dairy Cattle Physiology, National Dairy Research Institute, Karnal, Haryana 132001, India

Received 25 July 2002; accepted 25 July 2002

Abstract

The objective of this study was to develop and validate a direct simple and highly sensitive enzyme immunoassay (EIA) for

luteinizing hormone (LH) determination in buffalo plasma on microtiter plates using the biotin–streptavidin amplification

system and the second antibody coating. Biotin was coupled to LH and used to bridge between streptavidin-peroxidase and

immobilized antiserum in competitive assay. The EIA was carried out directly in 20 Al buffalo plasma. The LH standards

ranging 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

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 standard

curve even though a slight drop in the OD450 was seen with higher plasma volumes. A parallelism was carried out to compare

the endogenous buffalo plasma LH with bovine LH standards. For the biological validation of assay, 10 Murrah buffaloes were

used. These were administered (10 Ag im) with a synthetic analogue of gonadotropin-releasing hormone (GnRH) and blood

samples were collected at 15-min interval using indwelling jugular catheter beginning just prior to GnRH injection till 6 h and

thereafter 2-h interval for another 18 h. In all animals, sharp increases in LH concentrations were recorded post-GnRH

administration, which confirms the biological validation of the EIA. To record the LH peak during periestrus in a cycling

buffalo, the blood samples were collected at 2-h intervals from onset of behavioral estrus signs till ovulation. The LH peak was

observed after the initial behavioural estrus signs followed by the gradual decline in the levels towards the ovulation.

D 2002 Elsevier Science B.V. All rights reserved.

Keywords: Buffalo endocrinology; LH; EIA; Plasma

1. Background

The hypophysial hormone LH plays an important

role in ovulation and luteinization in females. The

control of ovulation is brought about by the interactions

between the pituitary gonadotropins, FSH and luteiniz-

ing hormone (LH) and intraovarian factors such as

steroids, cytokines and other growth factors (Findley et

al., 1996).Measurement of LH in peripheral circulation

of buffaloes is important for understanding the phe-

0022-1759/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved.

PII: S0022 -1759 (02 )00301 -0

Abbreviations: LH, luteinizing hormone; EIA, enzyme immuno-

assay; PBS, phosphate-buffered saline; BSA, bovine serum

albumin; GnRH, gonadotropin-releasing hormone.

* Corresponding author. Fax: +91-184-250042.

E-mail address: [email protected] (B.S. Prakash).

www.elsevier.com/locate/jim

Journal of Immunological Methods 270 (2002) 281–290

nomena limiting its fertility. LH measurements in

buffalo plasma are currently being carried out by

sensitive radioimmunoassay (RIA) procedures which

were established several years ago using 125I as the

label (Heranjal et al., 1976; Kaker et al., 1980; Galhotra

et al., 1981; Arora and Pandey, 1982; Rao and Pandey,

1983; Kanai and Shimizu, 1984; Avenell et al., 1985;

Singh, 1998). Although these methods are reliable and

accurate, they suffer from the problems associated with

the use of radioisotopes, which restricts their use to

specialized laboratories. The RIA procedure also suf-

fers from the disadvantage of using 125I as the label,

which has a short half-life. While enzyme immuno-

assay (EIA) procedures have been developed for

bovine LH (Mutayoba et al., 1990), GH (Hennies and

Holtz, 1993) and FSH (Prakash et al., 1999), no EIA

has so been established for buffalo LH. Hence, we

decided to develop a sensitive and convenient second

antibody EIA for LH determination in buffalo plasma

using the biotin–streptavidin-peroxidase amplification

system.

2. Type of research

1. Standardization and determination of LH in buffalo

plasma by EIA.

2. Biological validation of enzyme immunoassay by

measuring LH level after administration of gona-

dotropin-releasing hormone (GnRH) analogue.

3. Measurement of plasma LH during estrus in buffalo.

3. Time required

(1) Preparation of biotinyl–LH conjugate: 2 days.

(2) First coating of microtiter plates with goat

antirabbit IgG overnight.

(3) Second coating with 1% BSA in PBS 40 to 50 min.

(4) Immune reaction between antigen and antibody

overnight.

(5) Addition of biotinyl–LH conjugate and streptavi-

din-peroxidase 30 min for each step.

(6) Substrate reaction: 40 min.

(7) Addition of 4 N H2SO4 and reading of optical

density in Microtiter plate reader: 5 min.

(8) Microtiter plate washing (Four times each for 15

min).

4. Materials

4.1. Preparation of biotinyl–LH conjugate

Special equipment:

� Dialysis sack (250-7U, Sigma, USA).� Dialysis assembly (2 l beaker filled with PBS and

having a magnetic stirrer).

