Biomedical subjects
Publications and source records attributed to D K Hazra.
A patient presented with hyperthyroidism and a thyroid swelling, clinically thought to be malignant. Fine-needle aspiration of the thyroid nodule revealed microfilariae of Wuchereria bancrofti. After therapy the thyroid swelling subsided and the patient became euthyroid. The possible etiologic role of microfilariae in the genesis of the thyroid tumor and hyperthyroidism is discussed.
Earlier studies described the linkage of silver to antibodies using SH groups generated by the reduction of the SS groups using ascorbic acid (1) analogous to the Thakur and DeFulvio technique for linking technetium to antibodies. This work describes the linkage of silver to IgG after introducing SH groups by coupling the IgG to 2-imino thiolane. The protein was dissolved in sodium acetate buffer pH 4.5 containing 1 mM EDTA by dialysis/gel chromatography in a concentration of 20 mg/mL. 2-Imino thiolane dissolved in Tris-HCl acetate buffer, pH 8.2, 0.2M was added to give a final dilution of 0.2 mM 2-imino thiolane. The excess of 2-imino thiolane was removed by dialysis or G-25 Sephadex gel chromatography and then the protein was reacted with silver nitrate 0.1 mM. The unreacted SH groups were blocked by adding iodoacetamide to a concentration of 5 mM. The nonprotein reagents again were removed by dialysis or gel chromatography. The thiol groups were titrated using 1.5 mM 2 2-Py-SS-Py prior to and after addition of silver. It was observed that depending on the concentration of silver, 50-80% of the SH groups were coupled to silver. Higher concentrations of silver led to insoluble precipitates and should be avoided.
The impact of recent advances in the chemical and genetic engineering manipulations of antibodies on radioimmunotargeting is reviewed both in relation to radioimmunoscintigraphy and radioimmunotherapy. The resulting trends are: (1) the linking of parts of the mouse/rat and human antibody molecule; (2) the creation of molecules with dual antigen or multiple antigen recognition capabilities; (3) the making of smaller and smaller antigen recognition molecules; and (4) the development of molecules with dual capabilities, e.g. antigen recognition and enzyme activity. The various methods of creating antibodies in vitro are reviewed with reference to bacteria, using phage selection and a combinatorial library, mammalian cells, yeast cells and, finally, mice containing giant yeast artificial chromosomes. The advantages and disadvantages of smaller fragments as well as of the human anti-mouse antibody (HAMA) reaction are discussed and the need for early clinical evaluation and widespread availability of the newer antibodies is emphasized. It is envisaged that these immunotechnological advances will permit the large-scale production of precisely engineered humanized antibodies, and the specificity and affinity rate constant of these antibodies can be optimized using in vitro phage selection as well as by computer modelling where the stereo chemistry of the antigen is known precisely.
Radiosilver-111 and Radiogold-199 were proposed by us (1) as suitable isotopes for radioimmunotherapy in areas such as India by reason of their suitable half-lives and B-emissions (Ag-111 T1/2 = 7.45 d and Au-199 T1/2 = 3.15 d). Since silver is monovalent, it is difficult to link to conventional bifunctional chelates. We therefore explored the use of sulfur-based linkers (2). Encouraged by the Thakur and De Fulvio Technique (3) of linking technetium to disulfide groups in antibodies reduced by ascorbic acid that is eminently biocompatible, we have explored the linkage of silver to immunoglobulin reduced by ascorbic acid. The linkage of silver was assessed with stable Ag-108 using dialysis to quantify the free silver after the reaction of silver and reduced immunoglobulins in various molar ratios (1:1, 1:2, 1:5, 1:10). The silver quantity was estimated gravimetrically after precipitation as chloride. It was observed that using these molar ratios there was negligible silver efflux into the dialysate, suggesting stable linkage. We also assessed the linkage using Ag-110M as radiotracer. The comparative results with the two techniques are described.
Despite attempts to maintain asepsis, good manufacturing practices, and the use of terminal sterilization by millipore filtration, the nuclear practitioner is always worried about the possibility of endotoxin contamination. Methods, such as ion-exchange chromatography, have been tried for removing endotoxins during the preparation of radiolabeled antibodies, and so on. As suggested by Stevenson (1990), we evaluated the Issekutz technique (1) of endotoxin removal by affinity chromatography using a polymyxin cyanogen bromide (CNBr) Sepharose column. The endotoxin content of millipore filtrates of heat killed/sonicated suspensions of Pseudomonas pyocyaneus, E. coli were measured using a Sigma (St. Louis, MO) Endotoxin Assay Kit before and after filtration through such columns and compared with the results obtained using gel exclusion and ion-exchange columns of the same length and diameter. Reduction of endotoxin content to undetectable levels by the polymyxin column was observed. The use of such columns for terminal endotoxin removal analogous to terminal sterilization is advocated especially when developing a radiopharmaceutical such as radiolabeled antibodies for in house use.
Since tubercular lesions form an important differential diagnosis in our country, tuberculoma models in mice have been created and imaged using iodine-125 radiolabelled antimycobacterial monoclonal antibodies for the aetiopathological diagnosis.
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Thirty cases of viral hepatitis with and without coma and 15 normal healthy age and sex matched controls were studied for disseminated intravascular coagulopathy (DIC). Cases of viral hepatitis with coma showed strong evidence of DIC in the form of increased plasma FDP (240 +/- 190.64 dilution), diminished fibrinogen half life (48.1 +/- 15.3 hours) and increased fractional catabolic rate (46.13 +/- 14.7% per day) with normal Euglobulin clot lysis time and reduced fibrinogen (147.33 +/- 43.5%) and platelet count (1.27 +/- 0.324 lakhs/cm.). The possible role of heparin in the reversal of coagulation abnormalities have been discussed.
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