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Active Immunization and Evaluation against Luteinizing Hormone for Radioimmunoassay Technique in Human Serum ( 419 )

ABSTRACT

KEYWORDS

Active Immunization and Evaluation Against Luteinizing Hormone for Radioimmunoassay Technique in Human SerumEbeid, N.H.; Shafik, H.M.; Ayoub, S.M. and Mehany, N.L.

Received: 01/08/2013

Accepted: 10/03/2014

Available on line: 15/05/2014

E.mail:[email protected]

LH:BSA Immunogen, Anti- LH Polyclonal Antibody, Immunization, Radioimmunoassay.

J. Nucl. Tech. Appl. Sci, Vol. 2, No. 4, PP. 419 : 430 (2014)

Journal of

NUCLEARTechnology in Applied ScienceISSN 2314-8209 e-ISSN 2314-8217

1. Labelled Compounds Dept., Hot Labs Center, Atomic Energy Authority, Post No. 13759, Cairo, Egypt.

This study evaluated the antigenicity of luteinizing hormone con-jugate with Bovine Serum Albumin (LH-BSA). The conjugation of LH-BSA was carried out by 1-Ethyl-3-(3-Dimethylaminopropyl) Carbodi-imide HCl (ECDI). Three rabbits were immunized against LH-BSA. Two rabbits were immunized against nonconjugated LH and two rab-bits against BSA only. Immunization was carried out through primary injection and 4 boosters. The preparation of the radioiodinated 125I-LH was carried out using N- Bromo-Succinimide as oxidizing agent. The preparation of LH standards was carried out. The obtained LH antisera were characterized of titer, immunoresponse and displacement profile formulation, optimization and validation of the local liquid phase LH-Radioimmunoassay (RIA) system was carried out. The results provide a highly sensitive and accurate RIA system of LH-BSA. This technique could be used in measuring LH in human serum to investigate fertility especially disorders of the hypothalamic / pituitary / gonadal axis.

INTRODUCTION

Luteinizing Hormone (LH) is produced in both men and women from the anterior pituitary gland in response to Luteinizing Releasing Hormone LH-RH released by hypo-thalamus (Shiham and Inster, 1996). LH is a glycoprotein with a molecular weight of 30,000 Dalton approximately

(Wheller, 2006). It is composed of two non-covalently dissimilar amino acid chains, alpha and beta. The alpha chain is similar to that found in Thyroid Stimulating Hormone (TSH) and Follicle Stimulating Hormone (FSH). The β-subunits which display greater differences in the amino acid sequences among these hormones confer hormonal and immuno-

Ebeid, N.H. et al.( 420 ) J. Nucl. Tech. Appl. Sci., Vol. 2, No. 4

logical specificity (Boaz Hirshberg et al., 2003).

LH plays an important role in ovulation and luteinization in females and in male it stimulates in-terstitial cells to produce androgens and estrogens. Measurement of serum gonadotropin levels will allow for distinguishing between primary gonadal failure and deficient gonadal stimulation. If LH and FSH levels are elevated (hypergonadotropic hypogo-nadism), primary gonadal failure is present. On the other hand, if gonadotropin levels are low (hypogo-nadotropic hypogonadism), deficient gonadal stimu-lation has resulted in the hypogonadal state (Can, 1992).

The selection of a carrier protein is that it must be of a high molecular weight (greater than, 20,000) Dalton and phylogenically unrelated to the animal species in which the antisera raised. A wide variety of proteins have been employed in the synthesis of immunogens. These include serum albumins (Bo-vine and Human Serum Albumin, BSA and HSA respectively), ovalbumin, thyroglobulin, gamma globulins, Keyhole Limpet Haemocyanin (KLH), fibrinogen and the synthetic polypeptides poly-L-lysine and polyglutamic acid.

