Small Amounts Of Antibody Research Articles

Breastfeeding and treatment for multiple sclerosis.

Monoclonal antibody therapy is effective for multiple sclerosis, and only small amounts of antibodies are transferred to breast milk. Even though the approved product descriptions advise against breastfeeding during medicinal treatment, several of the most effective MS drugs are compatible with breastfeeding.

Intraventricular human immunoglobulin distributes extensively but fails to modify amyloid in a mouse model of amyloid deposition.

Intravenous immunoglobulin infusions into Alzheimer patients have been found to provide cognitive benefit over a period of 6 mo in open label studies. One suggestion has been that these preparations contain small amounts of antibodies directed against monomeric and oligomeric Aβ which underlie their effectiveness in patients. To test this hypothesis, we infused Gammagard, a version of intravenous immunoglobulin (IVIG), into the lateral ventricle of amyloid precursor protein (APP) transgenic mice with pre-existing amyloid deposits. Mice were infused over 4 weeks, and tested behaviorally for the last 2 weeks of treatment. Brains were analyzed for histopathology. We found widespread distribution of human-immunoglobulin G (h-IgG) staining in the mouse forebrain, including cerebral cortices and hippocampus. Some cortical neurons appeared to concentrate the h-IgG, but we did not detect evidence of amyloid plaque labeling by h-IgG. The IVIG-treated mice had no change in phenotype compared to saline-infused animals with respect to activity, learning and memory, or amyloid deposition. APP mice infused with an anti-Aβ monoclonal antibody did show some reduction in amyloid deposits. These data do not support the argument that anti-Aβ antibodies in IVIG preparations are responsible for cognitive benefits seen with these preparations.

Immunoproteomics Using Polyclonal Antibodies and Stable Isotope–labeled Affinity-purified Recombinant Proteins

The combination of immuno-based methods and mass spectrometry detection has great potential in the field of quantitative proteomics. Here, we describe a new method (immuno-SILAC) for the absolute quantification of proteins in complex samples based on polyclonal antibodies and stable isotope-labeled recombinant protein fragments to allow affinity enrichment prior to mass spectrometry analysis and accurate quantification. We took advantage of the antibody resources publicly available from the Human Protein Atlas project covering more than 80% of all human protein-coding genes. Epitope mapping revealed that a majority of the polyclonal antibodies recognized multiple linear epitopes, and based on these results, a semi-automated method was developed for peptide enrichment using polyclonal antibodies immobilized on protein A-coated magnetic beads. A protocol based on the simultaneous multiplex capture of more than 40 protein targets showed that approximately half of the antibodies enriched at least one functional peptide detected in the subsequent mass spectrometry analysis. The approach was further developed to also generate quantitative data via the addition of heavy isotope-labeled recombinant protein fragment standards prior to trypsin digestion. Here, we show that we were able to use small amounts of antibodies (50 ng per target) in this manner for efficient multiplex analysis of quantitative levels of proteins in a human HeLa cell lysate. The results suggest that polyclonal antibodies generated via immunization of recombinant protein fragments could be used for the enrichment of target peptides to allow for rapid mass spectrometry analysis taking advantage of a substantial reduction in sample complexity. The possibility of building up a proteome-wide resource for immuno-SILAC assays based on publicly available antibody resources is discussed.

Magnetic bead assisted labeling of antibodies at nanogram scale

There are currently several initiatives that aim to produce binding reagents for proteome-wide analysis. To enable protein detection, visualization, and target quantification, covalent coupling of reporter molecules to antibodies is essential. However, current labeling protocols recommend considerable amount of antibodies, require antibody purity and are not designed for automation. Given that small amounts of antibodies are often sufficient for downstream analysis, we developed a labeling protocol that combines purification and modification of antibodies at submicrogram quantities. With the support of magnetic microspheres, automated labeling of antibodies in parallel using biotin or fluorescent dyes was achieved.

