Nanobody-Based high-performance immunosorbent for selective beta 2-microglobulin purification from blood.

Removing ß2-microglobulin (ß2M) from blood circulation is considered to be the most effective method to delay the occurrence of dialysis-related amyloidosis (DRA). The ideal extracorporeal ß2M removal system should be cost-effective, highly specific and having a high capacity. However, the traditional technologies based on size exclusion do not have an adequate specificity, and alternative immunosorbents have limited applications due to low capacity and their high cost. Nanobodies (Nbs), the smallest functional recombinant antibody fragments, offer several advantages to overcome these obstacles. In this study, an anti-ß2M Nb with a C-terminal thiol-tag was successfully prepared from E. coli for site-directed and oriented immobilization and usage as capture ligand in a ß2M-selective immunosorbent. The prepared immunosorbent showed a high binding capacity of up to 7 mg ß2M per mL resin, which is 17 times higher than that of previous studies using single-chain variable antibody fragments (scFv). Furthermore, an exceptional high specificity has been demonstrated as other human serum proteins were not adsorbed during dynamic adsorption experiments. About 80% of the original binding capacity of the immunosorbent was restored after four consecutive easy regenerations, whereas 90% of the original capacity was retained after 1-month storage of the resin. Moreover, the mathematical model fitted very well the in vitro perfusion. The results with this pioneering immunosorbent confirm its possible clinical application and is expected to reach the required clinical effect of immunoadsorption therapy. STATEMENT OF SIGNIFICANCE: Dialysis-related amyloidosis (DRA), associated with the accumulation of ß2-microglobulin (ß2M), is a serious complication of end-stage kidney disease. Removing ß2M from blood circulation by extracorporeal blood purification is considered to be the most effective method to delay the occurrence of DRA. However, the existing methods are incapable to eliminate sufficient quantities of ß2M from circulation, either because of lack of specificity, high cost or for low capacity. In this manuscript, we provide a practical and economic immunosorbent based on anti-ß2M nanobody for DRA. The prepared immunosorbent was reusable and storable, and demonstrated high specificity and realized a high binding capacity of up to 7 mg ß2M per mL resin, which is 17 times higher than that of the previous studies.

One-step Preparation of a VHH-based Immunoadsorbent for the Extracorporeal Removal of ß2-microglobulin.

Dialysis-related amyloidosis (DRA), which has been widely recognized to be associated with the accumulation of ß2-microglobulin (ß2-m) in blood, is one of the most common complications in patients receiving long-term dialysis treatment. The most significant side-effect of existing hemodialysis sorbents for the removal of ß2-m from blood is the loss of vital proteins due to non-specific adsorptions. Although the traditional antibodies have the capability to specifically remove ß2-m from blood, high cost limits their applications in clinics. Single domain antibodies derived from the Camelidae species serve as a superior choice in the preparation of immunoadsorbents due to their small size, high stability, amenability, simplicity of expression in microbes, and high affinity to recognize and interact with ß2-m. In this study, we modified the anti-ß2-m VHH by the formylglycine-generating enzyme (FGE), and then directly immobilized the aldehyde-modified VHH to the amino-activated beads. Notably, the fabrication is cost- and time-effective, since all the preparation steps were performed in the crude cell extract without rigorous purification. The accordingly prepared immunoadsorbent with VHHs as ligands exhibited the high capacity of ß2-m (0.75 mg/mL). In conclusion, the VHH antibodies were successfully used as affinity ligands in the preparation of novel immunoadsorbents by the site-specific immobilization, and effectively adsorbed ß2-m from blood, therefore opening a new avenue for efficient hemodialysis.

Single Molecule Upconversion-Linked Immunosorbent Assay with Extended Dynamic Range for the Sensitive Detection of Diagnostic Biomarkers.

The ability to detect disease markers at the single molecule level promises the ultimate sensitivity in clinical diagnosis. Fluorescence-based single-molecule analysis, however, is limited by matrix interference and can only probe a very small detection volume, which is typically not suitable for real world analytical applications. We have developed a microtiter plate immunoassay for counting single molecules of the cancer marker prostate specific antigen (PSA) using photon-upconversion nanoparticles (UCNPs) as labels that can be detected without background fluorescence. Individual sandwich immunocomplexes consisting of (1) an anti-PSA antibody immobilized to the surface of a microtiter well, (2) PSA, and (3) an anti-PSA antibody-UCNP conjugate were counted under a wide-field epifluorescence microscope equipped with a 980 nm laser excitation source. The single-molecule (digital) upconversion-linked immunosorbent assay (ULISA) reaches a limit of detection of 1.2 pg mL-1 (42 fM) PSA in 25% blood serum, which is about ten times more sensitive than commercial ELISAs, and covers a dynamic range of three orders of magnitude. This upconversion detection mode has the potential to pave the way for a new generation of digital immunoassays.

