Monoclonal antibody (mAb) is an important biological macromolecule and widely used in immune detection, in vitro diagnostics, and drug discovery. However, the inherent properties of mAb restrict its further development, such as high molecular weight and complex structure. Therefore, there is an urgent need to develop alternatives for mAb. Various types of miniaturized antibodies have been developed, among which the variable domain of immunoglobulin new antigen receptor (VNAR) is very attractive. The shark single-domain antibody, also known as shark VNAR, is an antigen-binding domain obtained by genetic engineering technology based on the immunoglobulin new antigen receptor (IgNAR) that naturally exists in selachimorpha. It has a molecular weight of 12 kDa, which is the smallest antigen-binding domain found in the known vertebrates at present. Compared with mAb, the shark VNAR exhibits various superiorities, such as low molecular weight, high affinity, tolerance to the harsh environment, good water solubility, strong tissue penetration, and recognition of the hidden epitopes. It has attracted wide attention in the fields of immunochemical reagents and drug discovery. In this review, various aspects of shark VNAR are elaborated, including the structural and functional characteristics, generating and humanization techniques, affinity maturation strategies, application fields, advantages and disadvantages, and prospects.
Animals ; Antibodies, Monoclonal ; immunology ; Antibodies, Monoclonal, Humanized ; immunology ; Antigens ; Epitopes ; metabolism ; Protein Domains ; immunology ; Receptors, Antigen ; chemistry ; immunology ; Sharks

In order to optimize the fabrication of SiO2 tubes immobilized with antibody for hepatitis C virus antigen (HCAg) detection, we formed the activated amino on the surface of SiO2 tubes by using the activation of aminosilane. Then we immobilized the hepatitis C virus (HCV) monoclonal antibody on the surface of SiO2 tubes by using glutaraldehyde as a chemical cross-linker, followed by detecting HCAg. Sequence tests showed that when the SiO2 tubes were treated in 10% (V/V) aminosilane solution and 3% (V/V) glutaraldehyde solution for 3 hours and 2 hours, respectively, the HCV monoclonal antibody had high immobilization efficiency and low nonspecificity, and the HCAg was detected to 1 ng/mL. This experiment can provide principle and experimental data for establishment of HCAg magnetic immunoassay system.
Antibodies, Immobilized ; immunology ; Antibodies, Monoclonal ; chemistry ; immunology ; Hepatitis C Antibodies ; chemistry ; immunology ; Hepatitis C Antigens ; analysis ; immunology ; Humans ; Silicon Dioxide ; chemistry

We have constructed the immune microsphere against tumor necrosis factor-alpha (TNF-alpha) prospectively, hoping to establish the experiment groundwork in more researches which could be used in specific elimination of the TNF-alpha by blood purification method for the future. The recombinant human tumor necrosis factor-alpha monoclonal antibody (rHTNF-alpha McAb) was wrapped on the polystyrene microsphere (PSM) carrier connecting poly-L-lysine (PLL) beforehand. They were earmarked by the fluorescein isothiocyanate (FITC) respectively. The packing conditions were examined using the inversted and fluorescence microscopes and the spectrophotometer. The results showed that the best conditions for wrapping were 20 degrees C, pH9.5 and 60 minutes. The PLL content was not changed in the washing fluid after coating, which indicated the wrapping was quite firm. At the same temperature and same coating time, the rHTNF-alpha McAb coated on the PLL was obviously substantial when the concentration of glutaraldehyde solution was 0.2%. The findings demonstrated that the built immune microsphers can be used as a novel adsorption material. This method is simple and economic, and it offers a new approach to the related studies.
Antibodies, Monoclonal ; immunology ; Fluorescein ; chemistry ; Humans ; Microspheres ; Polylysine ; chemistry ; Polystyrenes ; chemistry ; Recombinant Proteins ; immunology ; Tumor Necrosis Factor-alpha ; immunology

OBJECTIVETo explore the methods for labeling CDTPA-coupled CD45 monoclonal antibody (mAb) with yttrium-90 ((90)Y) for potential acute myeloid therapy.

METHODSCD45 mAb was labeled with (90)Y by CDTPA and the labeling rate, radiochemical purity, final specific activity, and immunological activity of the mAb were detected.

RESULTSWith the optimal molar ratio of CDTPA/Ab at 20:1, the labeling rate was 95%, radiochemical purity 99.8%, and final specific activity 1.9 mCi/mg. This conjugate was stable in vitro with comparable immunological activity in comparison with unlabeled CD45 mAb.

CONCLUSION(90)Y-CDTPA-CD45 mAb possesses good properties as an ideal targetting therapeutic agent for acute leukemia.

