, usa . tricine , 2-mercaptoethanol , hynic were purchased from sigma - aldrich , usa , and stannous sulfate was purchased from merck . five hundred milligram of higg was dissolved in 10 ml distilled water and this solution was drawn onto slide - a - lyzer dialysis cell and dialyzed overnight at 4c using normal saline . the contents of dialysis cell were drawn through millipore filter ( 0.22 micron ) and 0.8 mg of hynic solution was added in 100 l dmso . the ph was adjusted to 6.4 with 1.5 m sodium phosphate buffer and dialyzed for overnight at 4c in a sterile dialysis cell using 1.5 m sodium phosphate buffer . finally , aliquots were dispensed into 15 ml vials , frozen overnight at -20c , lyophilized , sealed under vacuum and stored in a refrigerator until use . hundred milligram of tricine was dissolved in 8 ml of distilled water and acidified with 0.5 ml of 2.0 m hcl . 0.5 ml of stannous sulphate solution ( 20 mg / ml ) was added and ph adjusted to 5.5 using 2 m sodium hydroxide solution . the contents were filtered through 0.22  millipore filter and stored overnight at -20c , lyophilized sealed under vacuum and stored in a refrigerator until use . tc - hynic - higg was synthesized according to procedures described previously , with slight modification . in brief , vial containing higg - hynic was thawed at room temperature and 60 l of freshly prepared trycine kit was added to it followed by addition of 20 - 30 mci tc - perthecnetate . the contents were dissolved by swirling the vial gently and incubated at room temperature for 20 min to complete the labeling reaction . strips ( 112 cm ) of itlc - sg , ( gelman , usa ) were used as stationary phase and acetone was used as the mobile phase to separate free pertechnetate and tc - hynic - higg , which moved with the solvent . citrate buffer ( ph 5.5 ) was used as the mobile phase to separate tc - hynic - higg , and tc - hynic . one hundred microliters of the radiolabeled antibody was incubated in 1.9 ml of saline or human serum at 37c . small aliquots were withdrawn at different time intervals up to 24 h and radiolabeling efficiency was evaluated by itlc using citrate buffer as mobile phase . aliquots of 0.5 ml of tc - hynic - higg ( 100 l ) were challenged over a range of cysteine and dtpa concentrations ( 25100 mmol / l ) at 37c for 1 h. the percentage of tc displaced by cysteine and dtpa was determined by itlc - sg using normal saline as mobile phase . ( labeled - hynic - hynic , rf = 0.00.15 , free tc , rf = 0.751.0 and cystine / dtpa- tc , rf = 0.60.7 ) . hundred microliters of the radiolabeled antibody was mixed with 2.0 ml of dichloromethane and 1.9 ml of normal saline . the mixture was kept at room temperature for 1 h and radioactive counts of the two separated phases were taken using counter ( capintec , usa ) to determine per cent lipophilicity of the radiolabeled product . one hundred microliters of tc - hynic - higg , was mixed with 1.9 ml of fresh serum and the contents were kept at 37c for 1 h. 2 ml of 10% trichloric acetic acid was added to the mixture and the contents were centrifuged for 5 min at 3500 rpm . per cent radioactive counts in supernatant and precipitate fractions were noted using g - counter ( capintec , usa ) to determine extent of protein binding . all animal experiments were conducted in accordance with our institutional guidelines , and the experimental procedure was approved by the institutional animal ethical committee duly constituted for the purpose . biodistribution experiments were performed by intravenous administration of 100 l of tc- hynic - higg ( 1 ci ) to strain - a mice ( 2540 g ; n = 10 ) . organs of interest , namely , heart , liver , lungs , spleen , kidneys , stomach , intestine and muscle were removed , washed with saline and weighed , and respective radioactivity counts were determined with the help of a g - counter ( capintec , usa ) . 200 l of tc - hynic - higg ( 100 ci ) was intravenously administered to rabbits ( n = 5 ) through the ear vein . radioactivity per unit volume was calculated by counting a known volume from the sample at each time point . whole blood was considered as 7% of the body weight to calculate the percentage of administered radioactivity present in total body blood . a group of three male new zealand rabbits , weighing 23 kg were used in the immunogenicity studies . infection was induced by inoculation of e. coli ( atcc 22923 ) into the left thigh muscle . after 45 days , when gross swelling was apparent in the infected thigh , 3 mci of tc - hynic - higg was injected intravenously via ear vein . the anesthetized animal was placed in the supine position and scanned under the gamma camera . anterior whole body image and spot views of the lower extremities were obtained hourly for the first 2 h and subsequently at 24 h. acute aseptic inflammation