Preparation of Monoclonal Antibodies and a Simple Myeloperoxidase-Immunosorbent Assay for Detecting Human Myeloperoxidase.

Myeloperoxidase (MPO), a leukocyte hemoprotein released from neutrophils, is thought to be a potential participant in plaque formation and plaque rupture. Therefore, MPO is regarded as an early marker predicting the risk for atherosclerosis, especially for coronary artery disease and acute coronary syndrome. We generated hybridoma clones 1E3 and 3E8 secreting monoclonal antibodies (mAbs) specific to human MPO. BALB/c mice were immunized with MPO protein purified from human neutrophils. Splenocytes from these mice were fused with the mouse myeloma cell line SP2/0. Based on isotyping of the mAbs, both clones 1E3 and 3E8 were referred to the IgG1 subclass. The specificities of 1E3 and 3E8 were assessed by enzyme-linked immunosorbent assay (ELISA), and only 3E8 was confirmed by western blot. We developed a simple MPO-immunosorbent assay (MPO-ISA) on microplate based on both the immune activity and peroxidase activity of MPO. The mAb secreted by clone 3E8 was chosen as coating antibody to capture the plasma MPO without interfering with the peroxidase activity of MPO. Then, tetramethylbenzidine substrate was added to the microwell directly, catalyzed by captured MPO, and a colored product was formed. The simple MPO-ISA test has a sensitivity of 3.68 ng/mL. The linear concentration of MPO-ISA for commercial MPO standard ranged to 250 ng/mL. The average recovery rate is 101.02%. The imprecision within-day was <10% at three different MPO levels. The imprecision between-day was <10% at low and middle MPO levels and varied to 14.61% at the high MPO level. We found that the established MPO-ISA can detect the plasma MPO from human and cavy, but not from mouse and rat. Compared with the commercial human MPO ELISA assay, the MPO-ISA can be used to detect the natural human MPO protein, but not recombinant MPO polypeptides. The generated mAbs and MPO-ISA test may be useful tools to assess risk for inflammation and cardiac events.

Extracellular domains of the beta, gamma and epsilon subunits of the human acetylcholine receptor as immunoadsorbents for myasthenic autoantibodies: a combination of immunoadsorbents results in increased efficiency.

Myasthenia gravis (MG) is usually caused by autoantibodies against the human muscle acetylcholine receptor (AChR). Plasmapheresis offers a therapeutic option, but, as well as removing the pathogenic anti-AChR autoantibodies, it non-specifically removes indispensable immunoglobulins. An attractive alternative to plasmapheresis would be the extracorporeal specific removal of the autoantibodies using AChR-based immunoadsorbents. Previously, we used the N-terminal extracellular domain (ECD) of the AChR alpha subunit to immunoadsorb anti-alpha subunit autoantibodies from MG sera. In this study, we immobilised the beta -, gamma- and epsilon-AChR ECDs on Sepharose and tested them as immunoadsorbents on 50 MG sera. A given ECD removed a different percentage of autoantibodies from different sera and different ECDs removed different percentages from the same serum; on average, the beta-, gamma- and epsilon-ECDs removed 22%, 20% and 15.5% of the autoantibodies, respectively. Immunoadsorption was completed in 3 min, 1 mug of ECD removed approximately 2 pmol of autoantibodies, and the immunoadsorbent could be recycled approximately 4 times. The combined use of two (alpha+gamma) or four (alpha+beta+gamma+epsilon) ECDs in a single immunoadsorbent resulted in much higher (often additive) immunoadsorption. These results show that MG sera have autoantibodies against several AChR subunits, and suggest that the combined use of all AChR ECDs could provide the basis for a novel, antigen-specific therapy for MG.

Peritransplant immunoadsorption: a strategy enabling transplantation in highly sensitized crossmatch-positive cadaveric kidney allograft recipients.

