Fluorescence Polarization Immunoassay FPIA

1. Fluorescence Polarization Immunoassay FPIA

2. Fluorescence Polarization ImmunoassayFPIA • Fluorescence polarization is ideal for the study of small molecule fluorescent ligands binding to receptors. • In fluorescent polarization method of assay, polarized light excites a fluorescent label. As this label is bound, the degree of polarization of the fluorescent emission from the label increases.

3. FPIA reagent components • S: Antibody Reagent: Antiserum to analyte; • T: Tracer: Fluorescein-labeled analyte; • P: Pretreatment detergent: facilitate release of drug from serum proteins.

4. Key concepts in FPIA assay • FPIA utilizes three key concepts to measure specific analytes in a homogeneous format: fluorescence, rotation of molecules in solution, and polarized light. • Fluorescence: Fluorescein is a fluorescent label. It absorbs light energy at 490nm and releases this energy at a higher wavelength (520nm) as fluorescent light • fluorescence polarization immunoassays combine two technologes to determine analyte concentration: • competitive protein binding • fluorescence polarization

5. FPIA technology principle • ..\Fluorescence Polarization.mp4 • Polarized light can be used to produce a polarized flurorescent emission from the analyte-tracer. • The average polarization of the emitted fluorescence is related to the speed of rotation of the molecule. • The rate of molecule rotation in liquid is related to the size of the molecule. Small, unbound analytes rotate more rapidly than the larger analyte-antibody complex. • Axsym system undergo to this priniciple.

6. FPIA reaction sequence: • A typical FPIA reaction would occur as follows: • Sample, pretreatment reagent, and line diluent are combined and incubated at reaction temperature. An initial read is taken by the FPIA optics system as a blank for the assay.

7. Competitive binding occurs: • Antibody, tracer, line diluent and more sample are added to the reaction mixture and incubated. • The analyte from the sample competes with the analyte-tracer for binding sites on the antibodies. • If the sample contains a high concentration of the analyte, the sample analyte binds to the antibodies, leaving the analyte-tracer unbound. • If the sample contains a low concentration (or no concentration) of the analyte, then few or no sample analyte molecules bind to the antibodies, leaving the antibodies open for the analyte tracer.

8. If the sample contains a high concentration of the analyte, then many analyte-tracer molecules are left unbound. When excited by polarized vertical light, these small fluorescent molecules rotate rapidly, emitting light in many different planes. The result is a decrease in polarization. • If the sample contains a low concentration (or no concentration ) of the analyte, then the analyte-tracer binds to the antibodies, resulting in few unbound analyte-tracer molecules. These large molecules (complexes of analyte-tracer bound to antibody) rotate slower. Rotation is slowed enough so that light is emitted in a single plane. The result is an increase in polarization.

9. Change in polarization of emitted fluorescence indicates analyte concentration. • FPIA optics detect and measure the change in polarization of emitted fluorescence, which is inversely proportional to the concentration of the analyte in the specimen.

10. Advantages& disadvantages • FPIA is utilized to provide accurate and sensitive measurement of small toxicology analytes such as therapeutic drugs, and drugs of abuse, toxicology and some hormones • Disadvantages of this method are nonlinear assay when the concentration of the test compound is high • Also, this type of assay has a limiting assayed can be accurately measured.

13. FPIA results in an inverse dose response curve such that lower levels of patient analyte result in a higher signal (in this case, the signal is polarized light). High signal at low patient analyte levels results in a highly sensitive Assay.

14. Fertility testing Prolactin (PRL)

15. Introduction • Fertility denotes the ability of a man and woman to reproduce; conversely, infertility denotes the lack of fertility—an involuntary reduction in the ability to produce children. • When a couple has been engaging in regular, unprotected sexual intercourse for at least 1 year without conceiving, the couple is considered infertile. • In about one third of cases, a male factor is the predominant cause; • In another one third, the female factor predominates; and • In another onethird, no cause is found in either partner.