was induced in three other new zealand white rabbits , weighing 23 kg with turpentine . commercial grade turpentine liquid was sterilized by ultrafiltration using a 0.45 nm bio - filter . each animal received 0.1 ml of the sterile turpentine liquid by intramuscular injection into the right thigh muscle . the rabbits were then intravenously administered with 3.0 mci of tc - hynic - igg via ear vein . the anesthetized animal was placed in the supine position and scintigraphy was done under the gamma camera . normal biodistribution study was carried out using tc- hynic - higg scintigraphy in human volunteers . briefly , 10.0 mci tc - hynic - higg ( equivalent of 2.0 mg higg ) was administered in normal male volunteers ( n = 2 ) intravenously for the acquisition of imaging data . anterior and posterior whole - body images were obtained immediately at 6 h and 24 h post - injection using a dual head hawkeye gamma camera system ( ge , milwaukee , usa ) using the inbuilt software entegra version-2 . regions of interest ( roi ) were constructed at each time point for the whole body and all organs visualized . the geometric mean of activity in these regions was calculated and corrected for radioactive decay . organ radioactivity , as a fraction of the administered dose , was calculated by using the immediate whole body as 100% of the injected dose . from the imaging results , the residence time for tc - hynic - higg in the major source organs ( heart chambers , kidneys , liver , lungs , spleen , and the remainder whole body ) results of biodistribution experiments were statistically analyzed using a one - way anova followed by the dunnett post - hoc test . , usa . tricine , 2-mercaptoethanol , hynic were purchased from sigma - aldrich , usa , and stannous sulfate was purchased from merck . five hundred milligram of higg was dissolved in 10 ml distilled water and this solution was drawn onto slide - a - lyzer dialysis cell and dialyzed overnight at 4c using normal saline . the contents of dialysis cell were drawn through millipore filter ( 0.22 micron ) and 0.8 mg of hynic solution was added in 100 l dmso . the ph was adjusted to 6.4 with 1.5 m sodium phosphate buffer and dialyzed for overnight at 4c in a sterile dialysis cell using 1.5 m sodium phosphate buffer . finally , aliquots were dispensed into 15 ml vials , frozen overnight at -20c , lyophilized , sealed under vacuum and stored in a refrigerator until use . hundred milligram of tricine was dissolved in 8 ml of distilled water and acidified with 0.5 ml of 2.0 m hcl . 0.5 ml of stannous sulphate solution ( 20 mg / ml ) was added and ph adjusted to 5.5 using 2 m sodium hydroxide solution . the contents were filtered through 0.22  millipore filter and stored overnight at -20c , lyophilized sealed under vacuum and stored in a refrigerator until use . tc - hynic - higg was synthesized according to procedures described previously , with slight modification . in brief , vial containing higg - hynic was thawed at room temperature and 60 l of freshly prepared trycine kit was added to it followed by addition of 20 - 30 mci tc - perthecnetate . the contents were dissolved by swirling the vial gently and incubated at room temperature for 20 min to complete the labeling reaction . strips ( 112 cm ) of itlc - sg , ( gelman , usa ) were used as stationary phase and acetone was used as the mobile phase to separate free pertechnetate and tc - hynic - higg , which moved with the solvent . citrate buffer ( ph 5.5 ) was used as the mobile phase to separate tc - hynic - higg , and tc - hynic . one hundred microliters of the radiolabeled antibody was incubated in 1.9 ml of saline or human serum at 37c . small aliquots were withdrawn at different time intervals up to 24 h and radiolabeling efficiency was evaluated by itlc using citrate buffer as mobile phase . aliquots of 0.5 ml of tc - hynic - higg ( 100 l ) were challenged over a range of cysteine and dtpa concentrations ( 25100 mmol / l ) at 37c for 1 h. the percentage of tc displaced by cysteine and dtpa was determined by itlc - sg using normal saline as mobile phase . ( labeled - hynic - hynic , rf = 0.00.15 , free tc , rf = 0.751.0 and cystine / dtpa- tc , rf = 0.60.7 ) . hundred microliters of the radiolabeled antibody was mixed with 2.0 ml of dichloromethane and 1.9 ml of normal saline . the mixture was kept at room temperature for 1 h and radioactive counts of the two separated phases were taken using counter ( capintec , usa ) to determine per cent lipophilicity of the radiolabeled product . one hundred microliters of tc - hynic - higg , was mixed with 1.9 ml of fresh serum and the contents were kept at 37c for 1 h. 2 ml of 10% trichloric acetic acid was added to the mixture and the contents were centrifuged for 5 min at 3500 rpm . per cent radioactive counts in supernatant and