Chemicals and reagents:

� Bovine LH (USDA-bLH-B-6, Beltsville, USA).� PBS pH 7.4 (50 mM NaPO4, 0.15 M NaCl, pH

adjusted with 5 N HCl).� Biotinamidocaproate-N-hydroxysuccinimideester

(Biotin; Sigma, Germany).� 1 M NH4Cl solution in distilled water.� 1% bovine serum albumin solution in PBS (BSA;

Sigma, Germany).� Glycerol (Hi Media, India).

4.2. Preparation of affinity purified goat IgG anti-

rabbit IgG

� See Anandlaxmi and Prakash (2001).

4.3. EIA procedure: first coating with goat IgG anti-

rabbit IgG and second coating with 1% BSA

Special equipments:

� Microtiterplate shaker (Titertek, Flow Laboratories,

Germany).� Microtiter plates (Greiner, Labortechnik, Ger-

many).� Digital multichannel pipette (Flow TitertekR, Fin-

land).

Chemical and reagents:

� Goat IgG antirabbit IgG (Anandlaxmi and Prakash,

2001).� Coating buffer pH 9.6 (15 mM Na2 CO3, 35 mM

NaHCO3).� 1% bovine serum albumin in PBS (BSA, Sigma,

Germany).

B.S. Prakash et al. / Journal of Immunological Methods 270 (2002) 281–290282

4.4. Washing of coated microtiter plates

Special equipments:

� Automated microtiter plate washer (Model: EL50x8MS, USA).


� Washing solution (0.05% polyoxyethylenesorbitan

monolaurate, Tween 20 in distilled water, Sigma,

Germany).

4.5. Assay protocol

Special equipments:

� Dilutor dispenser (Hamilton, MicrolabR 500

series, Switzerland).� Digital multichannel pipette (Flow TitertekR).� Automated microtiter plate washer (Model: EL50x

8MS).


� Bovine LH standards (USDA-bLH-B-6).� Hormone free buffalo plasma having LH concen-

trations lower than the measurable limit collected

on day 4 of the parturition.� Rabbit polyclonal anti bovineLHantiserum(USDA-

309-684P, Beltsville, USA); very specific for LH

(USDA-bLH-B-6), as provided by the USDA the

cross-reactivity of the bLH antisera (USDA-309-

684P) with USDA-bFSH-B-1, USDA-bTSH-I-1,

USDA-bGH-B-1, and USDA-bPRL-B-1 was less

than 0.7%.� Assay buffer pH 7.4 (50 mM NaPO4, 0.15 M

NaCl, 0.02% thimerosal; pH7.4 adjusted with 5 N

HCl).� Biotinyl–LH conjugate.� Streptavidin-peroxidase (Sigma, Germany).

4.6. Substrate reaction

Special equipments:

� Microtiter plate reader (Model: ECIL, Microscan,

India).

� Graph pad PRISMR 2.01, software package.


� Substrate buffer pH 4.0 (0.05 M citric acid, 0.11 M

Na2HPO4, 0.05% ureum peroxide; pH4.0 adjusted

with 5 N HCl).� Substrate solution: 17 ml substrate buffer plus 340

Al 3,3V,5,5V-tetramethyl benzidene (Sigma, Ger-

many); 12.5 mg/ml dimethyl sulfoxide (Sigma,

Germany).� 4 N H2SO4 solution.

4.7. Biological validation of the buffalo plasma LH

enzyme immunoassay

Special equipment:

� Refrigerated centrifuge (IEC, India).


� Buserelin-Acetate (ReceptalR, Intervet, India).� Local anesthesia (XylocaineR 2%, Astra Zeneca,

India) and antibiotic (OxytetracyclineR, SarabhaiZydus, India) given during catheterization.

� Indwelling jugular catheter (60 cm surgical tubing

No. 51, Romsons, India; Three way valve with

stopper and sterilized 14-gauge stainless steel

needle).� 10 and 5 ml disposable syringes (Dispo vanR,

India).� 18-gauge stainless steel needle.� Heparin sodium salt (SRL, India) solutions con-

taining 200 IU/100 Al and 200 IU/ml in normal

saline.� 15 ml polypropylene tubes (Chemtron, India).� 2 ml Storage vials (Tarson, India).

5. Detailed procedure

5.1. Preparation of biotinyl–LH conjugate

(i) Add 40 Ag bovine LH (USDA-bLH-B-6)

dissolved in 200 Al of phosphate buffered saline

solution (PBS: pH7.4), 12 Al biotinamidocap-

B.S. Prakash et al. / Journal of Immunological Methods 270 (2002) 281–290 283

roate-N-hydroxysuccinimideester dissolved in

dimethyl sulfoxide (1 mg/ml) and immediately

vortex the mixture and incubate further for 3 h at

room temperature under constant agitation.