DEVELOPMENT AND VALIDATION OF A TWO-STEP FREE THYROXINE RADIOIMMUNOASSAY BASED ON ANTIBODY COATED TUBES

We describe a two-step radioimmunoassay procedure (RIA) for the measurement of free thyroxine (fT4) in human serum. A commercial antibody with the affinity of 5.5 × 109 L/M was used for this study. The anti-T4 antibody was immobilized on the inner walls of the polystyrene tubes by passive adsorption through normal rabbit IgG and anti-rabbit IgG as immunobridges. The method developed uses very small amounts of antibody with minimum dilution of the sample. The assay covers the range of 0 to 87.5 pmol/L with sensitivity of 0.9 pmol/L. The intra-assay precisions were 6.3% and 7% at 6.7 and 20.7 pmol/L, respectively, for 15 replicates. The inter-assay precision at 6.4 and 24.4 pmol/L were 13% and 11.6%, respectively. The normal range established by analyzing 54 healthy volunteers was 15.3–24.7 pmol/L and that of 69 ambulatory subjects was 14.8–23.3 pmol/L. Progressive dilution of euthyroid and hypothyroid samples up to 50-fold yielded virtually constant values, whereas a registered decrease in free T4 was observed with hyperthyroid samples. Free T4 estimated for 75 samples, including samples from 16 pregnant female subjects, correlated well with the values obtained by a reputed commercial kit with the linear correlation coefficient of 0.84.

Detection of Anti-Platelet Glycoprotein Antibodies Using MAIPA Method

AITP mostly occur in children accompanied by variable clinical sings including petechiae, purpura, ecchymosis and severe bleeding. This study has determined and characterized the antiplatelet glycoproteins in children with ITP. The aim of this study was to determinate antiplatelet glycoproteins (GPs) using MAIPA method. During 18 months 38 children with clinical signs of AITP were studied in Mofid children hospital. To determine anti-platelet antibodies by ELISA technique, washed O negative platelets were used as a source of platelet antigens. MAIPA method was used to detect antibodies against individual platelet membrane glycoprotein. The anti-platelet antibodies level above mean+ 3SD of control group was assumed as positive. The results indicated that the platelet count ranges was between 2×109/L and 95×109/L. 63.5 % out of 38 patients were anti-platelet antibodies positive with ELISA method. The correlation between the above patients with anti-platelet antibody positive and clinical signs was 0.4. Results for determination of antibody against platelet GPIIb/IIIa, GPIb/IX and GPIa/IIa using MAIPA method were 44%, 51% and 25% respectively. In conclusion the preference of MAIPA method is the detection of very small amount of antibody. Since MAIPA is the specific method for the detection of antibody against glycoprotein antigens, it has the advantage of differentiating immune and non-immune thrombocytopenia.

A dot-blot test using gold colloid cluster technology as a miniaturizable alternative to ELISA and hapten inhibition tests

A novel miniaturizable method to quantify antigens is described in form of a cluster linked immunosorbent assay (CLISA), using inexpensive nitrocellulose (NC) membranes as a support for dot-blotting the antigens. Antibodies for detection are labeled with gold-colloid clusters (GCC). After blocking of the membrane with non reacting protein and application of the GCC-labeled antibodies the signal is detectable by visual colorimetry and can be compared to a color scale prepared from a dilution series of known sample concentrations. The color reaction product is stable for a very long time and does not fade. The sensitivity of the method is comparable to that of ELISA if not better and furthermore needs only small amounts of antibody for detection or for GCC-labeling. This method is an alternative to the use of expensive enzyme-conjugated antibodies for a number of applications, such as tracking of antibodies during purification or hapten inhibition tests.

Hepatitis C immune globulin to prevent HCV recurrence after liver transplantation: Chasing windmills?

Hepatitis C immune globulin to prevent HCV recurrence after liver transplantation: Chasing windmills?

A novel method of Multiplexed Competitive Antibody Binning for the characterization of monoclonal antibodies

We have developed a novel method of high-throughput Multiplexed Competitive Antibody Binning (MCAB). Using only a small amount of antibody and antigen, this method enables the sorting of a large, complex panel of monoclonal antibodies into different bins based on cross-competition for antigen binding. The MCAB assay builds on Luminex® multiplexing bead-based technology to detect antibody competition. Because of its high sensitivity, the MCAB method is immediately applicable after identification of antigen-positive mAbs, providing information useful for advancing mAb candidates into further testing. The MCAB assay also can be used for sorting mAbs into binding groups after screening for functional activity.

