Sheep Anti-human IgG Research Articles

Novel Method for the Isolation of Proteins and Small Target Molecules from Biological and Aqueous Media by Salt-Assisted Phase Transformation of Their PEGylated Recognition Counterparts.

An efficient and simple method for the application of PEGylated affinity ligands in precipitative isolation of protein target molecules (TMs) from a biological fluid such as blood serum or small target molecules from an aqueous medium is presented for the first time. This approach is based on the high binding specificity of PEGylated recognition molecules (PEG-RMs) to their TMs and the unique physicochemical properties of PEG that result in their salt-assisted phase transformation. Addition of PEG-RM to blood serum results in the formation of an RM-specific macromolecular complex (PEG-RM + TM → PEG-RM.TM) that undergoes facile salt-assisted phase transformation to a separable semisolid with ammonium sulfate. PEG-RM.TM is then dissociated into its components by pH reduction or an increase of ionic strength (PEG-RM.TM → PEG-RM + TM). PEG-RM is salted out to afford pure TM in solution. The same phenomenon is observed when RM or TM are small molecules. The general applicability of the method was validated by PEGylation of two proteins (protein A, sheep antihuman IgG) and a small molecule (salicylic acid) used as model RMs for the isolation of Igs, IgG, and serum albumin from blood serum. The isolated protein TMs were shown to be pure and aggregate-free by gel electrophoresis and dynamic light scattering (DLS). IgG isolated by this method was further characterized by peptide mass fingerprinting. PEGylated protein A was used to demonstrate the recyclability and scale-up potential of PEG-RM. IgG isolated by this method from blood serum of a hepatitis C-vaccinated individual was tested for its binding to sheep antihuman IgG by UV spectroscopy, and its bioactivity was ascertained by comparison of its enzyme-linked immunosorbent assay (ELISA) result to that of a blood sample from the same individual. Reciprocity of RM and TM was ascertained using PEGylated salicylic acid to obtain pure serum albumin, and PEGylated serum albumin was utilized for near-exclusive isolation of one drug from an aqueous equimolar mixture of three drugs (salicylic acid, 91%; capecitabine, 6%; and deferiprone, 3%). Advantages of this approach, including target specificity and general applicability and celerity, over other affinity methods for the isolation of proteins are discussed at a molecular level.

Establishment and verification of detecting multiple biomarkers for ovarian cancer by suspension array technology

Objective: Establish and validation of combined detecting of CCL18, CXCL1, C1D, TM4SF1, FXR1, TIZ suspension array technology. Methods: (1)CCL18, CXCL1 monoclonal antibody and C1D, TM4SF1, FXR1, TIZ protein were coupled with polyethylene microspheres. Biotinylated CCL18, CXCL1 polyclonal antibody and sheep anti-human IgG polyclonal antibody were prepared simultaneously. The best packaged concentrations of CCL18, CXCL1 monoclonal antibody and C1D, TM4SF1, FXR1, TIZ antigens were optimized. The best packaged concentrations of CCL18, CXCL1 polyclonal antibodys and C1D, TM4SF1, FXR1, TIZ sheep anti-human IgG polyclonal antibody were optimized to establish a stable detected suspension array.(2)Sixty patients confirmed by pathological examination with ovarian cancer(ovarian cancer group)which treated in Affiliated Tumor Hospital of Guangxi Medical University, 30 patients with ovarian benign tumor(benign group)and 30 cases of healthy women(control group)were chosen between September 2003 and December 2003. Suspension array technology and ELISA method were used to detect expression of CCL18, CXCL1 antigen and C1D, TM4SF1, FXR1 and TIZ IgG autoantibody contented in 3 groups of serum, then to compare the diagnostic efficiency and diagnostic accuracy of two methods(coefficient of variation between batch and batch). Results: (1)This research successfully established stable detecting system of CCL18, CXCL1, C1D, TM4SF1, FXR1 and TIZ IgG autoantibody. The best concentration of CCL18, CXCL1 monoclonal antibody and C1D, TM4SF1, FXR1, TIZ antigen package were 8, 8, 12, 8, 4 and 8 μg/ml; the best detection of CCL18, CXCL1 biotin polyclonal antibody and C1D, TM4SF1, FXR1, TIZ sheep anti-huamne IgG polyclonal antibody were respectively 4, 2, 2, 4, 4 and 2 μg/ml.(2)Suspension array technology and ELISA method were used to detect CCL18, CXCL1 antigen and C1D, TM4SF1, FXR1, TIZ IgG autoantibody of three groups in serum were similar(P>0.05).(3)The comparison of two methods in the diagnosis of efficiency: the diagnostic accuracy of two methods were 99.2%(119/120)and 94.2%(113/120), the difference was statistically significant(P=0.031). The sensitivity of the diagnosis of ovarian cancer of two methods were 100.0%(60/60)and 93.3%(56/60), specific degrees were 100.0%(59/59)and 93.4%(57/61), positive predictive value was 100.0%(60/60)and 93.3%(56/60), negative predictive value was 98.3%(59/60)and 95.0%(57/60), the difference was statistically significant(P<0.05).(4)The detected results of CCL18, CXCL1 antigen and C1D, TM4SF1, FXR1, TIZ IgG autoantibody shown that the diagnostic accuracy of suspension array technology was superior to those of ELISA method(all P<0.05). Conclusion: The study has established the stable detection of suspension array technology, and the diagnostic efficiency and diagnostic accuracy was much better than that by ELISA.