The first of these, BSA, despite not being par-ticularly immunogenic (at least in rabbits and sheep) is probably the most commonly used carrier protein. This choice of carrier protein is widely available in pure form; it is inexpensive and well characterized (molecular weight of around 64000 with 60 primary amino groups for conjugation). It is also relatively resistant to denaturation which can be useful in some of the conjugation procedures which involve organic solvents. BSA conjugates are usually readily soluble which makes isolation and characterization easier (Erlanger, 1980 & Jenner and Law, 2005).

The use of carbodiimide to facilitate conjuga-tion of a carboxylic acid and amine (Good Friend et al., 1964) is one of the most widely used conjugation methods. The carbodiimide activates the carboxylic acid function for subsequent attack by the amine.

The carrier protein in aqueous solution is usu-

ally added to the reaction mixture after the carbodi-imide activation of the hapten. More commonly, the water soluble derivatives of this reagent, e.g., 1-Ethyl-3-(3-Dimethylamino-propyl) Carbodiimide (ECD) or 1-Cyclohexyl-3-(2-Morpholino-Ethyl) Carbodiimide metho-p-toluene-sulphnate (CMC or Morpho CDI), are used (Jenner and Law, 2005).

Immunoassays are one of the most powerful of all immunochemical technique, especially radio-immunoassay (RIA). They employ a wide range of methods to detect quantitative antigens or antibodies (Daci et al., 2003). The polyclonal antibodies and the radio-labeled tracer are the key important stone in RIA system.

MATERIALS AND METHODS

Luteinizing Hormone (LH), Bovine Serum Al-bumin (BSA), 1-Ethyl-3-(3-Dimethylaminopropyl) Carbodiimide HCl (ECDI), Complete and Incom-plete Freund’s Adjuvant, Polyethylene Glycol 8000 (PEG), anti-rabbit IgG (whole molecule) second an-tibodies developed in goat (R-1131) and N-Bromo-Succinimide were purchased from Sigma Chemical Co., (USA). Sodium Iodide Na-125I (5mCi/50µl) pH (7-11) was purchased from (Isotope, Hungary).

All other chemical reagents were analytical grade obtained from reputed manufacturers.

The research plan was achieved through the preparation of the following:

Preparation of Luteinizing Hormone-BSA Conju-gate (LH-BSA immunogen)

Luteinizing hormone (LH) was conjugated with bovine serum albumin (BSA) according to Grieger and Reeves (1990). 5 mg of LH antigen was dis-solved in 1 ml of distilled water. One µCi 125I-LH was added to LH solution. 5 mg of the protein carrier (BSA) was added to the preceding mixture. 12.5 mg of ECDI was dissolved in 1.5 ml of distilled water and added to the preceding mixture. This reaction was allowed to proceed overnight at room tempera-ture. Unreacted small molecules were removed by dialysis against 0.005 M phosphate buffer saline


(PBS) for 24 h at 4oC. The conjugation mixture was completed to 10 ml with phosphate buffer. Then one ml from conjugate mixture was counted/min. The ra-dioactivity of the total conjugation mixture was cal-culated. One µCi 125I-LH was diluted with 10 ml dis-tilled water and 1 ml from total 125I-LH was counted/ min. After that, the count of total 125I-LH was cal-culated. The % yield of LH which conjugated with BSA was calculated using the following equation

% Yield = Tc of conjugation mixture ∕ Tc of tracer where the Tc is the total count.

The final product was lyophilized and the pro-tein conjugation ratio (LH: BSA) was determined using UV spectrum analysis (Erlanger et al., 1959 & Elbanna and Ragab, 2000).

Animal and Treatments

Production of polyclonal anti-LH-BSA anti-body was undertaken through immunization of three male mature white New-Zealand rabbits (R1 – R3) weighing 2-3 Kg with luteinizing Hormone conju-gated with Bovine Serum Albumin (LH-BSA) im-munogen (Grieger and Reeves, 1990). Control rabbits were immunized with LH or BSA alone (R4 – R7).