Two expression vectors for the phage-displayed chicken monoclonal antibody

We previously reported the development of chicken monoclonal antibodies (mAb) against mammalian-conserved molecules by cell fusion and phage display using the mouse mAb expression vector pPDS. However, chicken hybridomas produce relatively small amounts of antibody when compared with mouse hybridomas, and application of the pPDS may be limited in two-antibody assays with a mouse mAb because it contains mouse Cκ as a detection tag. To circumvent the above problems, two expression vectors were established and used to produce a functional recombinant chicken mAb. These vectors, which were designed to accommodate a single chain fragment of the variable region (scFv) of the antibody, contained a chicken Cλ and FLAG with or without 6× histidine sequences in the 3′ terminus of the scFv to serve as detection and purification tags. In this study, a prion protein (PrP)-specific chicken mAb (HUC2-13) was expressed as phage-displayed and soluble scFv mAb forms by using these vectors. The scFv mAbs expressed by these vectors exhibited the same antigen-binding specificity to PrP as that of the original HUC2-13, could be purified with ease, and used in combination with a mouse mAb. These results indicate that the methods described herein offer an alternative to chicken mAb production from hybridomas and immunized chicken splenocytes, and may contribute to the use of chicken mAb reagents in numerous fields.

Functional Analysis of Peripheral Blood B Cells in Patients WithX-Linked Agammaglobulinemia

Nonoyama S, Tsukada S, Yamadori T, et alJ Immunol.161199839253929X-linked agammaglobulinemia (XLA) is classically described as a pure humoral immunodeficiency in which boys fail to produce mature B cells and as a consequence, make no immunoglobulin. With the advent of genetic testing for this disorder, it has become clear that there are boys with XLA who have small numbers of peripheral blood B cells and small amounts of antibody. This study evaluated the function of the peripheral blood B cells in boys with XLA.Five of 9 patients with XLA had detectable peripheral blood B cells. Four of the 5 patients had missense mutations of Btk identified. In the fifth patient, absence of Btk protein was demonstrated by Western blot.B cell function was evaluated using CD40 crosslinking and interleukin (IL)-4 stimulation. Proliferation, immunoglobulin E (IgE) production, and upregulation of CD23 were measured. In addition, patients were immunized to øX174.Proliferation, CD23 expression, and IgE production were all normal in the B cell cultures from XLA patients. Although antibody titers after immunization were not normal, XLA patients were capable of responding to the immunization and demonstrated an enhanced response after a secondary immunization suggesting immunologic memory is intact. Significantly, the patients did not undergo isotype switching to IgG on secondary immunization.CD40-mediated B cell function in XLA patients appears to be relatively normal. The authors suggest that this bypass strategy could be used as a potential therapy for XLA.The function of Btk is incompletely understood. Analysis has been complicated by the fact that murine models of XLA exhibit significant differences from the human disease. This study demonstrates that B cells from patients with Btk mutations are not intrinsically dysfunctional. This implies that Btk may be important in B cell maturation but is dispensible for some functions after maturation. This study also implies that there is a Btk bypass pathway that allows some B cells to mature. Using a Btk bypass therapeutic strategy would have significant theoretical risks but is certainly an approach worth consideration.