Response of primary vectors and related diseases to impoundment by the Three Gorges Dam

To investigate the impact of the Three Gorges Dam on the local ecological environment. We conducted a 3-year cross-sectional survey of natural focus infectious diseases in the area before and after the water level rose to 156 m to evaluate the dam's health impacts. Direct and indirect immunofluorescence methods were applied to detect rat antigen and antibody of haemorrhagic fever with renal syndrome (HFRS). Fresh rat kidneys were inoculated in Korth's culture medium to detect Leptospira. A group of captured crabs were ground to observe the metacercariae of Paragonimus. Serum samples were collected from healthy local individuals. ELISA kits were used to detect human antibody against HFRS and paragonimiasis. Human Leptospira infections were detected by a microscopic agglutination test. Upstream rodent density increased significantly with Rattus flavipectus and Apodemus agrarius as the major pathophoric genera. The infection rate of human HFRS and Leptospira in the upstream human population samples was significantly higher than in the downstream samples and correlated with the increase in rodent density. Paragonimus infection rates remained at a low level during the study. Culex pipiens fatigan and Armigeres obturbans were the dominant species of mosquito. The creation of the Three Gorges Dam changed the proliferation of intermediary agents of diseases, but not notably. However, the ecological effects on the environment may require a prolonged period of time to manifest themselves; thus, long-term and effective surveillance of vectors and related diseases needs to be established.

Therapeutic Apheresis in Myasthenia Gravis

Plasmapheresis has considerably improved the management of patients affected by myasthenia gravis (MG), particularly in case of severe acute worsening with bulbar impairment. Controlled trials against placebo are not available in this field, but evidences emerged from open-label studies are considered a strong confirmation of the clinical efficacy of plasmapheresis in MG. More recently, the techniques of selective apheresis based on the properties of protein A and sheep anti-human IgG have been introduced into clinical practice, allowing chronic apheresis in treatment-resistant MG patients.

Specific immunoadsorption therapy using a tryptophan column in patients with refractory heart failure due to dilated cardiomyopathy

Certain cardiac-specific autoantibodies found in patients with dilated cardiomyopathy (DCM) play a role in mediating myocardial damage and fatal ventricular arrhythmias resulting in sudden cardiac death. Immunoadsorption therapy (IA) is one of the therapeutic tools to remove such autoantibodies. Clinical studies from Germany have shown that nonspecific IA using columns loaded by sheep antihuman IgG or protein A improved hemodynamic data and affected favorably cardiac function and survival in patients with heart failure (HF) due to DCM. The goal of this study is to determine if IA therapy using the high-profile tryptophan column, which has high affinity for IgG3 subclass, affects favorably cardiac function in patients with severe HF who are refractory to conventional therapy. IA therapy was conducted in 16 patients with DCM (age 53 ± 4, male 8, New York Heart Association functional class III/IV, mean ejection fraction 18 ± 2%). Study subjects had autoantibodies directed against either β1-adrenergic or M2-muscarinic receptors. Plasma brain natriuretic peptide levels were significantly decreased after IA (P = 0.016). Plasma inflammatory cytokines including interleukin-6 and tumor necrosis factor-α did not change after each session of IA. Six-minute walk distance was significantly increased after IA (P = 0.01). Left ventricular ejection fraction increased by 3% 3 months after IA (P = 0.039). Our initial experience demonstrated safety and short-term efficacy of IA using a new IgG3-specific tryptophan column for patients with advanced HF due to DCM. Long-term follow-up is needed to confirm the effects on cardiac function and morbidity/mortality in such patients.