BACKGROUND: Kidney transplant recipients with a current positive complement-dependent cytotoxicity crossmatch (CDCXM) are at high risk for hyperacute rejection and graft loss. Immunoadsorption (IA) represents an efficient strategy to remove donor-specific alloantibodies. In this analysis, we evaluated effectiveness of peritransplant IA as an anti-humoral strategy to overcome a current positive CDCXM in presensitized renal allograft recipients. METHODS: Between 1999 and 2003, 40 high risk cadaveric kidney allograft recipients (median CDC panel reactive antibody [PRA] level, 77%; number of retransplants, n = 38) were subjected to peritransplant IA with protein A (one pretransplant IA session followed by a course of repeat posttransplant IA sessions) in addition to preemptive antilymphocyte antibody therapy. RESULTS: In nine of these patients, a current positive CDCXM was rendered negative by a single pretransplant IA session. Thirty-one recipients had a negative CDCXM already before pretransplant IA. No difference in graft survival was found between CDCXM-positive and CDCXM-negative recipients (3-year graft survival, 78% vs. 71%, P = 0.6). Comparable rates of immunological graft loss at 3 years were observed (11% vs. 13%, P = 0.8). Patient groups did not significantly differ with respect to median serum creatinine at 1 year (1.23 mg/dL [CDCXM-positive] vs. 1.57 mg/dl [CDCXM-negative], P = 0.07) and at the end of follow-up (median 32 months; 1.19 mg/dL vs. 1.63 mg/dL, P = 0.06). Moreover, patient groups showed similar rates of biopsy-proven cellular rejection (11% vs. 20%) or C4d-positive graft dysfunction (33% vs. 32%). CONCLUSION: Our results demonstrate that peritransplant IA enables successful cadaveric kidney transplantation in the context of a positive CDCXM.

Adsorption column for myasthenia gravis treatment: Medisorba MG-50.

Adsorption column Medisorba MG-50 (Kuraray Medical Inc.) for the treatment of myasthenia gravis (MG) is introduced. The adsorbent in this column is composed of cellulose beads as carrier material and covalent-bound synthetic peptide as a ligand that has a specific affinity to the pathogenic anti-acetylcholine receptor antibody of MG. The amino acid sequence of the peptide is modified from the segment of alpha 183-200 of the torpedo acetylcholine receptor (AChR) protein, and the segment is the acetylcholine binding site on AChR and the target site of anti-AChR antibody. The adsorbent showed specific adsorption characteristics to the anti-ACHR antibody (blocking antibody) in vitro. Clinically, MG-50 is used in plasma-perfusion therapy, and it is recognized that MG-50 specifically reduces blocking antibody titer and improves MG symptoms. MG-50 is approved in Japan.

32P-postlabelling of bulky human DNA adducts enriched by different methods including immunoaffinity chromatography.

DNA adducts in lymphocytes and granulocytes of men exposed occupationally and environmentally to high concentrations of aromatic compounds in air were measured by the 32P-postlabelling method. Adducts in the same samples were characterized using nuclease P1 enrichment, butanol extraction and immunoaffinity purification with an antiserum raised against benzo[alpha]pyrene diol epoxide (BPDE). Only part of the adducts found in human samples were extracted by butanol. It also seemed, that only a small part of them belonged to the group of polycyclic aromatic hydrocarbons (PAHs) recognised by the antibody. Relative content of hydrophobic adducts and those with a structure similar to PAHs was higher in winter samples (when exposure to aromatic chemicals in air was higher) in comparison to samples collected in summer.

Antibodies released from immunoadsorbents: effect of support, activation and elution conditions.

Immunoadsorption is an application of affinity chromatography, as a therapeutic method to specifically deplete biological fluids such as blood plasma from proteins in excess, or to extract a biomolecule from a complex mixture. However, the leakage of small amounts of antibodies covalently immobilized on the support hampers the practical use of this method. In fact, these released antibodies contaminate the purified proteins or depleted media and, when they are of animal nature, they may lead to immunization of patients, or cause an anaphylactic shock when a clinical use is concerned. It is therefore of prime importance that the immunoadsorbents exhibit a satisfactory stability over the whole range of chemical and biochemical conditions involved during their clinical handling. To determine optimal conditions for the preparation of stable immunoadsorbents designed to remove selectively Low Density Lipoproteins (LDLs) from the plasma of patients affected by familial hypercholesterolemia, various immunoadsorbents were prepared by covalent immobilization of goat anti-apolipoprotein B polyclonal antibodies on different supports (Sepharose CL-4B, Sepharose 6 Fast Flow, Sphérodex and Fractogel) previously activated by various chemical reagents (cyanogen bromide, divinyl sulphone, tresyl chloride and trichloro-s-triazine). Their adsorption capacity, specificity, stability and the amount of immobilized antibodies were compared in terms of the activation method and the support used. It turns out that the immunoadsorbents prepared with Sepharose 6 Fast Flow lead to optimal yield of coupling, adsorption capacity, and an excellent stability at neutral pH. TC-activated-Fractogel turns out as well to afford an excellent coupling yield, a good adsorption capacity and an optimal stability in the whole pH range tested.