Leukocyte depletion for leukaemia and multiple sclerosis

LeukoSite, Inc. (Cambridge, MA, USA), and ILEX Oncology, Inc. (San Antonio, TX, USA), have completed the enrolment for their joint, investigational clinical trial of a humanized monoclonal antibody, CAMPATH-1H, in the treatment of B-cell chronic leukaemia (BCLL).CAMPATH-1H (a humanized rat IgGI antibody) selectively targets the CD52 (CAMPATH-1) antigen, a small glycosylphosphatidylinositol (GPI)-anchored glycoprotein antigen of unknown function that is abundantly expressed on lymphocytes, but is absent from haemopoietic stem cells. `Unlike existing cytotoxic drugs and radiation therapy, which destroy many types of cell, CAMPATH-1H selectively depletes all lymphocytes but spares the haemopoietic stem cells that are needed to repopulate the immune system', says Herman Waldmann, professor of pathology at Oxford University (UK) and a member of LeukoSite's scientific advisory board. `The CAMPATH-1 antigen appears to be an unusually good target for cell lysis', explains Waldmann. The susceptibility of the CAMPATH-1 antigen as a target cannot simply be explained by its abundance—∼5×105 molecules per lymphocyte—because even small amounts of antibody are lytic. Waldmann speculates that the proximity of the antigen to the plasma membrane might make it very susceptible to the cytotoxic mechanisms of natural killer cells.The 96 patients taking part in the trial of intravenous CAMPATH-1H, which will take place in several centres throughout the USA and Europe, have BCLL that has failed to respond to standard second-line cytotoxic therapy (fludarabine, an antipurine). In two preliminary trials in which 54 BCLL patients received intravenous CAMPATH-1H, major responses were seen in 40% of patients. `The response rates were very, very encouraging, and survival improved significantly—to one or two years or more', says Chris Mirabelli, chairman and chief executive officer of LeukoSite.The preliminary trials of CAMPATH-1H in patients with BCLL were carried out by the former company Burroughs Wellcome, but it discontinued the development programme for commercial reasons, concentrating instead on developing the antibody for the treatment of patients with rheumatoid arthritis. `Although the antibody worked, this programme too was abandoned because of its adverse effects—mainly infection', says Mirabelli.Nevertheless, Mirabelli is confident that the marriage between LeukoSite's expertise in leukocyte biology and therapeutic antibodies and ILEX's experience with the clinical development of cancer drugs will lead to the successful development of CAMPATH-1H for the treatment of BCLL. He expects an application for regulatory approval and marketing will be made to the US Food and Drug Administration in mid-1999.Several other diseases might benefit from lymphocyte depletion with the CAMPATH-1 family of antibodies. Waldmann and Alastair Compston (University of Cambridge, UK) are about to publish the results of a preliminary trial of CAMPATH-1H in multiple sclerosis (MS). MS is thought to be an autoimmune disease that is mediated by CD4+ T cells that cross the blood–brain barrier and cause neuronal inflammation and demyelination, leading to axonal degeneration. In this trial, in which 37 patients with progressive MS were treated, CAMPATH-1H successfully stopped nerve inflammation (by magnetic resonance imaging analysis). However, half of the patients worsened clinically, indicating that axonal degeneration continued during treatment with CAMPATH-1H. Compston plans to try the drug in patients with very early MS in the near future.CAMPATH-1 antibodies have already been used extensively to prevent graft-versus-host disease in bone-marrow transplant recipients, and LeukoSite is considering using CAMPATH-1H not only as front-line therapy for BCLL, but also for non-Hodgkin's lymphoma.The effects of CAMPATH-1H on the immune system might vary from disease to disease and will depend on the degree of lymphocyte depletion; at the relatively small doses used in the MS study, increased susceptibility to infection is a rare side effect. `In lymphoma, where high doses are used and patients have already had drugs such as fludarabine, immune impairment is likely to last some weeks', cautions Waldmann. `BCLL is itself an immunosuppressive disease; the decision is really one of risk versus benefit.'

Novel surface plasmon resonance sensor chip functionalized with organic silica compounds for antibody attachment

A novel method was developed for the fabrication of a surface plasmon resonance (SPR) sensor chip. A glass chip was covered with a gold layer which was then functionalized with gamma-aminopropylethoxysilane. Biomolecules could then be immobilized on the surface of the sensor using standard techniques. The sensor chip was found to be useful for highly sensitive SPR measurements using even smaller amounts of antibody than usual. The chip was found to be useful for the immobilization of anti-human serum albumin (anti-HSA) and anti-atrazine antibody, and was used for the SPR measurement of HSA and enzyme-labeled atrazine. The chip showed higher responses at both 10 and 100 mg l −1 HSA than a commercially available model. The relative standard deviation (RSD) of the responses was 7% for both concentrations of HSA ( n=8). A monoclonal antibody for atrazine was immobilized on the chip and 0.05 μg l −1 atrazine was measured with a RSD of 7% ( n=5). Atrazine was determined after addition to river water; 0.05 μg l −1 atrazine was detected with a very similar response as a standard atrazine solution of the same concentration.

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.

Determination of drugs from urine by on-line immunoaffinity chromatography—high-performance liquid chromatography—mass spectrometry

A method for rapid extraction and identification of drugs in urine is described. The system utilizes a high-performance protein G immunoaffinity column coupled to a reversed-phase analytical column by use of a trapping column and switching valve. A small amount of antibody (5 μg drug-specific) is used for each analysis to extract either propranolol or lysergic acid diethylamide (LSD) from human urine. Urine diluted with phosphate-buffered saline is pumped directly through the protein G column thus eliminating time- and solvent-consuming sample preparation procedures. On-line ultraviolet or mass spectral analysis provides the means of drug detection and identification. With ultraviolet detection propranolol may be detected in spiked urine at the 250 pg/ml level. A Hewlett-Packard mass spectrometer modified for atmospheric pressure ionization and equipped with an ion spray source allows detection of propranolol in urine at 2.5 ng/ml and LSD at 500 pg/ml using single ion monitoring. The potential applicability of the technique for drug confirmations is discussed.