Abstract B138: Disappearance of “M” component connected with cow’s milk protein-free diet

Abstract B138 The presence of “M” component, as monoclonal gammopathy of undetermined significance (MGUS) in many patients with psoriatic arthritis and in 5% healthy people older than 70 years is well known. Besides, it was stressed out that patients with psoriatic arthritis and MGUS can sometimes develop multiple myeloma. Serum from patient 83 years old, with psoriatic arthritis diagnosed at 2000 was analyzed. Determination of the total IgG, IgA and IgM immunoglobulins was done by nephelometry using commercial (Binding Site) tests. Serum protein electrophoresis was done on gel electrophoresis in barbital buffer, pH=8.6; this was followed by immunofixation using sheep antihuman antibodies (Binding Site) to light and heavy chains of immunoglobulins. Determination of serum IgA and IgG immunoreactivity to gliadin in (IU/ml) was done using commercial Binding Site ELISA test, while serum immunoreactivity to cow’s milk protein (CMP) in (AU/ml) was done by home made ELISA test, using cows milk pasteurized powder (ICN Biomedicals, Inc.) as the antigen and sheep antihuman IgA and IgG, HRP labeled antibodies (Binding Site) as secondary antibodies. Blocker was 1% bovine serum albumin. Results obtained showed the enhanced IgA and IgG immunoreactivity to CMP. The analysis of serum by electrophoresis and immunofixation reveals the presence of monoclonal IgG (λ) immunoglobulin. Upon the knowledge that the enhanced immunoreactivity especially to CMP exists the patient decided to consume the food without cow’s milk proteins. About month and half after the start of CMP free diet he has done his blood for the analysis of serum proteins again. In that time of period the patient has not taken any immunosuppressive drugs. Electrophoresis of patient sera reveals that after the CMP free diet the “M“component disappeared. These data are supported by the findings that serum anti-CMP IgG dropped from 892 AU/ml to 0 AU/ml (anti-CMP IgA also dropped from 240 AU/ml to 0 AU/ml) Besides, concentrations of total serum proteins were diminished after the CMP free diet from 73 g/l to 69 g/l, while concentration of IgG was decreased from 17.8 g/l to 11.2 g/l. Our report shows that there could be a connection between diet restricted for immunogenic protein(s) and disappearance of MGUS. Our results are along with the data from only one report dealing with a possible link between disappearance of MGUS and gluten free diet. They open the question whether the special, appropriate diet restriction regime could help in the prevention of multiple myeloma. Citation Information: Cancer Prev Res 2008;1(7 Suppl):B138.