16. The workup for infertility starts with a complete history and physical exam for both the woman and the man, including their sexual history. • A rational approach is to put each partner through a series of tests that generally cover a vast majority of the contributing factors of infertility. These tests usually take 2 to 3 months to complete.

17. Standard pretest and posttest care for couples undergoing fertility testing includes the following: • Provide information and support. Be sensitive to the couple’s need for privacy and confidentiality. • Maintain a communication network about new procedures, tests, and treatments. • Help couples deal with feelings of sadness and loss. Assist couples to deal with the effects of stress and the financial burden during the diagnostic process. • Assist couples in arranging work and testing schedules with the least amount of disruption for the couple

18. Arrange for counseling with experts who understand the different ways infertility affects someone’s life. • Tests include evaluation of: • amenorrhea, • anovulation, • sperm count (oligospermia, Azoospermia) • hormone testing • hysterosalpingogram • laparoscopy • Hysteroscopy • fertiloscopy, • Semen analysis, • postcoital test • endometrial biopsy • chromosome karyotype to exclude Kallmann’s syndrome.

19. Hormonal testing • Rules pregnancy in or out, chorionic gonadotropin • Prolactin • Luteinizing hormone [LH] • Follicle-stimulating hormone [FSH] • Thyroid-stimulating hormone [TSH] • Estrogen testing.

20. Pregnancy trimesters Remember: • First Trimester (0 to 13 Weeks) • Second Trimester (14 to 26 Weeks) • Third Trimester (27 to 40 Weeks)

21. Prolactin

23. Synthesis and regulation

24. Prolactin • Prolactin (PRL, luteotropic hormone) is secreted from lactortrophs of the anterior pituitary gland in both men and woman. • It is a protein consisting of a single polypeptide biological action of the hormone is on the mammary gland where maintenance of milk production. • Molecular weight of approximately 23.000 Daltons. • The gender difference in prolactin does not occur until puberty, when increased estrogen production results in higher prolactin levels in females. • Women normally have slightly higher basal prolactin levels than men. • During and following pregnancy, prolactin, in association with other hormones, stimulates breast development and milk production.

25. Note: High levels of prolactin are normal during pregnancy and after childbirth while the mother is nursing.

26. why women who are breastfeeding (and thus have high levels of prolactin) usually don’t become pregnant??!. • High prolactin levels inhibit secretion of GnRH and FSH. Therefore, if your prolactin levels are high, your ovulation may be suppressed. • High levels of prolactin appear to inhibit steroidogenesis as well as inhibiting LH and FSH synthesis at the pituitary gland.

27. PRL role in fertility • Prolactin is a hormone that plays a role in fertility by inhibiting follicle stimulating hormone (FSH) and gonadotropin-releasing hormone (GnRH), the hormones that trigger ovulation and allow eggs to develop and mature. • It is unclear what role prolactin plays in men, but it is clearly linked to infertility.

28. Significance • The clinical usefulness of the measurement of prolactin hormone in ascertaining the diagnosis of hyperprolactinemia and for the subsequent monitoring the effectiveness of the treatment has been well established. • It is also useful in the management of hypothalamic disease • Monitoring the effectiveness of surgery, chemotherapy, and radiation treatment of prolactin-secreting tumors. • The prolactin test can also be performed if a woman is having infertilityproblems or irregular menstrual periods • Measurement of plasma prolactin also has been used as an index of response to the injection of TRH, which stimulates release of prolactin in addition to stimulating the release of TSH.

29. Hypersecretion of prolactin can be caused by pituitary tumors, hypothalamic diseases, hypothyroid, renal failure, acute exercise and several medications. • Hyperprolactinemialeads to hypogonadism in men and women with accompanying low FSH and LH levels. • Human Prolactin is similar in structure to human growth hormone(HGH), and both are good lactogenic.

30. Prolactinomas • High levels of prolactin can also be caused by pituitary tumors, which can be treated medically or surgically • Based on its size, a prolactinoma can be classified as a microprolactinoma (< 10 mm diameter) or a macroprolactinoma (>10 mm diameter). • Occur in both men and women but are more commonly diagnosed in women who are less than 50 years than in older women or men. • In cases of prolactinoma, the test is carried out regularly to check the tumor’s response to treatment.