Immunochemical comparison of transformation-associated protein and secreted phosphoprotein.

Transformation-associated protein (TAP) has been detected in MSV-M-transformed rat cell lines as glycosylated, weakly phosphorylated protein of molecular weight (Mr) 66,000 and 68,000. In the ts-MSV-M-transformed rat kidney cell line (6m2), the synthesis of TAP and the v-mos gene product is temperature-sensitive and accompanies the expression of transformation phenotypes. Therefore, TAP potentially plays a role in cellular transformation. On the other hand, SPP represents a family of glycosylated phosphoprotein with apparent Mr ranging from 42,000 to 69,000. SPP has been detected in osteoblasts and in avian and murine retrovirus-transformed rat and mouse epithelial cells. Therefore, the potential relatedness of TAP and SPP was studied. Using the 6m2 cells, we found that SPP was strongly phosphorylated and was synthesized at both the permissive (33 degrees C) and non-permissive (39 degrees C) temperatures. By contrast, TAP was weakly phosphorylated, and was synthesized, as we found previously, only at the permissive temperature of 33 degrees C. Furthermore, in 35S-methionine incorporation studies, TAP became heavily labelled whereas SPP was not (consistent with its amino acid composition having few methionine residues). Using 125I-TAP in both immunoprecipitation and radioimmunoassays, it was found that an antiserum raised against SPP did not cross-react with 125I-TAP. Additionally, SPP has now been found in many human and rodent cells, while TAP thus far has only been detected in MSV-transformed rat cells. These data suggest that structurally, TAP and SPP are not closely related phosphoproteins.

[Extracorporeal immunosorption in the treatment of atopic bronchial asthma]. / Ekstracorporal'naia immunosorbtsiia v lechenii atopicheskoi astmy.

The author presents information on the use and clinical efficacy of extracorporeal therapeutic methods in various diseases. The main difficulties in the assessment of the efficacy of these therapeutic methods have been unraveled. The results of the use of immunosorption in systemic lupus erythematosus, cancer, hemophilia, diabetes mellitus, ABO-incompatibility and bronchial asthma indicate the promise of this approach to the performance of specific therapeutic programs.

Extensive cross reactivity between Treponema pallidum and cultivable treponemes demonstrated by sequential immunoadsorption.

Extensive cross reactivity between Treponema pallidum and three nonpathogenic species of treponemes was demonstrated by the Western blot technique. Rabbit antiserum produced by adjuvant immunization with solubilized T. pallidum antigens reacted with 34 T. pallidum antigens and with approximately 30 antigens each of T. phagedenis biotype Reiter, T. noguchii and T. vincentii. Adsorption of the antiserum with T. phagedenis Reiter removed only about half of the cross-reacting antibodies. Sequential adsorption with all three nonpathogenic treponemes removed antibodies to all but three polypeptides of 36,000, 34,000 and 27,000 daltons.

Envelope proteins of avian infectious bronchitis virus: purification and biological properties.

Immunoadsorbents, made with monoclonal antibodies, were used to purify the spike and membrane proteins of infectious bronchitis virus (IBV). The purified proteins were inoculated into rabbits to produce antisera. The rabbit anti-spike sera neutralized the infectivity of the virus whereas the anti-membrane sera did not. IBV-infected chickens produced antibodies to both the spike and membrane proteins. Both these antibodies were at their highest concentration about 9-11 days after inoculation, whereas neutralizing antibodies were present only at very low concentrations at that time. Neutralizing antibodies were at their highest concentration 21 days after inoculation. A second inoculation of virus at 42 days induced an anamnestic antibody response to the spike and membrane proteins and also for the neutralizing antibodies. The neutralizing, anti-spike and anti-membrane antibodies all reached highest concentrations 7-11 days after this inoculation. The advantages of purifying viral proteins using affinity chromatography with monoclonal antibodies are discussed.

Introduction of bacterial components in postadsorbed plasma during adsorption with Staphylococcus aureus.