precipitate fractions were noted using g - counter ( capintec , usa ) to determine extent of protein binding . all animal experiments were conducted in accordance with our institutional guidelines , and the experimental procedure was approved by the institutional animal ethical committee duly constituted for the purpose . biodistribution experiments were performed by intravenous administration of 100 l of tc- hynic - higg ( 1 ci ) to strain - a mice ( 2540 g ; n = 10 ) . organs of interest , namely , heart , liver , lungs , spleen , kidneys , stomach , intestine and muscle were removed , washed with saline and weighed , and respective radioactivity counts were determined with the help of a g - counter ( capintec , usa ) . 200 l of tc - hynic - higg ( 100 ci ) was intravenously administered to rabbits ( n = 5 ) through the ear vein . radioactivity per unit volume was calculated by counting a known volume from the sample at each time point . whole blood was considered as 7% of the body weight to calculate the percentage of administered radioactivity present in total body blood . a group of three male new zealand rabbits , weighing 23 kg were used in the immunogenicity studies . infection was induced by inoculation of e. coli ( atcc 22923 ) into the left thigh muscle . after 45 days , when gross swelling was apparent in the infected thigh , 3 mci of tc - hynic - higg was injected intravenously via ear vein . the anesthetized animal was placed in the supine position and scanned under the gamma camera . anterior whole body image and spot views of the lower extremities were obtained hourly for the first 2 h and subsequently at 24 h. acute aseptic inflammation was induced in three other new zealand white rabbits , weighing 23 kg with turpentine . commercial grade turpentine liquid was sterilized by ultrafiltration using a 0.45 nm bio - filter . each animal received 0.1 ml of the sterile turpentine liquid by intramuscular injection into the right thigh muscle . the rabbits were then intravenously administered with 3.0 mci of tc - hynic - igg via ear vein . the anesthetized animal was placed in the supine position and scintigraphy was done under the gamma camera . normal biodistribution study was carried out using tc- hynic - higg scintigraphy in human volunteers . briefly , 10.0 mci tc - hynic - higg ( equivalent of 2.0 mg higg ) was administered in normal male volunteers ( n = 2 ) intravenously for the acquisition of imaging data . anterior and posterior whole - body images were obtained immediately at 6 h and 24 h post - injection using a dual head hawkeye gamma camera system ( ge , milwaukee , usa ) using the inbuilt software entegra version-2 . regions of interest ( roi ) were constructed at each time point for the whole body and all organs visualized . the geometric mean of activity in these regions was calculated and corrected for radioactive decay . organ radioactivity , as a fraction of the administered dose , was calculated by using the immediate whole body as 100% of the injected dose . from the imaging results , the residence time for tc - hynic - higg in the major source organs ( heart chambers , kidneys , liver , lungs , spleen , and the remainder whole body ) results of biodistribution experiments were statistically analyzed using a one - way anova followed by the dunnett post - hoc test . the potential value of radiolabeled higg as an imaging agent for detecting focal infection has been previously reported . an indirect   in - labelling via chelating group attached to the antibody has been exploited successfully ; this labeling approach is not preferred due to the variable binding affinity of in to the antibody 's amino groups , which may partly be located in the hypervariable region of the antibody molecule , thereby altering its immunoreactivity . the radiolabeling procedure being reported here for labeling higg with tc-99 m using hynic gives consistently high ( > 96% ) radiolabeling efficiency with respect to the radiolabeled product [ table 1 ] . stability of higg kit ( shelf life ) was also evaluated by storing the kit for different time periods . results indicate that the kit is stable up to three months with only 1.6% reduction in radiochemical purity of the tc-99 m labeled product [ table 2 ] . in vitro stability study of radiolabeled higg was studied in saline at room temperature and in human serum at 37c till 24 h [ table 3 ] , up to 4 h , no free pertechnetate was detected irrespective of the storage condition , indicating no leaching of tc-99 m from the product . even at 24 h the free pertechnetate content were only 5.1% and 9.5% in case of serum and saline , respectively . high binding affinity of tc - higg was ascertained by allowing the tagged protein to incubate with dtpa and cysteine at different molar ratios [ table 4 ] . dtpa at a molar