Unappreciated HLA Antibodies in Adult Immune Thrombocytopenic Purpura

Immune thrombocytopenic purpura (ITP) is an autoimmune disease. Platelet refractoriness is frequently seen in patients with ITP. Platelets express platelet-specific antigens and human leukocyte antigens (HLA). Platelet antibodies to platelet-specific antigens and HLA may be present, but HLA antibodies in patients with ITP have rarely been reported. Sera from 44 adult patients with ITP were screened for platelet antibodies by two flow cytometric assays. In method I, platelets from normal donor platelets were used as target cells to screen both platelet-specific antibodies and HLA class I antibodies. In method II, the FlowPRA Class I Screening Test kit was used to screen HLA class I antibodies. Fluorescein isothiocyanate (FITC)-conjugated sheep anti-human IgG Fc was used as the staining reagent in both methods. The negative serum control was from one of the normal males with AB blood group who had never received a transfusion. Sera from a pool of five highly sensitized patients were used as the positive control. Of the 44 sera from patients with ITP, 31 (70.5%) were method I positive, and 28 (63.6%) were method II positive. There was no significant difference between the results of method I and method II (p = 0.439). The distribution of the results of these two tests was: both tests positive in 22 sera, method I positive and method II negative in nine sera, method I negative and method II positive in six sera, and both tests negative in seven sera. The mean platelet counts of patients with positive (41.0 +/- 40.0 x 10(9)/L) and negative (40.4 +/- 26.8 x 10(9)/L) tests by method I did not differ significantly (p = 0.643). The mean platelet counts of patients with (36.7 +/- 31.5 x 10(9)/L) and without (48.1 +/- 43.6 x 10(9)/L) HLA class I antibodies did not differ significantly (p = 0.59). HLA class I antibodies are frequently found in ITP. The screening of platelet antibodies including platelet-specific antibodies and unappreciated HLA class I antibodies is warranted in patients with ITP.

Screening for Platelet Antibodies in Adult Idiopathic Thrombocytopenic Purpura: A Comparative Study Using Solid Phase Red Cell Adherence Assay and Flow Cytometry

Idiopathic thrombocytopenic purpura (ITP) is an autoimmune disorder caused by antiplatelet autoantibodies. In this study, we compared 2 methods for screening serum platelet antibodies in patients with ITP. A total of 44 adult patients were clinically classified with ITP. We used 2 indirect tests to detect human leukocyte antigen antibodies and/or platelet-specific antibodies in their sera. In method I, we used solid phase red cell adherence (SPRCA) assay. In method II, by flow cytometry, platelets from plateletpheresis components were used as target cells, and fluorescein isothiocyanate-conjugated sheep anti-human IgG Fc was used as the staining reagent. Positive results were defined as any test with the percentage of fluorescence exceeding the reference range by 3% or more in method II. Direct tests detecting platelet-associated IgG on platelets of patients with ITP were done by flow cytometry. Serum specimens from 44 adult patients with ITP (28 female, 16 male) were tested. SPRCA assay could only detect platelet antibodies in 22 patients (50%). By method II, 31 serum specimens (70.5%) yielded positive results. There was a difference between the results of the SPRCA test and method II, with a high degree of significance (p < 0.001) by the McNemar test. No significant difference in platelet counts was observed for patients with and without discernible platelet antibodies by SPRCA assay (p = 0.90). The direct test was positive in 12 patients (66.7%) out of 18 ITP patients tested. Flow cytometry is more sensitive than SPRCA assay for detecting platelet antibodies. Detection of platelet antibodies is useful in explaining the immune mechanism and platelet transfusion refractoriness in ITP.

Frequencies of the expression of main protein antigens fromHelicobacter pyloriisolates and production of specific serum antibodies in infected patients