Antibody exchange immunochemistry.

Antibodies are found to transfer rapidly between antigen samples attached to separate solid supports. The half-times for this antibody exchange at 20 and 37 degrees C were 7 and 1 h, respectively. Antibody exchange was exhibited by all 17 polyclonal and monoclonal antibodies tested and was readily detected by enzyme-linked immunosorbent assay, immunoblotting, or immunocytochemical assays. Taking advantage of this phenomenon, antibodies can be affinity-purified using less than a picomole of antigen and small amounts of antibody.

Efficacy of Influenza Haemagglutinin and Nucleoprotein as Protective Antigens against Influenza Virus Infection in Mice

Influenza nucleoprotein (NP)-specific cytotoxic T lymphocytes (CTL) stimulated by immunization of mice with VV-PR8-NP6, a recombinant vaccinia virus expressing A/PR/8/34 NP, did not protect mice against challenge with A/PR/8/34 4 days later. Neither were secondary NP-specific CTL stimulated by reimmunization able to protect mice. These results contrast with the ability of transferred, in vitro-cultured and stimulated, NP-specific CTL to protect recipient mice from challenge with A/PR/8/34. Immunization of mice with a recombinant vaccinia virus expressing A/PR/8/34 HA protected mice challenged 4 days later, either via the small amount of antibody already present, or via HA-specific CTL that would have to be more efficient than NP-specific CTL in either trafficking to the infected lung or in effector function.

Immunochemical Properties of Native and Heat Denatured Ovalbumin

ABSTRACTAffinity antibodies purified against native ovalbumin were found more reactive (higher avidity) against heat‐denatured ovalbumin than against the native molecule by three different immunochemical methods. Quantitative immunoprecipitation in soluble phase revealed that more antigen‐antibody complexes were insoluble with native ovalbumin than with heat‐denatured ovalbumin; 25% of antibodies were still present in supernatant at equivalence as measured by ELISA. At the same conditions with heat‐denatured ovalbumin, very small amounts of antibodies were precipitated while almost no activity was found in the supernatant. Classical ELISA or competitive ELISA test allowed detection down to 100 ng/mL of native ovalbumin and 10 ng/mL of denatured ovalbumin.

A genotype-dependent hippocampal dynorphinergic mechanism controls mouse exploration

Following microinjections with two dilutions of anti-dynorphin B antiserum into the hippocampal CA3 region, adult male mice from the inbred strains DBA/2 and C57BL/6 were individually tested for various exploratory behaviors in a novel environment and compared to preimmune serum control animals. Treatment augmented vertically-oriented exploratory acts in strain DBA/2 and reduced the scores in strain C57BL/6 so that strain differences originally present between the controls were reversed or eliminated after antiserum. These opposite effects indicate that a hippocampal dynorphinergic mechanism is involved in the regulation of novelty-induced behavior in mice and that its modulatory function depends on the genotype. It is concluded that DBA/2 animals exposed to novelty, as compared to C57BL/6, are characterized by an over-release of hippocampal dynorphin B which is neutralized in part by small amounts of antibody.

Inhibition of development in Myxococcus xanthus by monoclonal antibody 1604.

Monoclonal antibody (mAb) 1604 is directed against a cell surface antigen of Myxococcus xanthus. Purified antibody 1604 inhibited development of M. xanthus under conditions of submerged culture procedure otherwise leading to fruiting body formation. Intact molecules of mAb 1604, as well as its Fab fragments, inhibited developmental aggregation, autolysis, fruiting body formation, and sporulation. The addition of relatively small amounts of antibody every 4 hr was much more effective than a single large dose given at the onset of development. The inhibitory action of mAb 1604 on development was reversible after prolonged incubation of the antibody with cells; this was probably due to proteolytic degradation of the antibody. The effect of mAb 1604 on submerged bacterial development was neutralized by affinity-purified 1604 cell surface antigen. Another antibody, mAb 2788, directed against an M. xanthus cell surface antigen, did not block development. These data suggest that 1604 cell surface antigens is involved in contact-mediated cell interactions in M. xanthus.