concentration of 25 and 50 times that of higg decreased labeled higg by only 3.1% and 7.2% respectively . however , when dtpa was increased 100 times , the labeled percentage of higg was reduced by 11.83% . in case of transchelation with cysteine the reduction of the labeled higg was more pronounced . even at a molar ratio of 25 times the decrease was about 5.8% , which increased to 23.7% at 100 times cysteine concentration . the above observations suggested a high binding affinity of higg  sh group for tc-99 m . lipophilicity study of the labeled product showed good lipophilic character of the molecule [ table 5 ] . optimization of radiolabeling efficiency of tc - higg with different concentration of reducing agent using itlc method of quality control stability of higg kit after storing these kits for different time period stability of 99m - higg when incubated at room temperature in saline and at 37c in human serum , respectively , using itlc method of quality control different molar concentration of dtpa and cysteine were mixed with tc - higg . each value is the mean of three separate experiments of the same preparation lipophilicity profile of the 99mtc - hynic - higg biodistribution of 99mtc - hynic - higg in mice at 4 h and 24 h after intravenous administration is shown in figure 1 . among the various organs studied , significant accumulation of the radiotracer was found in liver ( 4.9% ) , kidney ( 3.7% ) and intestine ( 2.1% ) at 4 h interval . by 24 h interval the remaining organs ( heart , lungs , spleen , stomach and muscles ) had very low accumulation of the radiotracer . low activity in the stomach , suggest an insignificant amount of free pertechnetate in the tc - higg preparation , further confirming the in vivo stability of the radiolabeled higg molecule . significant amount of radioactivity in liver , intestine and kidneys suggest hepatobiliary as well as renal route of clearance for tc - hynic - higg . blood clearance data in rabbits after intravenously administering 2 - 3 mci of tc - hynic - higg exhibited slow and biphasic clearance [ figure 2 ] . at 1 h , 15.1% of the injected radioactivity was present in blood , which reduced to 4.93% by 24 h. biodistribution of tc - hynic - higg . the animal were intravenously administered with 1 uci tracer and the radioactivity in various organ was measured at 4 and 24 h. each value is the mean of 6 mice expressed in % dose administered per organ blood clearance of tc - hynic - higg in rabbit after administering it intravenously . each value is the mean of three independent experiments anatomical delineation of the extent of focal inflammation is critical to clinical management of infectious processes , both for diagnosis and for monitoring the response to therapy . in our experiments , acute inflammatory lesion was developed within 3 days following the injection of sterile turpentine liquid . an opaque creamy whitish lesion developed at the site of injection surrounded by inflammatory tissue . no pus was found in the inflamed tissue during autopsy and the entire inflamed thigh muscle tissue measured about 3 cm in size . the site of inflammation appeared as an area of diffuse uptake of tc- hynic - higg during scintigraphy . in animals that were injected with e. coli to generate an infection model , abscess formation occurred within 78 days post inoculation of e. coli . a small firm mass measuring less than 1 cm in size was found in the infected thigh with noticeable gross swelling . the anterior whole body and spot scintigraphy images at 2 h and 24 h showed increased uptake of tc - hynic - higg , with the area seen as a focal hot spot , indicating good localization of the radiolabeled higg at the site of infection [ figure 3 ] . the image of an inflammatory lesion of patient [ figure 4 ] obtained using a spect camera after administering tc - hynic - higg indicates not only the quality of the kit and radiopharmaceutical preparation , but also clinical usefulness . ( a ) 2 h and ( b ) 24 h anterior and posterior view showing 99mtc - higg accumulation in rabbit . right thigh shows the acute infection developed by sterile turpentine while right thigh of rabbit shows the site of abscess formation by e. coli tc - hynic - higg distribution in human volunteer at 24 h the present method of radiolabeling higg with hynic is a simple , easy to perform radiolabeling procedure that gives high radiolabeling yield with minimum colloid formation . our experimental observations indicate good accumulation of tc - hynic - higg at the infection and/ or inflammation site . tc - hynic - higg imaging data indicated higher lesion to background ratio in the infected foci as compared to the inflammatory foci the technique may therefore have a good diagnostic utility in the differentiation of infection from inflammation in clinical situations .