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hCG, PRL, LH, FSH CLIA Kit

Reaktivität: Human Chemiluminescent Sandwich ELISA
Produktnummer ABIN504775
  • Target
    hCG, PRL, LH, FSH
    Reaktivität
    Human
    Nachweismethode
    Chemiluminescent
    Methodentyp
    Sandwich ELISA
    Applikation
    ELISA
    Verwendungszweck
    Immunoenzymometric assay (Types 3 and 4): The essential reagents required for an immunoenzymometric assay include high affinity and specificity antibodies (enzyme and immobilized), with different and distinct epitope recognition, in excess, and native antigen. In this procedure, the immobilization takes place during the assay at the surface of a microplate well through the interaction of streptavidin coated on the well and exogenously added biotinylated specific monoclonal antibody. Upon mixing monoclonal biotinylated antibody, the enzyme-labeled antibody and a serum containing the native antigen, reaction results between the native antigen and the antibodies, without competition or steric hindrance, to form a soluble sandwich complex.
    Analytische Methode
    Quantitative
    Produktmerkmale
    The Quantitative Determination of HCG, PRL, LH and FSH Concentration in Human Serum and Plasma by a Microplate Chemiluminescence assay (CLIA). Measurements of these hormones are used as an aid in the determination and therapeutic monitoring of reproductive endocrinologies.
  • Applikationshinweise
    All products that contain human serum have been found to be non-reactive for Hepatitis B Surface Antigen, HIV 1&2 and HCV Antibodies by FDA required tests. Since no known test can offer complete assurance that infectious agents are absent, all human serum products should be handled as potentially hazardous and capable of transmitting disease. Good laboratory procedures for handling blood products can be found in the Center for Disease Control / National Institute of Health, "Biosafety in Microbiological and Biomedical Laboratories," 2nd Edition, 1988, HHS Publication No. (CDC) 88-8395.
    Kommentare

    Sample Volume: LH/FSH: 50 µL Two Step (Sequential), 25 µL One Step (Equilibrium)

    Plattentyp
    Pre-coated
    Protokoll

    Specimien Collection and Preparation:

    The specimens shall be blood, serum in type and the usual precautions in the collection of venipuncture samples should be observed. The blood should be collected in a plain redcap venipuncture tube without additives. Allow the blood to clot. Centrifuge the specimen to separate the serum from the cells. Samples may be refrigerated at 2_x001E_8oC for a maximum period of five (5) days. If the specimen(s) can not be assayed within this time, the sample(s) may be stored at temperatures of _x001E_20oC for up to 30 days. Avoid repetitive freezing and thawing. When assayed in duplicate, 0.100ml of the specimen is required for LH and FSH. For prolactin and hCG, 0.050ml of sample is needed. _x000C_

    Reagent Preparation:

    1. Wash Buffer Dilute contents of Wash Concentrate to 1000ml with distilled or deionized water in a suitable storage container. Store at room temperature until expiration date printed on concentrate label. 2. Working Signal Reagent Solution - Store at 2 - 8(C. Determine the amount of reagent needed and prepare by mixing equal portions of Signal Reagent A and Signal Reagent B in a clean container. For example, add 1 ml of A and 1ml of B per two (2) eight well strips (A slight excess of solution is made). Discard the unused portion if not used within 36 hours after mixing. If complete utilization of the reagents is anticipated, within the above time constraint, pour the contents of Signal Reagent B into Signal Reagent A and label accordingly. TEST PROCEDURE (HCG, LH & FSH): Before proceeding with the assay, bring all reagents, serum references and controls to room temperature (20 - 27( C). 1. Format the microplates wells for each serum reference, control and patient specimen to be assayed in duplicate. Replace any unused microwell strips back into the aluminum bag, seal and store at 2-8(C. 2A. For hCG: Pipette 0.025ml (25l) of the appropriate serum reference, control or specimen into the assigned well. 2B. For LH and FSH: Pipette 0.050ml (50l) of the appropriate serum reference, control or specimen into the assigned well. 3. Add 100 l of the appropriate tracer reagent to each well. It is very important to use the right Tracer Reagent for each assay for correct results. 4. Swirl the microplate gently for 20-30 seconds to mix and cover. 5. Incubate 45 minutes at room temperature for LH and/or FSH or 20 minutes for hCG. 6. Discard the contents of the microplate by decantation or aspiration. If decanting, tap and blot the plate dry with absorbent paper. 7. Add 350l of wash buffer (see Reagent Preparation Section), decant (tap and blot) or aspirate. Repeat two (4) additional times for a total of five (5) washes. An automatic or manual plate washer can be used. Follow the manufacturers instruction for proper usage. If a squeeze bottle is employed, fill each well by depressing the container (avoiding air bubbles) to dispense the wash. Decant the wash and repeat four (4) additional times. 8. Add 100 l of Working Signal Reagent to all wells. 9. Incubate in the dark for five (5) minutes. 10. Read the Relative Light Units (RLU) in each well using microplate luminometer. The results should be read within thirty (30) minutes of adding the working signal reagent. Note: It is very important to dispense all reagents in the center of the coated well. Always add reagents in the same order to minimize reaction time differences between wells.