Rabbits were kept under the same hygienic con-ditions, well balanced diet and water was supplied ad libitum. The local production of polyclonal anti-body was carried out through primary immunization and four boosters. For primary immunization each rabbit (R1–R3) received 140 µg LH-BSA immunogen (which contain 100 µg LH antigens) /250 µl saline emulsified with 500 µl Freund’s Adjuvant Complete (FAC). Control rabbits were immunized with uncon-jugated LH or unconjugated BSA (100 µg LH or 100 µg BSA /250µl saline) per 500 µl FAC. Freund’s ad-juvant incomplete (FAI) was used for booster doses instead of FAC (Grieger and Reeves, 1990 and Me-hany et al., 2009).

Emulsification was performed using Hamilton double hub syringes connected to each other with narrow metallic tubing. The aqueous solution was

placed in one syringe and the adjuvant in the other one. Each rabbit received a single primary subcu-taneous injection followed by booster injections at 3 weeks intervals. Blood samples were obtained a week after each injection. After clotting overnight at 4oC, blood was centrifuged and serum was collected and stored at −20 oC.

Anti-sera were assessed in terms of titer, dis-placement and immunoresponse. A collected pool of anti-LH-BSA with highest titer and displacement was subjected to purification using caprylic acid pre-cipitation method according to Steinbuch and Au-dran (1969).

Preparation of LH Standards

The preparation of LH standards was carried out by diluting the stock LH antigen using tris-ac-etate-NRS (non-immunized Rabbit Serum) and di-luted to obtain the dilution ranged from 1.5 to 300 mIU/ml (Hichens et al., 1979).

Iodination of LH Antigen

The preparation of radioiodinated 125I-LH was performed using N-Bromo- Succinimide oxidation method described by Reay (1982). To an eppendorf tube containing 3 µg LH antigen/ 10 µl saline, 10 µl 0.5M phosphate buffer pH 7.4 was added followed by 5 µl of Na 125I (250 µCi, 9.25 MBq). The reaction was started by addition of 10 µl 0.05M phosphate buffer (pH 7.4) containing 5 µg N-Bromo-Succin-imide. The reaction was allowed to proceed for one minute and quenched by the addition of 10 µl phos-phate buffer 0.05 M (pH 7.4) containing 20 µg so-dium metabisulfite followed by the addition of 20 µg KI in 100 µl phosphate buffer 0.05 M (pH 7.4) as a carrier. The product was purified by gel filtration using PD-10 column. The radiochemical yield was estimated using paper electrophoresis.

The tracer obtained was diluted with 0.05 M phosphate buffer containing 0.1% Sodium Azide. The 125I-LH tracer produced was characterized in terms of radiochemical yield, specific activity, maxi-mum binding and non-specific binding.


Assay design

Radioimmunoassay (RIA) of luteinizing hor-mone (LH) was carried out as follows: 100 µl of LH standards or unknown samples and 100 µl of 125I-LH tracer were incubated 3 hours with 100 µl of anti-LH at 37oC. The separation of bound and free fractions was carried out by adding 100 µl goat anti-rabbit IgG, 100 µl non-immunized rabbit serum (NRS) and 500 µl polyethylene glycol (PEG) (8000, 8%) into all assay tubes and incubated for 15 min (Mehany et al., 2009 and Hichens et al., 1979). The supernatant was decanted carefully after centrifugation and the bound fraction was counted using gamma counter (Auto Gamma Counter, Cobra II, Packard Instru-ment, USA) and the results were calculated using logit-log graph paper.

Validation tests of liquid phase RIA system

Some studies were carried out to assure the va-lidity of the LH RIA system such as sensitivity, pre-cision, accuracy and method comparison.

RESULTS AND DISCUSSION

In the present study chemical conjugation reac-tion of the LH antigen with BSA as a protein carrier (LH: BSA) used the water soluble coupling agent, 1-ethyl-3-dimethylaminopropyl) carbodiimide (ECDI). The % yield obtained for Luteinizing Hor-mone (LH) with BSA was 58%. The ratio for LH: BSA was (2.5:1). These data are in good agreement with Butt et al. (1983).