Anhydrides ; chemistry ; Antibodies, Monoclonal ; chemistry ; immunology ; Humans ; Immunoconjugates ; chemistry ; immunology ; Isotope Labeling ; methods ; Leukocyte Common Antigens ; immunology ; Pentetic Acid ; chemistry ; Radiopharmaceuticals ; chemical synthesis ; chemistry ; immunology ; Yttrium Radioisotopes ; chemistry

Heavy metal leftover on farm and stock products has become a big threat to human. It is necessary to develop some fast and efficient detection methods. Heavy metal immunoassays are new methods for detection of heavy metal ions. Compared to the traditional chemical methods, immunoassays are not only fast, cheap, simple, but also reasonably portable, highly sensitive and selective. It can be used as preliminary screening for rapid determination of heavy metal ions. Except chemical chelators, phytochelatin and metallothionein can also be used for preparing immunogen, both of them can chelate heavy metal ions to carrier protein. There are two prototype assays: polyclonal antibody immunoassay and monoclonal antibody immunoassay. The former includes fluorescence polarization immunoassay; the latter includes indirectly competitive ELISA, one-step competitive immunoassay and KinExA immunoassay. Among these assays, indirectly competitive ELISA which was used for determining heavy metal ions in the early days was easy to be interfered and showed false positive. Fluorescence polarization immunoassay which used polyclonal antibody for determining heavy metal ions was simple and cheap. KinExA instrument could be functioned as an immunosensor for environmental samples. One-step immunoassay which avoided to the addition of second antibody and chromogenic substrate was simple and sensitive. Colloidal gold enhanced immunochromatography assay is a semi-quantitation for determining heavy metal ions. As an adjunctive way for chemical methods, it has the potential application in rapid determination of heavy metal ions.
Animals ; Antibodies, Monoclonal ; immunology ; Gold ; chemistry ; Humans ; Immunoassay ; methods ; Metals, Heavy ; analysis ; immunology

BACKGROUNDWe previously reported that iodine-131((131)I)-labeled anti-pro-gastrin-releasing peptide (ProGRP(31-98)) monoclonal antibody D-D3 could selectively accumulate in the tumor sites of nude mice bearing small cell lung cancer (SCLC) xenografts. However, (131)I-D-D3 was cleared slowly from the body, and the best radioimmunoimaging time for SCLC was 72 - 96 hours after injection. The aims of this study were to radiolabel anti-ProGRP(31-98) D-D3 monoclonal antibody with technetium-99m ((99m)Tc) and to investigate the biodistribution of this antibody in healthy ICR mice.

METHODSD-D3 was labeled with (99m)Tc via the 2-mercaptoethanol reduction method. (99m)Tc-D-D3 was purified by the gel column separation method. The labeling efficiency and radiochemical purity were measured by thin-layer chromatography. The immunological activity of (99m)Tc-D-D3 was determined with cell conjugation assays. (99m)Tc-D-D3 was injected into healthy ICR mice via a tail vein, and all the healthy ICR mice were sacrificed by cervical dislocation at a designated time. Then, the blood and major organs were removed and weighed, and counted in a gamma scintillation counter to determine the percentage of the injected dose per gram (%ID/g).

RESULTSThe labeling rate and the radiochemical purity of (99m)Tc-D-D3 were (73.87 ± 2.89)% and (94.13 ± 4.49)%, respectively. The immunobinding rates of (99m)Tc-D-D3 to the human small cell lung cancer NCI-H446 cell line and lung adenocarcinoma A549 cell line were (81.2 ± 2.37)% and (24.3 ± 1.46)%, respectively. The distribution data of normal ICR mice demonstrated that (99m)Tc-D-D3 was mainly distributed in the liver, kidney and lung, and less in the brain tissue and muscle.

CONCLUSIONS(99m)Tc-D-D3 antibody not only had high radiochemical purity, but also had good stability both in vitro and in vivo, and maintained good immunological activity. (99m)Tc-D-D3 was metabolized mainly in the kidney and liver, and the blood radioactivity decreased rapidly. Thus, (99m)Tc-D-D3 is conducive to the radioimmunoimaging of SCLC.

Animals ; Antibodies, Monoclonal ; chemistry ; immunology ; metabolism ; Female ; Male ; Mice ; Mice, Inbred ICR ; Peptide Fragments ; immunology ; Recombinant Proteins ; immunology ; Technetium ; chemistry

OBJECTIVETo assess the binding ability of microbubbles targeted to alphavbeta3-integrin (MBp) for thrombus-targeted contrast-enhanced ultrasound.

METHODSTargeted microbubbles were prepared by conjugating the monoclonal antibody against alphavbeta3-integrin to lipid shell of the microbubble via the avidin-biotin bridges. Equivalent isotype control microbubbles (MB) or targeted ultrasound microbubbles (MBp) were randomly added into the flow chamber. After a 30-min incubation with the thrombus fixed in an agarose flow chamber model, the thrombus was washed with a continuous flow of PBS solution (15 cm/s) for 2, 4, 6, 8 and 10 min, followed by thrombus imaging using contrast-enhanced ultrasound and measurement of the video intensity (VI) values of the images.