(ii) Stop the coupling reaction by the addition of 20

Al NH4Cl (1 M) and incubate the reaction

mixture further for 30 min before addition of 2

ml of a solution of 1% BSA in PBS pH7.4.

(iii) Biotin–LH conjugate is isolated by dialysis of

mixture in dialysis sack overnight at 4 jC with

four changes in PBS.

(iv) After dialysis, the conjugate is mixed with an

equal volume of glycerol to prevent freezing and

preserved at � 20 jC in 1 ml aliquots.

5.2. Preparation of affinity purified goat IgG anti-

rabbit IgG

For preparation of affinity purified goat IgG anti-

rabbit IgG see Anandlaxmi and Prakash (2001). The

brief procedure is detailed below:

(i) About 40 ml plasma from a goat immunized

against rabbit IgG containing 20 IU heparin/ml of

blood is vortexed with rabbit IgG agarose and

loaded onto a small column.

(ii) First non-specific proteins are eluted with PBS

(10 mM NaPO4, 0.5 M NaCl, pH7.2) buffer.

(iii) Proteins bound specifically are eluted with 15 ml

of 0.1 M glycine–HCl (pH2.0).

(iv) All steps are performed at room temperature.

(v) The eluted fractions (3 ml each) are collected in

vials containing 0.2 ml of 1 M Tris–HCl (pH

8.0).

(vi) The eluted IgG is dialyzed overnight against PBS

and the protein content determined by measuring

the absorbance spectrophtometrically at 260 nm

and 280 nm, and extrapolated from a normo-

graph.

5.3. EIA procedure

a. First coating with goat IgG antirabbit IgG:

(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

plates are subsequently incubated overnight at 4

jC.

b. Second coating with 1% BSA:

(ii) For blocking the remaining binding sites, add 300

Al of 1% BSA in PBS to all the wells and

incubate for 40 to 50 min at room temperature

under constant shaking.

c. Washing of coated microtiter plates:

(iii) The coated plates are washed twice with 350 Al/well of washing solution (0.05% Tween 20) using

an automated microtiterplate washer.

5.4. Assay protocol

(i) Duplicate of 20 Al of unknown buffalo plasma

sample or bovine LH standards (USDA-bLH-B-

6; prepared in hormone free plasma collected on

day 4 of parturition) ranging from 6.25 to 400 pg/

20 Al/well are simultaneously pipetted into

respective wells along with 100 Al of LH anti-

body diluted 1:160,000 in assay buffer (pH7.4)

with the aid of a dilutor dispenser.

(ii) Thereafter, the plates are incubated overnight at

room temperature after 30 min constant agitation.

(iii) The next day plates are decanted and washed

twice with washing solution before addition of

100 Al of biotinyl–LH conjugate diluted 1:400 in

assay buffer.

(iv) The plates are further incubated for 30 min with

constant agitation, decanted and washed four

times with washing solution.

(v) Then add 20 ng streptavidin-peroxidase in 100 Alof assay buffer to all the wells and wrap the plates

in aluminum foils and incubate further for 30 min

under constant agitation.

(vi) All steps are performed at room temperature.


(i) The plates are then washed five times with

washing solution and incubated further in the

dark for 40 min after addition of 150 Al of

substrate solution per well.

(ii) Stop the reaction by the addition of 50 Al 4 N

H2SO4 and measure the colour produced at 450

nm with a 12-channel microtiter plate reader.

(iii) Calculate LH concentration in buffalo plasma

samples from the graph; plotted LH concen-

tration against absorbance at 450 nm by using

Graph pad PRISMR 2.01, software package.


5.6. Biological validation of the buffalo plasma LH

enzyme immunoassay

(i) For the biological validation of the assay, 10 non-

lactating cycling Murrah buffaloes maintained at

the National Dairy Research Institute farm are

used.

(ii) These are administered (10 Ag im) with a

synthetic analogue of GnRH (Buserelin-Acetate)

and blood samples (4 ml) are collected at 15-min

interval using indwelling jugular catheter begin-

ning just prior to GnRH injection till 6 h and

thereafter 2-h interval for another 18 h.

(iii) All experimental protocols and animal care met

IACUC regulations. Before catheterization, the

local anesthesia (XylocaineR) is given and after

removal of catheter the animal is treated with

antibiotic (Oxytetracycline) for 3 days.