For liquid phase RIA technique, three compo-nents were prepared to obtain valid and accurate as-say system. These components were anti-LH poly-clonal antibody, 125I-LH tracer and LH standards.

In the present study, the production of poly-clonal anti-LH was undertaken through the active immunization of three white New-Zealand rab-bits, (R1 - R3). Each rabbit was injected with 140 µg LH-BSA immunogen which contain 100 µg LH antigens. Control rabbits were injected with 100 µg unconjugated LH and 100 µg unconjugated BSA, re-spectively.

The data of the present study showed that: R1, R2 and R3 gave highest displacement percent and successful immunoresponse. The result obtained re-vealed that R1 showed the highest displacement per-cent (61.8%) and maximum immunoresponse after approximately three weeks from the fourth booster immunization at dilution 1/8000, whereas R2 and R3 showed displacement percent of 52.5% and 49.8%, respectively and maximum immunoresponse after approximately three weeks from the fourth booster immunization at the same dilution (1/8000). The control rabbits: R4 and R5 (immunized with LH anti-gen) gave low displacement percent (18.5%) and im-munoresponse at dilution (1/1000). The other con-trol rabbits: R6 and R7 (immunized with BSA) failed to immunoresponse.

05

101520253035404550556065

Dilution of anti-LH antiserum 1:100 1:500 1:1000 1:2000 1:4000 1:8000 1:16000 1:32000 1:64000

Bl1 Bl2 Bl3 Bl4 Bl5

Disp

lace

men

t, %

Fig. (1): Titre and displacement of LH-polyclonal anti-body for rabbit (1).

Fig. (2): Immunoresponse of rabbit (1) immunized with LH-BSA immunogen.

0 2 4 6 8 10 12 14 160

10

20

30

40

50

60

70

Disp

lecem

ent,

%

Period of immunization schedule (weeks)


The data of the displacement percent and im-munoresponse of the present study are in good agreement with previous reports (Mehany et al., 2009 and Hichens et al., 1979).

In the present study, the highest displacement and best immunoresponse of LH polyclonal anti-body at dilution titre1/8000 for R1 after three weeks from the fourth booster was selected as a basic com-ponent for this liquid phase RIA system (Figs. 1, 2).

The results obtained of IgG content of the puri-fied anti-LH were 16.64 mg/ml. The concentrations of total IgG in serum ranged from 5 to 20 mg/ml and the very strong polyclonal antiserum might con-tain 1 - 3 mg/ml of specific antibody or occasionally

even more (Goding, 1986). The purification step of neat antibody leads to improvement of the percent binding and it is essential to get antibodies with sat-isfactory specifications without denaturation of its components.

In the present study, the preparation of LH stan-dards was carried out using LH antigen in tris-buffer used as a standard matrix.

Factors affecting iodination of LH antigen

Effect of amount of oxidizing agent

It was found that the optimum amount of N-Bromo-Succinimide was 5 µg to obtain maximum yield and maximum binding as shown in Table (1).

Table (1) Effect of the amount of oxidizing agent on radioiodination of LH.

Table (2) Effect of reaction time on radioiodination of LH.

Amount of oxidizing agent(µg) Yield % Maximum

binding %Non-specificbinding %

3 30.9 25.0 1.05 39.8 31.7 1.110 38.7 30.3 1.320 26.3 16.5 1.650 20.0 9.1 2.3

Effect of reaction time

As illustrated in Table (2), it was found that the optimum reaction time for iodination of LH by N-

Bromo-Succinimide was one minute. This result is in agreement with another report (Sallam and Me-hany, 2012).