    Test Procedure:

    (HCG, LH & FSH): Before proceeding with the assay, bring all reagents, serum references and controls to room temperature (20 - 27( C). 1. Format the microplates wells for each serum reference, control and patient specimen to be assayed in duplicate. Replace any unused microwell strips back into the aluminum bag, seal and store at 2-8(C. 2A. For hCG: Pipette 0.025ml (25l) of the appropriate serum reference, control or specimen into the assigned well. 2B. For LH and FSH: Pipette 0.050ml (50l) of the appropriate serum reference, control or specimen into the assigned well. 3. Add 100 l of the appropriate tracer reagent to each well. It is very important to use the right Tracer Reagent for each assay for correct results. 4. Swirl the microplate gently for 20-30 seconds to mix and cover. 5. Incubate 45 minutes at room temperature for LH and/or FSH or 20 minutes for hCG. 6. Discard the contents of the microplate by decantation or aspiration. If decanting, tap and blot the plate dry with absorbent paper. 7. Add 350l of wash buffer (see Reagent Preparation Section), decant (tap and blot) or aspirate. Repeat two (4) additional times for a total of five (5) washes. An automatic or manual plate washer can be used. Follow the manufacturers instruction for proper usage. If a squeeze bottle is employed, fill each well by depressing the container (avoiding air bubbles) to dispense the wash. Decant the wash and repeat four (4) additional times. 8. Add 100 l of Working Signal Reagent to all wells. 9. Incubate in the dark for five (5) minutes. 10. Read the Relative Light Units (RLU) in each well using microplate luminometer. The results should be read within thirty (30) minutes of adding the working signal reagent. Note: It is very important to dispense all reagents in the center of the coated well. Always add reagents in the same order to minimize reaction time differences between wells. (PROLACTIN): 1. Format the microplates wells for each serum reference, control and patient specimen to be assayed in duplicate. Replace any unused microwell strips back into the aluminum bag, seal and store at 2-8(C. 2. Pipette 0.025 ml (25l) of the appropriate serum reference, control or specimen into the assigned well. 3. Add 100l of the sequential PRL Biotin Reagent to each well. It is very important to use the right Tracer Reagent for each assay for accurate results. 4. Swirl the microplate gently for 20-30 seconds to mix and cover. 5. Incubate 30 minutes at room temperature. 6. Discard the contents of the microplate by decantation or aspiration. If decanting, tap and blot the plate dry with absorbent paper. 7. Add 350l of wash buffer (see Reagent Preparation Section), decant (tap and blot) or aspirate. Repeat two (4) additional times for a total of five (5) washes. An automatic or manual plate washer can be used. Follow the manufacturers instruction for proper usage. If a squeeze bottle is employed, fill each well by depressing the container (avoiding air bubbles) to dispense the wash. Decant the wash and repeat four (4) additional times. 8. Add 100l of the sequential Prolactin Tracer to each well. 9. Incubate 30 minutes at room temperature. 10. Follow steps 6 through 10 as outlined in the procedure for HCG, LH & FSH above.
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  • Target
    hCG, PRL, LH, FSH
    Hintergrund
    Human chorionic gonadotropin (hCG) concentration increases dramatically in blood and urine during normal pregnancy. hCG is secreted by placental tissue, beginning with the primitive trophoblast, almost from the time of implantation, and serves to support the corpus luteum during the early weeks of pregnancy. hCG or hCG similar glycoproteins can also be produced by a wide variety of trophoblastic and nontrophoblastic tumors. The measurement of hCG, by assay systems with suitable sensitivity and specificity has proven great value in the detection of pregnancy and the diagnosis of early pregnancy disorders. hCG is detectable as early as 10 days after ovulation, reaching 100 mIU/ml by the first missed period. A peak of 50,000 to 100,000mIU/ml is attained by the third month, then a gradual decline is observed (2, 3,18). Prolactin hormone (PRL), secreted from the lactotrophs of the anterior pituitary, is a protein consisting of a single polypeptide chain containing approximately 200 amino acids. The primary biological action of the hormone is on the mammary gland