(iv) In another experiment designed to measure the

levels of LH during periestrus, blood samples are

collected at 2-h intervals by jugular venipuncture

from the onset of behavioural estrus till 2 h of the

ovulation.

(v) The blood samples are collected in heparinized

polypropylene tubes and immediately kept in ice-

box (4 jC) and then centrifuged at 3000 rpm for

20 min at 4 jC, plasma separated out is stored at

� 20 jC till assayed for LH.

6. Results

6.1. Standardization of enzyme immunoassay for

buffalo plasma LH determination

Titration of biotinyl–LH antiserum: A two dimen-

sional titer determination for the optimum dilution of

LH label and the antiserum was carried out. Antibody

dilutions ranging from 1:5000 to 1:640,000 and the

biotinyl–LH dilutions of 1:100 to 1:1600 were tested.

The antibody titer of 1:160,000 and the biotinyl–LH

conjugate titer of 1:400 were found to be the most

suitable and achieved an OD450 of around 1.5.

6.2. Assay validation

6.2.1. Assay interference and sensitivity

To determine the possible interference of plasma

with the assay sensitivity, bovine LH standards in va-

rious amounts of plasma (10, 20 and 40 Al) were run in

Fig. 1. Influence of different volumes of 10, 20 and 40 Al of buffalo plasma on optical density displacement in LH standard curve. Along with

different volumes of plasma, the standards were also prepared in assay buffer. Optical density was measured at 450 nm.


assay. There was no difference in the absolute binding

sensitivity between 10 and 20 Al plasma volumes,

which were similar to that observed in buffer standards;

however, a slight decrease in sensitivity was seen when

standards taken in 40 Al plasma were run in the assay.

Increasing plasma volumes also showed a slight reduc-

tion in the final colour development and a drop in OD

were registered (Figs. 1 and 2). Keeping these aspects

Fig. 2. Influence of different volumes of 10, 20 and 40 Al of buffalo plasma on percentage binding in LH standard curve. Along with different

volumes of plasma, the standards were also prepared in assay buffer. Optical density was measured at 450 nm.

Fig. 3. LH (meanF S.E.) profile in Murrah buffaloes after GnRH treatment (n= 10). GnRH analogue (10 Ag) was administered intra-muscularly

and blood samples were collected at 15-min intervals for 6 h of GnRH administration and at 2-h intervals for another 18 h.


in view, standards were subsequently prepared in

hormone free plasma and run along with the unknowns

in the test. The nonspecific binding using both the

volumes of plasma was low (OD450) ranging from

0.114 to 0.145. All assays were hence conducted taking

20 Al of unknown plasma samples and standards per

well in duplicates. The lowest LH detection limit

significantly from zero concentration was 6.25 pg/20

Fig. 4. LH profile after onset of estrus in a buffalo (Mu4420). Blood samples were collected at 2-h intervals after the initial expression of estrus

signs by the buffalo till the 2 h of ovulation. Ovulation was observed by rectal palpation at 2-h intervals.

Fig. 5. Parallelism for bovine LH standards with serially diluted different volumes of 1.25, 2.5, 5.0, 10.0 and 20.0 Al of buffalo plasma.

Standards for bovine LH were ranging from 6.25 to 400 pg/well/20 Al.


Al plasma which corresponded to 0.31 ng/ml plasma.

The 50% relative binding (B/B0) sensitivity was 50 pg/

20 Al plasma/well which corresponded to 2.5 ng/ml

plasma.

6.2.2. Intra- and inter-assay precision

Intra- and inter-assay coefficients of variations

determined using pooled plasma containing 0.62 and

5.0 ng/ml in 11 assays were 7.73% and 16.0% and

3.21% and 10.89%, respectively.

6.3. Biological validation

Mean LH concentrations in blood samples col-

lected from the 10 non-lactating cycling Murrah

buffaloes after GnRH injection are presented in (Fig.

3). The LH concentration rose sharply to a peak mean

value of 13.78F 3.74 ng/ml after 2 h of the GnRH

analogue administration. Subsequently, the hormone

concentration declined to reach basal levels (0.31 ng/

ml) at about 6 h post-treatment and remained basal

thereafter. An LH peak was also recorded in a buffalo

post-estrus with the peak value of 14.9 ng/ml, which

was followed by ovulation (Fig. 4).

6.4. bLH parallelism with buffalo plasma

The homology between bovine LH standards used

and endogenous LH in buffalo plasma was assessed

by conducting parallelism. To serve the purpose,

buffalo plasma sample containing high level of endog-

enous LH was serially diluted (containing 20, 10, 5,

2.5 and 1.25 Al buffalo plasma sample size) and run

along with the bovine LH standards (in buffer) in an

assay. When plotted with the increasing plasma vol-

umes and increasing standard concentrations a parallel

drop in relative percent binding was observed (Fig. 5).