Reaction time (minute) Yield % Maximumbinding % Non-specific binding %

0.5 35.2 24.6 1.01 39.7 32.1 1.13 38.5 30.0 1.35 34.9 25.7 1.710 29.0 19.5 2.5

Effect of pH

The iodination method was carried out at differ-ent pH values ranging from 4 - 8. The data obtained are summarized in Table (3). It was observed that pH 7.4 is the optimum pH value. This can be explained

by the fact that between pH 7, 8, the Ortho-position in the aromatic ring of tyrosine of peptides and pro-teins is activated for electrophilic attack, owing to the electron donating effect of the neighboring hy-droxyl group (Salacinski et al., 1981).


Table (3) Effect of pH on the radioiodination of LH.

Table (4) Effect of quantity of LH on its iodination by 125I.

pH Yield % Maximumbinding % Non-specific binding %

4 15.4 9.1 1.76.3 27.7 23.7 1.57.4 39.4 32.6 1.18 32.7 12.6 1.9

Effect of LH content

As shown in Table (4), the yield and maximum binding were independent of the antigen content

when the antigen amount was in the range of 3 – 10 µg, so 3 µg were taken for all experiments.

Quantity of LH (µg) Yield % Maximumbinding % Non-specific binding %

3 45.8 32.9 1.05 40.6 31.0 1.27 39.4 29.0 1.510 37.5 28.5 1.9

The purification step is mandatory to separate the labeled protein monomer from the free iodide, other reactants and damaged materials. Several tech-niques are currently available like ion-exchange, adsorption, electrophoresis and HPLC but the most widely technique is gel filtration (Brown et al., 1983).

The elution pattern of 125I-LH using gel chro-matography on PD-10 column was shown in Fig.(3).

0 2 4 6 8 10 12 14 16 180

10

20

30

40

50

125I (19.8%)

125I-LH (40.6%)

Activ

ity, µ

Ci

Number of fractions

Fig. (3): Elution pattern of 125I-LH using gel-chromatogra-phy PD-10 column.

The figure shows 2 peaks, the first peak cor-responding to 125I-LH (40.6%) and the second peak corresponding to free 125I (19.8%). These results are in good agreement with other reports (Sallam and Mehany, 2012 and Salacinski et al., 1981). The specific activity of 125I-LH was 38.3 µCi/ µg. The maximum binding and non-specific binding in ap-plying local liquid phase RIA systems were 32.9% and 1.0%, respectively.

Liquid phase RIA system

The development of a radioimmunoassay sys-tem based on local production of primary reagents for the measurement of LH hormone in human se-rum is described. For achieving reliable assay, the general principals of the assay optimization must be followed. Optimization of RIA system for LH was carried out by studying the effect of sample volume (50-200 µl), incubation time (1-24 h) and precipitat-ing agents.

Sample volume

The assay procedure was performed using vari-able sample volumes (50, 100 and 200 µl). The data obtained in Table (5) shows that the highest differ-


Table (5) Effect of sample volume on liquid phase RIA system for LH.

Table (6) Effect of incubation time of liquid phase RIA system for LH.

Fig. (4): Optimized standard curve for LH using liquid phase RIA system.

ences in binding between the concentration of LH (1.5, 30, 200 mIU/ml) were demonstrated using 100

µl as sample volume while using an excess of sample volume resulted in decrease in binding efficiency.

LH Standard(mIU/ml)

Samples volume50 µl 100 µl 200 µl

1.5 91.7 85.3 70.030 78.5 50.8 49.5200 29.5 14.7 12.5

Displacement % 67.8 82.8 82.14The data are presented as % bound (B/Bo).

The data are presented as % bound (B/Bo).

Incubation time

The assay was performed using different incu-bation times 1, 3 and 24 hr. three standard concentra-tions were assayed (1.5, 30, 200 mIU/ml). The data

in Table (6) show that the binding percent was in-creased with increasing the incubation time and the displacement percent between different standards were acceptable at 3 hours.