RESULTSThe VI of the thrombus in MBp group was reduced by 28%-66%, while that in control MB group was decreased by 87%-94%, and the VI values of the thrombus group were significantly greater in former group at each of the time points (P<0.05).

CONCLUSIONMBP has good targeting ability to the thrombus with resistance to the shear stress after adhesion to the thrombus. In vitro evaluation of the thrombus-binding capability of the targeted microbubble (MBp) by simulating the shear stress in vivo can be helpful for predicting the in vivo effects of ultrasonic molecular imaging using MBp.

Antibodies, Monoclonal ; chemistry ; immunology ; Contrast Media ; chemistry ; Humans ; Integrin alphaVbeta3 ; immunology ; metabolism ; Microbubbles ; Sepharose ; Thrombosis ; diagnostic imaging ; Ultrasonography

The N protein of the rinderpest virus (RPV) was analyzed topologically and antigenically by using anti-N monoclonal antibodies (Mabs). Ten Mabs were raised against the N protein of the RPV. At least six non-overlapping antigenic sites (sites A-F) were delineated by competitive binding assays using biotinylated Mabs. Of them 5 sites (A, C, D, E and F) on the N protein were recognized by RPV-specific Mabs in ELISA and IFA while site B was recognized by Mabs reacting with both RPV and PPRV. Non- reciprocal competition was found among sites C, D and E. Recombinant RPV N protein after exposure to 0.2% SDS exhibited higher ELISA titers in all Mabs recognizing 6 sites. Four sites (A, B, E and F) on 2% SDS-treated N protein lost completely reactivity with Mabs while the remaining sites (C and D) on the protein retained their antigenicity to some degree. It indicates that two sites (C and D) were sequential. Six representative Mabs bound to each site exhibited competition with rinderpest antibodies in a blocking ELISA, indicating that the sites were actively involved in antigenicity in cattle.
Antibodies, Monoclonal/*immunology ; Antigens, Viral/chemistry/*immunology ; Binding, Competitive ; Enzyme-Linked Immunosorbent Assay ; Epitopes/immunology ; Nucleocapsid Proteins/chemistry/*immunology ; Rinderpest virus/*immunology

With the application of monoclonal antibody technology more and more widely, its production technology is becoming more and more perfect. Small molecule monoclonal antibody technology is becoming a hot research topic for people. The application of traditional Chinese medicine small molecule monoclonal antibody technology has been more and more widely, the technology for effective Chinese medicine component knockout provide strong technical support. The preparation of monoclonal antibodies and small molecule knockout technology are reviewed in this paper. The preparation of several steps, such as: in the process of preparation of antigen, hapten carrier coupling, coupling ratio determination and identification of artificial antigen and establishment of animal immunization and hybridoma cell lines of monoclonal antibody, the large-scale preparation; small molecule monoclonal antibody on Immune in affinity chromatography column method is discussed in detail. The author believes that this technology will make the traditional Chinese medicine research on a higher level, and improve the level of internationalization of Chinese medicine research.
Animals ; Antibodies, Monoclonal ; chemistry ; genetics ; immunology ; Humans ; Hybridomas ; metabolism ; Immunologic Techniques ; methods ; trends

OBJECTIVETo obtain monoclonal antibodies (McAbs) against severe acute respiratory syndrome (SARS) associated coronavirus (SARS-CoV) nucleocapsid (N) protein to develop diagnostic test for SARS and study the pathogenesis of the disease.

METHODSBALB/c mice were immunized with purified N protein of SARS-CoV. Hybridoma cell lines secreting monoclonal antibodies against SARS-associated coronavirus nucleocapsid were established after cell fusion with mouse splenic cells and SP2/0 cells. The specificity of the McAbs obtained was examined by Western blot and indirect fluorescence assay. Epitopes reacted with the McAbs were preliminarily located through Western blot by expressing truncated N proteins.

RESULTSAfter cell fusion and three rounds of cell cloning, six hybridoma cell lines secreting monoclonal antibodies specifically against SARS-CoV nucleocapsid were obtained. Western blot and indirect fluorescence assay showed that the McAbs reacted specifically with nucleocapsid protein and SARS-CoV. Among the six McAbs, three recognize the epitopes located in the N-terminus of the protein, whereas the others reacted with those located in the C-terminus.

CONCLUSIONThe anti-SARS-CoV nucleocapsid McAbs were developed and these McAbs may be useful in the development of diagnosis assays and basic research of SARS.

Animals ; Antibodies, Monoclonal ; biosynthesis ; immunology ; Antibodies, Viral ; biosynthesis ; immunology ; Antibody Specificity ; Female ; Hybridomas ; secretion ; Mice ; Mice, Inbred BALB C ; Nucleocapsid Proteins ; immunology ; isolation & purification ; SARS Virus ; chemistry ; immunology