31. For the interpretation of the finding of an increased plasma (prolactin), it is important to enquire about the intake of drug. • Many centrally acting drugs (phenothiazines, methyldopa, imipramine) inhibit the release of prolactin release-inhibiting hormone. • Oestrogens and oral contraceptive may also cause raised plasma prolactin

32. Sample collection and manipulation: • Serum: • Blood should be drawn using standard veinpuncture techniques and serum separated from blood cells as soon as possible. Samples should be allowed to clot for one hour at room temperature, centrifuged for 10 minutes (4°C) and serum extracted. • Avoid grossly hemolytic, lipemic or turbid samples. • Serum samples to be used within 24-48 hours may be stored at 2-8°C otherwise samples must be stored at -20°C to avoid loss of bioactivity and contamination. Avoid freeze-thaw cycles.

33. When performing the assay slowly bring samples to room temperature. • It is recommended that all samples be assayed in duplicate. • DO NOT USE HEAT-TREATED SPECIMENS. • This test cannot be made for mother at lactation period. • Patient should be avoid Emotional stress and strenuous exercise.

34. Procedure 1. Ensure that the patient fasts for 12 hours before testing. Obtain a 5-mL venous blood sample (red-topped tube). Serum is used. 2. Procure specimens in the morning, between 8:00 and 10:00 a.m. Draw in chilled tubes, and keep specimen on ice. 3. Observe standard precautions. Place specimen in a biohazard bag. The only result of prolactin deficiency in pregnancy is the absence of postpartumlactation.

35. Interfering Factors 1. Increased values are associated with newborns, pregnancy, postpartum period, stress, exercise, sleep, nipple stimulation, and lactation (breast-feeding). 2. Drugs (eg, estrogens, methyldopa, phenothiazines, opiates) may increase values. 3. Dopaminergic drugs inhibit prolactin secretion. Administration of L-dopa can normalize prolactin levels in galactorrhea, hyperprolactinemia, and pituitary tumor. 4. Increased levels are found in cocaine abuse, even after withdrawal from cocaine. 5. Macroprolactin can falsely increase test results.

36. Reference range • Male >18 years (4.0-15.2 ng/ml) • Female>18 years (4.8-23.3 ng/ml) • Levels >200 ng/mL or >200 μg/L in a nonlactating female indicate a prolactin-secreting tumor; • however, a normal prolactin level does not rule out pituitary tumor.

37. Thyroid dysfunction and infertility • Thyroid dysfunction is a condition known to reduce the likelihood of pregnancy. Additionally, abnormal thyroid hormones disturb the normal menstrual pattern. • Measurement of prolactin and thyroid hormones, especially thyroid stimulating hormone (TSH), has been considered an important component of infertility work up in women. • Thyroid dysfunctions interfere with numerous aspects of reproduction and pregnancy. • Several articles have highlighted the association of hyperthyroidism or hypothyroidism with menstrual disturbance, anovulatory cycles, decreased fertility and increased morbidity during pregnancy

39. Thyroid stimulation by chorionic gonadotropin: • The placentae of humans and other primates secrete huge amounts of a hormone called chorionic gonadotropin (in the case of humans, human chorionic gonadotropin or hCG) which is very closely related to LH. • TSH and hCG are similar enough that hCG can bind and transduce signalling from the TSH receptor on thyroid epithelial cells. Toward the end of the first trimester of pregnancy in humans, when hCG levels are highest, a significant fraction of the thyroid-stimulating activity is from hCG.

40. During this time, blood levels of TSH often are suppressed. The thyroid-stimulating activity of hCG actually causes some women to develop transient hyperthyroidism. • The net effect of pregnancy is an increased demand on the thyroid gland. In the normal individuals, this does not appear to represent much of a load to the thyroid gland, but in females with subclinical hypothyroidism, the extra demands of pregnancy can precipitate clinical disease.

41. The End