LH Standard (mIU/ml)Incubation time

1 h 3 h 24 h1.5 79.0 85.0 86.5

30 50.0 48.0 49.4

200 22.0 15.5 14.8

Displacement % 72.2 81.8 82.8

From the above results, it could be concluded that the most appropriate conditions from the techni-cal point of view were obtained when the assay was carried out using 100µl of the purified anti-LH with initial dilution 1:8000 (R1), 100µl of LH standards or samples and 100 µl of LH tracer. The assay tubes were vortexed and incubated overnight at 37oC. The NSB tubes contain all reagents except the first anti-body. After that separating agent (2nd Abs) (100 µl goat anti-rabbit IgG 1/100), 100 µl of normal rab-bit serum 1/200 and 500 µl of PEG-8000 (8%) were added to all tubes except (Tc) tubes and incubated 30 min at room temperature. The tubes were centri-fuged at 4000 rpm for 20 min at 4oC. All the tubes were decanted and counted, using multi-crystal gam-ma counter. The optimized standard curve is illus-trated in Fig. (4).

Validation tests

To assure the validity of the assay some studies were carried out. These studies were as follows:


Sensitivity

Sensitivity is the smallest concentration of the ligand which can be detected in a particular assay system or can be distinguished from zero doses. For this purpose, zero standard tube was set up in sev-eral replicates along with other standards. The mean and standard deviation of counts were calculated. The dose corresponding to mean −2SD counts was read off the standard curve. The data showed that the detection limit of the assay was approximately 0.25mIU/ml. These data are in good agreement with Sallam et al., (2012).

Precision

Precision is a statistical index of the ability of an assay to yield the same result when it is repeated on the same sample.

- Intra-assay precision

Intra-assay precision was determined using 10 replicates of three human serum samples of LH (low, medium and high) on one run. The results of intra-assay are presented in Table (7).

- Inter-assay precision

Inter-assay precision was determined using 10 replicates of three human serum samples of LH on separate runs. The results are presented in Table (7).

Table (7) Intra-assay and inter-assay precision for LH using liquid phase RIA system.

Table (8) Recovery assessment for LH using liquid phase RIA system.

SampleIntra-assay Inter-assay

MeanmIU/ml SD C.V% Mean

mIU/ml SD C.V%

Low 3 0.25 8.3 3.2 0.3 9.4Medium 18 1.64 9.1 18.5 1.87 10.1

High 80 6.96 8.7 78.0 8.1 10.4

The data of the intra-assay and inter-assay pre-cision of this study revealed the consistency of the results obtained by this method. These results are in accordance with the results obtained by Pillai and Bhandarkar (1998) and El-Kolaly et al. (2001). They reported that the CV% of the intra-assay should be less than 10% while in the inter-assay less than 15%.

Accuracy Assessment

Accuracy is defined as the degree of agreement between the measured value and the true value.

To check the accuracy of the local assay for es-timation of LH in human sera, recovery and dilution tests were undertaken.

Recovery

As shown from the results in Table (8), it can be observed that % recovery test ranged from 95.6 % to 108.6 %. The recovery data for LH are in good agreement with the data of (Pillia and Bhanderkar, 1998). Who reported that the recovery in RIA sys-tem should be 100±15%. Also they reported that standard and sample should be chemically identical and their matrix should be the same.

Sample LH (mIU/ ml) LH standard Expected E Observed (O) Recovery (O/E)%

1 21.530200

1.7516.0101.0

1.915.3102.0

108.695.6

100.99

2 101.530200

5.7520.0105.0

6.120.5103.5

106.1102.598.6

3 751.530200

38.2552.5137.5

37.554.0139.0

98.0102.86101.10


Table (9) Dilution factor for LH using liquid phase RIA system.

Dilution

Dilution is demonstrated by diluting a sam-ple or standard with an appropriate diluent and running the assay (El-Bayoumy, 2012). The di-lution test results of the present study in Table (9) reveal the concentrations of three levels of LH in human serum samples at various dilu-tions to assess the linearity of the assay. The results obtained show that the technique of the present study maintained good linearity under dilution.