To investigate the frequencies of the expression of main protein antigens of Helicobacter pylori (H pylori) isolates, such as UreB, VacA, CagA1, HpaA, NapA, FlaA and FlaB and the production of specific antibodies in sera from H pylori-infected patients, and to understand the correlations among the different clinical types of chronic gastritis and peptic ulcer and the infection and virulence of H pylori. H pylori strains in biopsy specimens from 157 patients with chronic gastritis and peptic ulcer were isolated and serum samples from the patients were also collected. The target recombinant proteins rUreB, rVacA, rCagA1, rHpaA, rNapA, rFlaA and rFlaB expressed by the prokaryotic expression systems constructed in our previous studies were collected through Ni-NTA affinity chromatography. Rabbit antisera against rUreB, rVacA, rCagA1, rHpaA, rNapA, rFlaA and rFlaB were prepared by using routine subcutaneous immunization. By using ultrasonic lysates of the isolates as coated antigens, and the self-prepared rabbit antisera as the first antibodies and commercial HRP-labeling sheep anti-rabbit IgG as the second antibody, expression frequencies of the seven antigens in the isolates were detected by ELISA. Another ELISA was established to detect antibodies against the seven antigens in sera of the patients by using the corresponding recombinant proteins as coated antigens, and the sera as the first antibody and HRP-labeling sheep anti-human IgG as the second antibody respectively. Correlations among the different clinical types of chronic gastritis and peptic ulcer and the infection and virulence of H pylori were statistically analysed. In the 125 isolates of H pylori, the positive rates of UreB, VacA, CagA1, HpaA, NapA, FlaA and FlaB were 100%, 65.6%, 92.8%, 100%, 93.6%, 100% and 99.2% respectively. In the 125 serum samples from the H pylori-infected patients, the positive rates of antibodies against recombinant UreB, VacA, CagA1, HpaA, NapA, FlaA and FlaB were 100%, 42.4%, 89.6%, 81.6%, 93.6%, 98.4% and 92.8% respectively. H pylori strains were isolated from 79.6% (125/157) of the biopsy specimens, but no close correlations among the H pylori infection frequencies and different types of chronic gastritis and peptic ulcer could be found (P>0.05, chi2 = 0.01-0.87). The VacA positive rate (82.40%) in the strains isolated from the specimens of patients with peptic ulcer and the anti-VacA positive rate (54.3%) in the sera from the patients were significantly higher than those (51.5%, 32.3%) from the patients with chronic gastritis (P<0.01, chi2 = 13.19; P<0.05, chi2 = 6.13). When analysis was performed in the different types of chronic gastritis, the VacA in the strains isolated from the specimems of patients with active gastritis showed a higher expression frequency (90.0%) than those from superficial (47.9%) and atrophic gastritis (30.0%) (P<0.05, chi2 = 5.93; P<0.01, chi2 = 7.50). While analysis was carried out in the strains isolated from the specimens with superficial (93.8%) and active gastritis (100%), NapA showed a higher expression frequency compared to that from atrophic gastritis (60.0%) (P<0.01, chi2 = 8.88; P<0.05, chi2 = 5.00). The types of chronic gastritis and peptic ulcer and their severity are not associated with H pylori infection frequency but closely related to the infection frequency of different virulent H pylori strains. The optimal antigens for developing vaccine and diagnostic kit are UreB, FlaA, HpaA, FlaB, NapA and CagA1, but not VacA.

Cerebroside Sulfotransferase Deficiency Ameliorates L-selectin-dependent Monocyte Infiltration in the Kidney after Ureteral Obstruction

Mononuclear cells infiltrating the interstitium are involved in renal tubulointerstitial injury. The unilateral ureteral obstruction (UUO) is an established experimental model of renal interstitial inflammation. In our previous study, we postulated that L-selectin on monocytes is involved in their infiltration into the interstitium by UUO and that a sulfated glycolipid, sulfatide, is the physiological L-selectin ligand in the kidney. Here we tested the above hypothesis using sulfatide- and L-selectin-deficient mice. Sulfatide-deficient mice were generated by gene targeting of the cerebroside sulfotransferase (Cst) gene. Although the L-selectin-IgG chimera protein specifically bound to sulfatide fraction in acidic lipids from wild-type kidney, it did not show such binding in fractions of Cst(-/-) mice kidney, indicating that sulfatide is the major L-selectin-binding glycolipid in the kidney. The distribution of L-selectin ligand in wild-type mice changed after UUO; sulfatide was relocated from the distal tubules to the peritubular capillaries where monocytes infiltrate, suggesting that sulfatide relocated to the endothelium after UUO interacted with L-selectin on monocytes. In contrast, L-selectin ligand was not detected in Cst(-/-) mice irrespective of UUO treatment. Compared with wild-type mice, Cst(-/-) mice showed a considerable reduction in the number of monocytes/macrophages that infiltrated the interstitium after UUO. The number of monocytes/macrophages was also reduced to a similar extent in L-selectin(-/-) mice. Our results suggest that sulfatide is a major L-selectin-binding molecule in the kidney and that the interaction between L-selectin and sulfatide plays a critical role in monocyte infiltration into the kidney interstitium.

Enhancement of the sensitivity of surface plasmon resonance biosensor with colloidal gold labeling technique

In our experiment colloidal gold was used to enhance the signal obtained from the surface-plasmonresonance biosensor. Fab′ fragments of human IgG molecules were immobilized on the gold surface and reacted with the sheep-anti-human IgG(SAH-IgG) molecules in solution. It was found that when the colloidal gold labeling technique was introduced the sensitivity of surface plasmon resonance instruments increased by a factor of about 300.

