Free light chains in the CSF in multiple sclerosis

Summary The presence of free light chains (FLC) was investigated in 32 patients with clinically definite or laboratory supported definite multiple sclerosis (MS), 2 patients with neurosyphilis and 10 normal controls. The detection of FLC in unconcentrated cerebrospinal fluid (CSF) was performed by means of agarose isoelectric focusing, followed by transfer of proteins to nitrocellulose membranes, double immunofixation, avidin-biotin amplification and peroxidase staining. Bands due to FLC were clearly demonstrated in the CSF of 28 MS patients; 3 of them showed only kappa FLC, 10 only lambda FLC, while 15 had both kappa and lambda FLC. The CSF of 4 MS patients was FLC negative. In both cases of neurosyphilis FLC bands were observed. FLC were never found in normal CSF. Among the indexes of intrathecal immunological activity (IgG oligoclonal bands, FLC, IgG index, intra-blood-brain barrier IgG synthesis rate, pleocytosis) the FLC proved to be the second most frequent abnormality in MS CSF, the presence of IgG oligoclonal bands being the first. In one MS case an FLC band was found, while all the other indexes of intrathecal IgG production were negative. A high correlation was found between an elevated number of FLC and pleocytosis. The presence of FLC in MS CSF seems to indicate a recent immunological stimulation leading to increased synthesis of FLC within the CNS.     

In vivo imaging of chromogranin A-positive endocrine tumours by three-step monoclonal antibody targeting

The detection of chromogranins (Cg) by immunohistochemistry and serology represents a new in vitro diagnostic tool for endocrine tumours. We have recently reported on the feasibility of targeting chromogranin A (CgA) for in vivo detection of pituitary adenomas by immunoscintigraphy (ISG). The scintigraphic procedure, based on an anti-CgA monoclonal antibody and on the avidin-biotin three-step method (Cg-3S-ISG), was evaluated on a group of 29 consecutive patients with known or suspected endocrine tumours other than pituitary adenomas, i.e. medullary thyroid carcinoma, carcinoid, insulinoma and parathormone- or ACTH-producing tumours. Primary tumours (10) and recurrences (16) were visualised in 26 patients, whereas conventional imaging techniques (planar radiography, computerised tomography, magnetic resonance imaging and ultrasonography) failed to detect the tumour sites in ten of the same (Cg-3S-ISG-positive) patients. Therefore, these preliminary results indicate that Cg-3S-ISG, the first immunological method able to detect endocrine tumours in vivo, has a higher diagnostic accuracy than conventional imaging techniques (93.1% compared with 65.5%).     

Avidin bioconjugate with a thermoresponsive polymer for biological and pharmaceutical applications

A thermoresponsive polymer, N-isopropylacrylamide-co-acrylamide (Mn 6 kDa) with a lower critical solution temperature (LCST) of 37 °C, was activated and conjugated to avidin to yield a derivative with 200 kDa molecular weight. Gel permeation analysis demonstrated that the new bioconjugate possessed an apparent size corresponding to a 220 kDa globular protein. Photon correlation spectroscopy and turbidometric studies showed that the bioconjugate underwent temperature dependent phase transitions. The protein–co-polymer bioconjugate displayed the same onset phase transition temperature (LCST) as the original synthetic co-polymer. Nevertheless, the aggregation profile of the bioconjugate shifted at higher temperature as compared to the original polymer. This indicated that the aggregation behaviour coil-to-globule transition of the co-polymer was modified by anchoring to the protein surface. Circular dichroism analysis showed that the co-polymer conjugation did not alter the protein tertiary structure tertiary the aromatic amino acid environment. The bioconjugate maintained 85 ± 3% of native avidin affinity for biotin and biotin-Mab, and high affinity was maintained after three heating cycles. Pharmacokinetic studies demonstrated that the co-polymer bioconjugation increased the avidin residence time in the bloodstream. The distribution phase of avidin-co-polymer was longer than the native protein by a factor of 20. The co-polymer conjugation decreased by three-fold the distribution extent of avidin and reduced significantly its up-take to the liver.     

亲和素促排用于大肠癌放免显像的实验研究

目的提高99Tcm-CL3放免显像对大肠癌的诊断效果。方法对荷瘤裸鼠注射99Tcm-CL3-Bt4.14MBq(20μg)，6h后注射不同剂量亲和素(Av)，注射Av后不同时间显像，以ROI技术计算并比较剂量组间及不同促排时间组间肿瘤/本底(T/NT)及肝/本底(L/NT)比值。结果随Av用量增加及促排时间延长，软组织影减退，肿瘤及肝影增强。当用60μgAv促排2h，肿瘤影清晰，T/NT比值(4.7±0.8)明显高于促排前(1.2±0.2，P＜0.002)。结论Av促排能缩短显像时间，改善显像质量。     

Functional characterization of avidins in amphioxus Branchiostoma japonicum: Evidence for a dual role in biotin-binding and immune response