Method comparison

The validity of the assay was tested by compar-ing the results of LH of 20 serum samples obtained by commercially available kit reagents (Siemens, IRMA Kits Medical Solutions Diagnostics, Los An-geles, USA) with that obtained by the local method. The results are comparable with a correlation coef-ficients of r = 0.998 as shown in Fig.(5). The sta-tistical analysis showed good correlation between the results obtained from the present system and the commercially available kit (Feldkamp and Smith 1987).

Sample LH (mIU/ml) Dilution factor Expected E Observed (O) Recovery (O/E)%

1 121:21:41:8

6.03.01.5

6.52.91.4

108.396.793.3

2 301:21:41:8

15.07.53.8

14.28.04.1

94.7106.7109.3

3 801:21:41:8

40.020.010.0

38.621.09.5

96.5105.095.0

0 50 100 150 200 2500

50

100

150

200

250Y= A+B * XA= 0.705B= 1.008r = 0.998

Prep

ared

tech

niqu

e, m

IU/m

l

Siemens, mIU/ml

d e m o d e m o d e m o d e m o d e m o






Fig. (5): Correlation coefficient (r) between LH values obtained by Siemens method and the prepared technique.

CONCLUSION

Immunization of animals against endogenous hormones (LH) requires that the hormone be cou-pled to a carrier protein in order to evoke an effective immune response. Immunization against LH alone results only in a low immunological response, indi-cating that the immune system responds more effi-

ciently against a carrier protein hormone complex.

The technical simplicity of this sensitive, pre-cise and accurate method may suggest that this LH-RIA technique should be suited for routine labora-tory uses and can be used effectively as a decisive diagnostic tool for hypogonadism, hypergonadism in human serum.

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املناعى التحليل بتقنية وتقديره )الليوتينى( األصفر للجسم احملفز للهرمون النشط التمنيع اإلشعاعى فى مصل الدم البشرى

ناهد حسن عبيد – حنان حممد شفيق - سامية حممود أيوب – ناجى حلظى مهنى

LH تضمن هذا البحث دراسة التمنيع النشط للهرمون احملفز للجسم األصفر (الليوتينى) هرمونAnti-LH. وذلك عن طريق ارتباط اهلرمون وذلك النتاج األجسام املضادة عديدة النسيلة للهرمون بزالل املصل البقرى (BSA) باستخدام 1-إيثيل-3-3-ثنائى ميثيل أمينوبروبيل كارباميد عن طريق حتصني عدد ثالث أرانب باهلرمون املرتبط LH-BSA وأرنبني باهلرمون فقط وكذلك أرنبني بزالل تنشيطية بأربعة جرعات باحلقن أولية متبوعًا البقرى فقط وذلك من خالل احلقن جبرعة املصل

متتالية.

وقد مت حتضري القانى املشع هلرمون (الليتوتينى) باستخدام اليود-125 وكذلك حتضري احملاليل العيارية. وقد مت وضع تقنيه لنظام التحليل املناعى اإلشعاعى (LH-BSA-RIA) ذات الطور السائل وقد أوضحت النتائج مدى حساسية هذه التقنية الستخدامها فى قياس هرمون الليتوتينى لتشخيص

حاالت العقم ومشاكل اإلجناب.

)2014( ، 430 : جملد 2 ، عدد 4 ، ص 419

مجــــلة

التقنيــات النــوويــة فى العلوم التطبيقية

يصدرها

اجلمعية امل�شرية للعلوم الإ�شعاعية وتطبيقاتها

املوقع اإللكتروني www.esrsa.com

البريد اإللكتروني [email protected]

مجـــــــــلد 1

عدد 1 (2013)

قسم املركبات املرقمة – مركز املعمل احلارة – هيئة الطاقة الذرية –ص.ب 13759 القاهرة-مصر.. 1

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