In Vivo Clearance of Ternary Complexes of Vitronectin-Thrombin-Antithrombin Is Mediated by Hepatic Heparan Sulfate Proteoglycans

Thrombin is inhibited by its cognate plasma inhibitor antithrombin, through the formation of covalent thrombin-antithrombin (TAT) complexes that are found as ternary complexes with vitronectin (VN-TAT). To determine whether the metabolism of VN-TAT ternary complexes is different from that previously reported for binary TAT complexes, plasma clearance studies were done in rabbits using human VN-TAT. 125I-VN-TAT was shown to be cleared rapidly from the circulation (t1/2alpha = 3.8 min) in a biphasic manner mainly by the liver. 125I-TAT had a similar initial clearance (t1/2alpha = 5.3 min) but had a significantly faster beta-phase clearance (t1/2beta = 42.8 min versus 85.4 min for VN-TAT; p = 0.005). Protamine sulfate and heparin abolished the rapid initial alpha-phase of 125I-VN-TAT clearance and reduced its liver-specific association and in vivo degradation. Heparin also reduced the alpha-phase clearance of 125I-TAT and was associated with the appearance of high molecular weight complexes, suggesting enhanced complex formation between VN and TAT. 125I-VN-TAT binding to HepG2 cells was reduced by competition with VN-TAT or heparin but to a much lesser extent in the presence of TAT. The binding of VN-TAT to HepG2 cells was not inhibited by competition with the low density lipoprotein receptor-related protein ligand, methylamine-alpha2-macroglobulin. 125I-VN-TAT binding was also inhibited by treating HepG2 cells with heparinase or by growing the cells in the presence of beta-D-xyloside. Finally, both heparin and chloroquine, but not methylamine-alpha2-macroglobulin, reduced the internalization and degradation of VN-TAT by HepG2 cells. Taken together, these data indicate the importance of VN in TAT metabolism and demonstrate that VN-TAT binds to liver-associated heparan sulfate proteoglycans, which mediate its internalization and subsequent intracellular degradation.

Antigen endocytosis and presentation mediated by human membrane IgG1 in the absence of the Ig(alpha)/Ig(beta) dimer.

Membrane immunoglobulin (mIg) M and D heavy chains possess minimal (KVK) cytoplasmic tails and associate with the Ig alpha/Ig beta (CD79) dimer to achieve surface expression and antigen presentation function. In contrast, the cytoplasmic tail of mIgG is extended by 25 residues (gamma ct). We have tested the possibility that mIgG can perform antigen capture and presentation functions independently of the Ig(alpha)/beta dimer. We show that CD4/(gamma)ct chimeras are efficiently endocytosed partially dependent on a tyrosine residue in (gamma)ct. In addition, human mIgG was expressed on the surface of Ig(alpha)/Ig(beta)-negative non-lymphoid cells and mediated antigen capture and endocytosis. Antigen-specific human mIgG targeted antigen to MIIC-type vesicles in the Ig(alpha)/beta negative melanoma Mel JuSo and augmented antigen presentation 1000-fold, identical to the augmentation seen in Ig(alpha)/beta-positive B-cells expressing the same transfected mIgG. Thus, unlike mIgM, mIgG has autonomous antigen capture and presentation capacity, which may have evolved to reduce or eliminate the BCR's dependence on additional accessory molecules.

N‐(β‐Carboxypropionyl)luminol as a new chemiluminescence label in immunoassay

AbstractA new chemiluminescence label N‐(β‐carboxpropionyl)luminol (CPL) was used to label sheep anti‐human IgG (SaHIgG). The labeled antibody waa stable and could be detected at least dawn to 10−17 ∼10−16 mol. The molar incorporation ratio was estimated to be 0.26 mol of CPL per mol of SaHIgG. There were no apparent changes in the immunoreactivity of the labeled SaHIgG and in the quantum efficiency of the CPL after labeling.