In vitro plasma adsorption over either protein A-positive Staphylococcus aureus Cowan I (SAC) or protein A-negative S. aureus Wood 46 (SAW) led to leaching of bacterial biomolecules in the postadsorbed plasma. Presence of bacterial moieties was demonstrated in the postadsorbed plasma by more than one method: (1) using radiolabeled bacteria for adsorption with plasma and detecting radioactivity in the postadsorbed plasma, (2) gel filtration of pre- and post-adsorbed plasmas over Sephadex G-200 column and detecting additional peak(s) in the postadsorbed plasma, and (3) immunoelectrophoretic analysis of pre- and postadsorbed plasmas and their column fractions against rabbit anti-SAC antisera and demonstrating new precipitin bands in postadsorbed plasma. Using an extracorporeal plasma adsorption procedure in mongrel dogs, with radiolabeled SAC as the adsorbent, we have demonstrated the presence of radioactivity in both the adsorbed and filtered (0.2 micron) blood entering into the body, and the adsorbed blood that passed out of the body to reenter into the extracorporeal circuit. These data suggest that components of S. aureus origin enter into the host circulation during both in vitro and ex vivo plasma adsorption, although the exact nature of those extracted staphylococcal components remains unknown. This observation is of much significance since it can possibly help elucidate the mechanism of tumor regression observed following perfusion of plasma over SAC or SAW, followed by its reinfusion to the host.

Monoclonal antibodies to bovine somatotropin: immunoadsorbent reagents for mammalian somatotropins.

Twenty-nine stable hybridoma cell lines secreting monoclonal antibodies to bovine somatotropin (bST) have been produced and characterized. Five of the monoclonal antibodies bind porcine and human somatotropins as well as bST. One of these antibodies was used as a reagent in immunoadsorbent chromatography of recombinant bST or pituitary bST from cell extracts. Following chromatography, the bST preparations retained activity in a rabbit liver radioreceptor assay and in a radioimmunoassay. The immunoadsorbent reagent bound human and porcine somatotropins as well as bovine.

Fine-specificity of the lambda and chi L chains associated with antibodies directed to alpha (1 leads to 3) glucosyls in dextran.

The function of BALB/c primary B precursors, responding to dextran (dex) B-1355, and the fine-specificity of the B-1355-binding, lambda and chi, monofocal antibody (Ab) generated by the precursors have been examined. In splenic fragments from Limulus polyphemus haemocyanin (LPH)-primed, lethally irradiated, euthymic or nu/nu BALB/c mice cultured with thymus-independent (TI) dex B-1355, B-1355-lipopolysaccharide (LPS), B-1355-LPS-LPH, or thymus-dependent (TD) dex B-1355-LPH, the lambda 1 precursors responded with B-1355-binding Ab substantially equally with respect to precursor frequency, rate of Ab production, and range of fine-specificity, but not with respect to frequency of the IdX and IdI isotypes related to the VH and DH associated with the lambda 1. The lambda 2 contributed minimally to the repertoire. The chi precursors responded with B-1355-binding Ab at a rate nearly equal to the lambda 1 only under TD stimulus in euthymic fragments. A comparison of the lambda 1 and chi Ab fine-specificity, by inhibition of binding with dex differing in epitope contents and configurations, showed marked restriction in the chi relative to the lambda 1. Only approximately 10% of the relatively more abundant chi TD response showed fine alpha (1 leads to 3) specificity similar to that of the lambda 1. The lambda 1 fine-specificity diversity resided mainly in the IdI fraction.

Antigen-coated immunoadsorbents utilized for in vivo depletion of antibodies and lymphocytes with specificity for the antigen.

Hyperacute rejection of xenografts is thought to be triggered by humoral antibodies. It is known that natural antibodies against dog blood cells have been identified in sheep serum. Dog kidney antigen immunoadsorbent columns were placed in the extracorporeal lymph circuit of two sheep. In vivo studies have shown that these immunoadsorbent columns were effective in depleting the thoracic duct lymph of all antibodies with specificity for the dog kidney antigen. However, these columns were not effective in depleting the thoracic duct lymphocytes bearing surface receptors for the dog kidney antigen. When in vitro studies were carried out, the immunoadsorbent columns were effective in the depletion of both lymphocytes and antibody for the dog kidney antigen. Thus, since a humoral antibody response is thought to be responsible for the hyperacute rejection seen in xenografts, it is possible that these columns might be effective in prolonging xenograft survival.

Antigen-pulsed macrophage monolayers as specific immunoabsorbents: selective absorption of murine T cells committed to soluble protein antigen.

Antigen-pulsed macrophage (MO) monolayers in petri dishes were successfully employed as immunoabsorbents, for both the depletion and enrichment of T cells which proliferated upon exposure to soluble ovalbumin (OA). This procedure was shown not to deplete T cells reactive to a second antigen, and similarly MO monolayers pulsed with other antigens failed to deplete the T-cell preparation of OA-reactive cells. Employing brief incubation with trypsin/ethylenediamine tetracetic acid (EDTA), up to 2 X 10(6) OA-binding T cells could be recovered from each petri dish.