where it is involved in the growth of the gland and in the induction and maintenance of milk production. There is evidence to suggest that prolactin may be involved in steroidogenesis in the gonad, acting synergistically with luteinizing hormone (LH). High levels of prolactin appear to inhibit steroidogenesis as well as inhibiting LH and follicle stimulating hormone (FSH) synthesis at the pituitary gland (4, 24, and 25). The clinical usefulness of the measurement of prolactin hormone (PRL) in ascertaining the diagnosis of hyperprolactinemia and for the subsequent monitoring the effectiveness of the treatment has been well established (26). Luteinizing hormone (LH) is a glycoprotein consisting of two subunits with a molecular mass of 30,000 daltons. The (-subunit is similar to other pituitary hormones (follicle stimulating hormone (FSH), thyroid stimulating hormone (TSH) and chorionic gonadotropin (CG)( while the (-subunit is unique. The (-subunit confers the biological activity to the molecule. The (-subunit consists of 89 amino acid residues while the (-subunit contains 129 amino acids. The carbohydrate content is between 15% and 30%. (6, 8, 9). Follicle Stimulating hormone (FSH) is a glycoprotein consisting of two subunits with an approximate molecular mass of 35,500 daltons. The (-subunit is similar to other pituitary hormones (luteinizing stimulating hormone (LH), thyroid stimulating hormone (TSH) and chorionic gonadotropin (CG)( while the (-subunit is unique. The (-subunit confers the biological activity to the molecule. Stimulation by gonadotropin-releasing hormone (GnRH) causes release of FSH, as well as LH, from the pituitary and is transported by the blood to their sites of action, the testes or ovary. The clinical usefulness of the measurement of luteinizing hormone (LH) in ascertaining the homeostasis of fertility regulation via the hypothalamic - pituitary - gonadal axis has been well established (4,6). In addition, the advent of in vitro fertilization (IVF) technology to overcome infertility associated problems has provided the impetus for rapid improvement in LH assay methodology from the technically demanding bioassay to the procedurally simple and rapid immunoenzymometric assays. In men, FSH acts on the Sertoli cells of the testis, stimulating the synthesis of inhibin, which appears to specifically inhibit further FSH secretion, and androgen-binding protein. Thus, it indirectly supports spermatogenesis (9,10). _x000E_In women, FSH acts on the granulosa cells of the ovary, stimulating steroidogensis. All ovulatory menstrual cycles have a characteristic pattern of FSH, as well as LH, secretion. The menstrual cycle is divided into a follicular phase and a luteal phase by the midcycle surge of the gonadotropins (LH and FSH). As the follicular phase progresses, FSH concentration decreases. Near the time ovulation occur, about midcycle, FSH peaks (lesser in magnitude than LH) to its highest level (8). In this method, hCG/PRL//LH/FSH (referred to as antigens, in the Product Insert) combination calibrator, patient specimen or control is first added to a streptavidin coated well. Biotinylated monoclonal and enzyme labeled antibodies (directed against distinct and different epitopes of the hormones) are added and the reactants mixed. Reaction between the various antibodies (specific to the respective hormones) and native hormones forms a sandwich complex that binds with the streptavidin coated to the well. In the PRL procedure, a sequential method of antibody addition is followed. That is, the biotinylated antibody is introduced first, and after an appropriate reaction period, the plate is washed. Than an enzyme liked antibody, directed against a different epitope is added and the plate is processed as the other antigens.. After the completion of the required incubation period(s), the antigen antibody enzyme bound conjugate is separated from the unbound enzyme antigen conjugate by aspiration or decantation. The activity of the enzyme present on the surface of the well is quantitated by reaction with a suitable substrate to produce light. The employment of several serum references of known hormone levels permits construction of a dose response curve of activity and con_x001F_centration. From comparison to the dose response curve, an unknown specimen's activity can be correlated with the specific hormone concentration.
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