I= SOCIO – ECONOMIC CONSEQUENCES OF RETURN MIGRATION AND THE ADJUSTMENT PROBLEMS IN PUNJAB Title of Thesis


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Title of Thesis

THE DEVELOPMENT AND STANDARDIZATION OF MAGNETIZABLE SOLID STATE REAGENTS FOR RIA PROCEDURES AND THEIR DIAGNOSTIC APPLICATIONS FOR THE EVALUATION OF THYROID FUNCTION IN TUBERCULAR

Author(s)

Khan Mohammad Sajid

Institute/University/Department Details

Bahauddin Zakariya University, Multan / Chemistry

Session

March, 2000

Subject

Materials Science

Number of Pages

279

 

Keywords (Extracted from title, table of contents and abstract of thesis)

Magnetizable Solid State Reagents, Thyroid Function, Tubercular, Thyroid Hormones,Tsh, Tsh-Ria, Tsh-Irma, Magnetic Particles (Cellulose-Iron Oxide), DAMP (Double Antibody Magnetic Particles), Hypothyroid, Hyperthyroid, PTB (Pulmonary Tuberculosis)font>

 

Abstract

In order to replace expensive ready-made commercial radioimmunoassay kits with high quality local techniques a three phases study was conducted. Phase-1: Development of magnetic solid phase immunoassays. The work was started with the development of antisera and proceeded through preparation and standardization of reagents to end at in house techniques. Phase-2: The evaluation of locally developed techniques. Intrassay, interassay and interlaboratory quality control was established. Phase-3: The assays were applied to normal people and tuberculous patients. The clinical study on TB patients was based on the hypothesis that in tuberculosis the release of thyroid hormones known to regulate basal metabolic rates is decreased due to decrease in sensitivity to TSH as has been reported in many other infections1, 2. Estimation of titre of T3 antisera in immunized rabbits showed that 3 out of 4 immunized rabbits developed significant quantities of antibodies with a maximum titre of 1:8500. Radioiodination of T3 to produce radiotracers (125I- T3 and 125I- T4) by chloramines-T method showed that 31 % radioactivity was bound to T3 and 50% to T4 with specific activities of 300-400 uCi/ug and 50-60 uCi/ug respectively. RIA of T3 using 15%PEG precipitation was imprecise at low concentration levels (working range: 4-30 nmol/l), although it worked well at higher levels. Results of T3-RIA using PEG assisted second antibody precipitation were improved (working range: of 0.4 to > 10 nmol/l) and showed good correlation with commercial techniques (ICN, USA; corr.coeff=0.93). Four out of 12 rabbits immunized to develop T4 antibody titres responded well to the immunogen and a maximum titre of 1:9700 was achieved with a working dilution from 1: 1 000 to 1: 10000. Second antibody for T3 and T4 RIAs was raised in two sheep. Results of optimization of second antibody showed a working dilution of 1:30 for T3-RIA and 1:60 for T4-RIA. Double antibody T4- RIA showed more than 7% error in, the concentrations below 10 nmol/l with high precision at higher concentrations. The results correlated well with the commercial techniques. The TSH-RIA was also developed using 2nd antibody precipitation. The standards were prepared in phosphate buffered saline containing BSA using a commercial TSH preparation (SIGMA, USA). The radiotracer produced by chloramines-T method was of specific activity 400 uCi/ug with 90% of radioactivity tagged to the hormone. TSH-RIA using double antibody precipitation gave some serious problems. %NSB (non-specific binding) increased with time whereas radiochemical purity decreased. The tracer was of short shelf life and the procedure was very long. It was therefore decided to work on TSH-IRMA. For magnetizable solid phase T3 and T4-RIAs the second antibody raised in sheep was coated onto the magnetic particles (cellulose-iron oxide) to prepare DAMP (double antibody magnetic particles). Magnetic solid phase T3-RIA employing DAMP was very precise with wide working range <0.2 to >50 nmol/l) and low drift. Similar work on development of T4-RIA produced a very precise system with a working range of (5 to >1000 nmol/l). To develop magnetic solid phase TSH-IRMA, anti-TSH antibody was first raised in three sheep and an antibody titre of 1:90000 was achieved. IgG was isolated from antiserum by chromatographic filtration and coated on the cellulose-iron oxide. The TSH-IRMA