Avidin is well known for its high affinity to biotin and has been found in many egg-laying vertebrate species. However, little is known about avidin in invertebrate species to date. Here we clearly showed the presence of two avidin genes, Bjavidin1 and Bjavidin2, in the amphioxus Branchiostoma japonicum, the first ones in non-vertebrate animals. We also showed that the expression of both Bjavidin1 and Bjavidin2 were inducible by progesterone, LTA and LPS. Moreover, we demonstrated for the first time that in addition to biotin-binding, the recombinant proteins rBjAVIDIN1 and rBjAVIDIN2 were not only able to interact with Gram-positive and negative bacteria as well as their conserved surface components LTA and LPS but also to enhance phagocytosis of bacteria by macrophages, suggesting that BjAVIDIN1 and BjAVIDIN2 both function as pattern recognition receptors and opsonins. It is thus clear that avidin may play a dual role in biotin-binding and immune response.     

Avidin chase reduces side effects of radioimmunotherapy in nude mice bearing human colon carcinoma

AIM: To evaluate the influence of avidin chase on the side effects of radioimmunotherapy (RIT) in nude mice bearing human colon carcinoma and therapeutic outcome. METHODS: Purified anti-CEA monoclonal antibody (McAb) was biotinylated with NHS-biotin, and then radiolabeled with 188Re by the direct method. 188Re-labeled biotinylated anti-CEA McAb (188Re-CEA McAb-Bt) was intravenously injected followed by intravenous injection of avidin after 24 h. SPECT imaging and biodistribution study were performed at 28-48 h after the injection of 188Re-CEA McAb-Bt. Three groups of nude mice subcutaneously grafted with human colon carcinoma were treated 7 d after the graft. Mice in the avidin chase group received intravenous injection of 188Re-CEA McAb-Bt (11.1 MBq/ 20 μg) followed by intravenous injection of cold avidin (80 μg) after 24 h. Mice in the control group (treated group without avidin chase) only received the injection of 188Re-CEA McAb-Bt (11.1 MBq/20 μg), another control group (non-treated group) only received 0.1 ml normal saline solution. Toxicity was evaluated on the basis of change of body weight and peripheral WBC counts, and therapy effects were determined by variation in tumor volume. Histological analysis of tumors was also performed. RESULTS: Avidin chase markedly accelerated the clearance of 188Re-CEA McAb-Bt from the blood and normal tissues. The tumor uptakes of 188Re-CEA McAb-Bt at 28 h were 5.90 and 6.42% ID/g, respectively, in chase group and in non-chase group, while the tumor-to-background (T/NT) ratios were 3.19 and 0.56, respectively. The tumor uptake was slightly decreased by avidin chase, but the T/NT ratios were increased. In treated groups the growth rate of body weight and the number of WBC decreased after injection of 188Re-CEA McAb-Bt, and the WBC counts recovered earlier in the group with avidin chase than in the group without avidin chase. Compared to the nontreated group, treated groups with and without avidin chase showed significant anti-tumor effects. CONCLUSION: Avidin chase can effectively reduce the side effects of RIT, and improve therapeutic efficacy.     

Synthesis of bifunctional saxitoxin analogues by biotinylation

Saxitoxin (STX) contaminates seafood and freshwater catchments worldwide. Conjugation of STX with biotin would enable new biochemical methods to quantitate STX and its analogues as well as diversify its utility as a research tool. We conjugated biotin at the region of the toxin normally occupied by a carbamoyl and this conjugate could concurrently bind both avidin/streptavidin and saxiphilin. Increasing the length of the linker between biotin and the STX portion of the semisynthetic analogue increased potency of saxiphilin binding of the STX moiety.     

Endogenous biotin staining in a subset of spinal neuronal cell bodies: a potential confounding factor for neuroanatomical studies

Biotinylated compounds are commonly used to label neuronal cell bodies via intracellular filling or retrograde tracing. Endogenous concentrations of biotin within a subset of neuronal cell bodies would pose a problem for interpreting such experiments. Here I report that a subset of turtle spinal cord neuronal cell bodies strongly stains for biotin, using the avidin-biotin-horseradish peroxidase (ABC) reaction, in the absence of any exogenous biotinylated compound.     

Sensitive ELISA for Determination of Serum E2 Using a New Tracer E2-Biotin

Abstract

A new tracer conjugate of E₂-Biotin, with different spacers, was synthesized at position 3 in the estradiol molecule for first time. Immunoreactivity of the tracer was determined by reacting with the anti-E₂ monoclonal antibody. The monoclonal antibodies raised against E₂ were characterized for its use in ELISA detection systems of serum E₂. The purified antibody has a high affinity and specificity for E₂. The antibody and tracer were used for establishing a competitive ELISA for estradiol (E₂). The experimental results showed that the dose-response curve of the assay covered a range of 33–20,000 pg/mL (n = 8). The detection limit is 28.3 pg/mL (S/N = 3). The intra- and inter-assay coefficients of variation for the assay of serum samples ranged from 5.7 to 13.2% and from 5.3 to 10.6%, respectively. Precoated microtiter plates were dried at 4°C and they were stable for up to 3 months.