Both curves, i.e. for increasing plasma volumes and

bovine LH standards, were lying almost parallel to

each other, thereby, confirming the actual LH estima-

tion in buffalo plasma.

7. Discussion

The method described here is the first report using

the second antibody technique and the LH–biotin–

streptavidin system for buffalo plasma LH EIA. The

use of the second antibody for coating the wells instead

of the hormone specific antibody is preferred as it

reduces assay variability associated with uneven bind-

ing of the latter antibody to the wells and further

reduces the amount of hormone specific antibody

needed in the EIA (Meyer, 1986).

The high degree of parallelism in the concentrations

plotted from hormone values obtained from serial

dilutions of a blood sample from buffalo containing

high LH, and the standard curve of bovine LH (Fig. 5)

indicates considerable homology between buffalo LH

and bovine LH used in the assay.

To obtain a high degree of sensitivity in direct EIA,

less sample volume is desirable to reduce the non-

specific binding and plasma matrix effects (Mutayoba

et al., 1990). This requires the use of highly specific

antibody, a very efficient amplification system and

optimum ligand antibody dilutions at suitable incuba-

tion temperature. In our EIA there was a decrease in

optical density with increasing plasma volumes

although the sensitivities and the relative binding

percentage did not change when 10 and 20 Al plasma

were taken along with standards (Figs. 1 and 2). In

order to compensate for this effect, it is necessary to

use the same plasma volumes for standards and

unknowns. A high assay sensitivity of 6.25 pg/well

LH was obtained when 20 Al of plasma was taken for

estimation. This was sufficient to determine the low

physiological baseline LH concentrations as well as

distinctly observe the LH release in 10 cycling Murrah

buffaloes after GnRH analogue injection. The appli-

cation of short period of incubation in EIA for

samples with expected high LH levels (>10 ng/ml)

is a helpful tool for rapid confirmation of estrus where

LH surge is the dominating indicator of heat and

ovulation in cattle and buffaloes, as well as in several

biomedical reproductive studies modifying the func-

tion of hypothalamo-hypophyseal axis. The assay

described here require less expensive instrumentation

and reagents when compared to RIA, and can be

adopted in developing countries where financial con-

straints limit the adoption of RIA. Highly purified LH

preparations from cattle and other species of animals

are available, and biotinylation of LH is not difficult

as compared to iodination procedures. Biotin and

streptavidin-peroxidase of good quality are also com-

mercially available at rather cheaper costs than 125I

preparations.


8. Quick procedure

8.1. Enzyme immunoassay procedure

(i) First coating with 0.63 Ag of goat antirabbit IgG

dissolved in 100 Al of coating buffer per well of

the microtiter plate and incubate overnight at 4

jC.(ii) Second coating or blocking the remaining sites

with 300 Al of 1% BSA in PBS per well and

incubate at room temperature for 40 to 50 min

under constant shaking.

(iii) Wash the coated plates twice with 350 Al per wellof washing solution.

8.2. Assay protocol

(i) Pipette out duplicates of 20 Al buffalo plasma

samples or bovine LH standards along with 100

Al LH antibody diluted 1:160,000 in assay buffer

in respective wells of coated microtiter plates.

(ii) Incubate overnight at room temperature after 30

min constant agitation.

(iii) Decant and wash twice with washing solution

before addition of 100 Al biotinyl LH conjugate

(1:400 in assay buffer).

(iv) Incubate further for 30 min with constant shaking

and then decant and wash four times with

washing solution.

(v) Add 20 ng streptavidin-peroxidase in 100 Al assaybuffer per well and wrap the plate in aluminum

foil and incubate for 30 min with constant

shaking.


(i) Wash the plate five times and then add 150 Alsubstrate solution per well and incubate further in

dark for 40 min.

(ii) Stop the reaction with addition of 50 Al 4 N

H2SO4 per well.

(iii) Measure the absorbance of colour developed at

450 nm.

(iv) Plot the standard curve; plotting LH concentration

against absorbance obtained and calculates con-

centration of LH in buffalo plasma samples by

using Graph pad PRISMR 2.01 software.

Acknowledgements

The authors wish to thank the United States

Department of Agriculture, Animal Hormone Program,

Beltsville, USA for the generous gift of reference LH

standards and bovine LH antiserum. The funds

provided by National Agricultural Technology Project

PSR No. 47 for this study is duly acknowledged. We

are also grateful to Director, NDRI, Karnal for pro-

viding all necessary facilities during the course of this

study.

References

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