established using magnetic particles was a very precise system with a working range of 0.15 to >60 uIU/ml). The qualitative comparison of magnetic solid phase assays with commercial techniques and analysis of passive internal QC data collected on 219 T3 assay batches, 219 T4 assay batches and 241 TSH assay batches showed that our assays were finally applicable in every respect. Some systematic trends in our house magnetic immunoassays, which were detected the beginning were removed with experience. Data collected under EQAS (external quality assurance scheme) showed an agreement of our results with other laboratories. The reference ranges determined in 713 normal persons (males 274, females 439 age: 10-66 years) were, T3=0.8-3.2 nmol/l (Mean=2.03), T4=52-175 nmol/l (Mean=113.1), TSH=0.5-7.1uIU/ml (Mean=3.15). The reference ranges determined in 453 clinically proven patients of hypothyroidism and 481 patients of hyperthyroidism were, (a). Hypothyroid patients: T3=0-1.0 nmol/l (Mean=0.6), T4=2.2-57 nmol/l (Mean=29.61), TSH=5.8-160 uIU/ml (Mean=48.2). (b). Hyperthyroid patients: T3=2.8-15.4 nmol/l (Mean=6.01), T4=163--455 nmol/l (Mean=245), TSH= 0.1-1.0 uIU/ml (Mean=0.3). Comparison of these values with normal persons showed that there is an overlapping of values of these patients with the normal persons at the borders but the number of the patients giving results equivocal with the normal persons is small. This showed that our results are completely applicable to the patients with altered thyroid function. The diagnostic application of our assays to 266 cases of confirmed PTB (pulmonary tuberculosis) cases (males 176, females 90, age 11-65 years) patients started with selection and collection of samples. The sensitivities of assays applied were T3=0.01 nmol/l, T4=2.5nmol/l, TSH=0.1 uIU/ml. The observed ranges in these patients were T3=0.1-1.8 nmol/l, T4=13-l75 nmol/l, TSH=0.5-7.3uIU/ml. The T3 values in TB patients were significantly decreased compared to normal persons (p<0.00I) with 50% subjects in hypothyroid range. The T4 values lied mostly within normal limits and only 11.6% of total patients showed values in hypothyroid range. The comparison of T4 values with normal population however showed a shift towards the lower side indicating suppressing effect of the disease. Comparison of T4 levels of males and females also showed that levels are more affected in males than females. The observed ranges for TSH values in TB patients suggest almost no effect of the disease on TSH levels. 31 patients who showed both T3 and T4 levels in hypothyroid range were requested to donate two more samples, one during the course of treatment (3 months after start of treatment) and one after the completion of treatment. The comparison of results of three measurements showed that the T3 and T4 levels reached to normal values after completion of treatment. There is almost no significant effect of treatment on TSH values. It is therefore confirmed that release of T3 and T4 is decreased in pulmonary-tuberculosis with almost no effect on TSH secretion.

 

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    Table of Contents  

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1 1 GENERAL INTRODUCTION TO THE PROPOSED WORK 3

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2 2 DEVELOPMENT OF T3RADIOIMMUNOASSAY 6

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3 3 DEVELOPMENT OF T4 RADIOIMMUNOASSAY WITH PEG 41

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4 4 DEVELOPMENT OF TSH RADIOIMMUNOASSAY USING SECOND ANTIBODY 60

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5 5 REVIEW OF SEPARATION TECHNIQUES AND DEVELOPMENT OF MAGNETIC

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6 6 QUALITATIVE COMPARISON OF IN HOUSE MAGNETIC SOLID PHASE

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7 7 INTERNAL AND EXTERNAL QUALITY CONTROL OF IMMUNOASSAYS EMPLOYING

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8 8 THYROID HORMONE ECONOMY IN NORMAL PERSONS AND PATIENTS WITH

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9 9 DIAGNOSTIC APPLICATIONS OF MAGNETIZABLE SOLID STATE REAGENTS FOR

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10 10 REFERENCES

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