Evaluation of Human IgG Subclass Assays on Beckman Array

Automated immunonephelometric assays were developed to measure human IgG subclasses in serum, on the Beckman Array Protein System (APSR), with sheep antihuman IgG subclass antisera. The interassay imprecision was judged to be satisfactory in each case (CV: IgG1: 2.3%; IgG2: 2.9%; IgG3: 4.0% and IgG4 5.4%). The standard curves were found to be linear in the ranges 1.5-30.0 g/L for IgG1, 1.5-15.0 g/L for IgG2, 0.1-1.0 g/L for IgG3 and 0.1-1.0 g/L for IgG4. The lower limit for quantification of the immunephelometric assays was 0.5 g/L for IgG1, 0.04 g/L for IgG2, 0.06 g/L for IgG3 and 0.03 g/L for IgG4. We found no antigen excess to at least 60.0 g/L for IgG1 and IgG2, 3.5 g/L for IgG3 and 2.0 g/L for IgG4. Nephelometric results correlated with those of radial immunodiffusion (r = 0.96 for IgG1, r = 0.93 for IgG2, r = 0.90 for IgG3 and r = 0.96 for IgG4). The reaction monitored by the Beckman Array Protein System is easy to perform, rapid and precise. The performance makes the immunonephelometric method suitable for the determination of IgG subclass concentrations in routine analysis.

Removal Of Baboon And Human Antiporcine Igg And Igm Natural Antibodies By Immunoadsorption

The safe and effective removal of xenoreactive antibodies in the peritransplant period is likely to be critical for the clinical application of xenotransplantation involving disparate donor species, such as the pig. In an effort to develop an improved method for antibody removal in xenotransplantation, we have studied reusable antihuman antibody (Ig) columns in vitro and in vivo. Two types of columns were tested: (1) an antihuman Ig column containing polyclonal sheep antihuman IgG (heavy- and light-chain-specific) conjugated to sepharose CL-4B (Ig-Therasorb), and (2) an antihuman Ig column using polyclonal antihuman IgM (mu-chain-specific) conjugated to sepharose. Passage of human or baboon plasma through the Ig-Therasorb column resulted in 97.5% and 78.4% mean reductions in total IgG and IgM, respectively. Reductions in total IgG and IgM correlated with lowering of antipig IgG (54-486 fold) and IgM (9-54 fold) antibody titers as assessed by pig endothelial cell ELISA. The ability of the Ig-Therasorb to significantly reduce IgM may be attributed to the light chain specificity of this column. With the anti-IgM column, marked reductions in total (82.6-83.9%) and antipig (27-54 fold) IgM in human and baboon plasma occurred, while levels of total and xenoreactive IgG were slightly affected. Other than a dilutional effect, neither column resulted in significant reduction in albumin, fibrinogen, factor 5, and factor 8. Repeated in vivo use of either column in baboons achieved reductions in IgG and IgM that closely followed the results of our in vitro studies. No subject morbidity or mortality occurred. Use of the Ig-Therasorb column with immunosuppression in two baboons receiving pig renal xenografts achieved sustained reductions in antipig antibodies and prevented hyperacute rejection. Subjects were sacrificed at 11 and 13 days posttransplant with functioning xenografts and were found to have no evidence of vascular xenograft rejection. We conclude that anti-Ig columns represent a safe and effective method for antibody removal, without several of the limitations of other antibody removal techniques. Also, columns appear to be safe for repeated antibody removal in the posttransplant period.

Potential for targeting hepatoma with anti-ferritin IgG-daunomycin immunoconjugate

Sheep anti-human ferritin IgG (AHFI) and nonspecific normal sheep IgG (NSIgG) were conjugated with Daunomycin (Dau) separately via a mediator Dextran T40. The conjugates (AHFI-Dex-Dau and NSIgG-Dex-Dau) were labelled with125I and given i. v. into Balb/c nude mice bearingan hepatocellular carcinoma (HCC). The biodistribution of the two conjugates in different organs or tissues 3 days after injection of radiolabels were measured. Selective tumor accumulation of AHFI-Dex-Dau was observed. The specific localization index was at least fourteen times higher in case of the specific AHFI-Dex-Dau when compared to another control conjugate (NSIgG-Dex-Dau) in liver tissues.In vivo treatment experiment, the results demonstrated that conjugation of Dau to IgG could greatly reduce its nonspecific chemical toxicity and significantly increase its ability to inhibit the engrafted tumor growth.

Continuous liquid-phase piezoelectric biosensor for kinetic immunoassays.

The reactions of immunoglobulins with protein A and, subsequently, of antibodies to these immunoglobulins were continuously monitored by a piezoelectric biosensor. AT-cut crystals, with a fundamental resonant frequency of 10 MHz, were mounted in a Plexiglas holder, and one side was directly coated with protein A. Upon exposure to solutions containing rabbit or human IgG the resonant frequency was followed continuously and its decrease due to the binding of IgG to protein A observed. Subsequent addition of sheep antihuman IgG to the now immobilized human IgG caused a specific 3-fold further decrease in resonant frequency. We observed a frequency change of approximately 1 Hz fore each 10 ng of added immunoglobulin. Decreasing the pH to 3 released the bound IgG but not the protein A and permitted reuse of the crystal for further IgG binding.

Proteins associated with activity of Fc receptors on isolated human placental syncytiotrophoblast microvillous plasma membranes.

To identify Fc receptors from human placental microvilli, proteins that were liberated by detergents from human placental synctiotrophoblast microvillous membranes (StMPM) were characterized by their abilities to bind human IgG in immune complexes with sheep or goat anti-human IgG and to monomeric rabbit anti-dinitrophenol (DNP) IgG bound to DNP-lysine Sepharose. Three placental IgG-binding proteins coprecipitated with immune complexes (Mr = 68,000, 52,000-56,000, 40,000) and were designated pIBP68, pIBP56 and pIBP40, respectively. Of the three proteins only pIBP56 bound to immobilized monomeric rabbit IgG. It was isolated from detergent lysates of StMPM and LDS/phenol glycoprotein extracts of placental plasma membranes suggesting that pIBP56 was a glycoprotein FcR previously reported (Mikulska et al, 1982). The binding specificities of pIBP56 and pIBP40 appeared to be detergent dependent. Photoaffinity crosslinking of StMPM surface proteins in situ to monomeric rabbit derivatized with N-succinimidyl(4-azidophenyl)-I, 3-dithiopropionate identified IgG-binding proteins identical in size to pIBP56 and pIBP40. Crosslinking further suggested that monomeric IgG covalently bound to a complex of StMPM proteins with a total size of 110,000-120,000 Mr. The findings suggest that pIBP68, pIBP56 and pIBP68 are responsible for IgG binding activity of placental StMPM.

Enzyme-linked immunosorbent assay for human metallothionein: correlation of induction with infection.

Very little information is available concerning the relationship between metallothionein and disease in humans. Recently, investigators have used the Cd-heme method to measure metallothionein levels in human liver samples obtained from autopsy. This assay, however, is not sensitive enough to measure metallothionein in small tissue biopsy specimens. As an alternative, we report the development of a human metallothionein enzyme-linked immunosorbent assay (ELISA). This assay used high-performance liquid chromatography-purified human metallothionein-1 and purified sheep anti-human metallothionein-1 IgG. A limiting antigen coating concentration of 100 ng/ml and a minimal antibody dilution of 1:4000 were chosen. The sensitivity of the ELISA extended to 300 ng/ml (15 ng). The coefficients of interassay and intraassay variation were 15.4% and 4.2%, respectively. Human livers obtained from autopsy were assayed by this method and the values compared with values obtained by the Cd-heme method. The livers were separated by their autopsy reports into four groups: normal, immunosuppression, cancer, and infection. Livers from the infection group (ELISA 2979 micrograms/gm, Cd-heme 1201 micrograms/gm) contained significantly more metallothionein than those from the normal (ELISA, 1035 micrograms/gm, Cd-heme 245 micrograms/gm) and the immunosuppression (ELISA 1272 micrograms/gm, Cd-heme 221 micrograms/gm) groups (p less than 0.05). The cancer group (ELISA 2415 micrograms/gm) also had significantly elevated liver metallothionein levels. We conclude that this ELISA is sensitive enough for the measurement of tissue samples. Furthermore this assay is comparable to the Cd-heme assay in its ability to reflect metallothionein values among various treatment groups. We postulate that hepatic metallothionein induction is mediated by disease-related mechanisms such as interleukin-1, glucocorticoid secretion, or both.