Detection of IgG antibody to measles virus by radioimmunoassay technique

A highly sensitive procedure of solid-phase radioimmunoassay (RIA) was developed for the detection of measles IgG antibody. HeLa cells persistently infected with measles virus were used as a solid-phase antigen. This technique was applied to the detection of measles IgG antibody in patients with subacute sclerosing panencephalitis (SSPE) and multiple sclerosis. Normal subjects having experienced natural measles or measles vaccination and patients with various neurological diseases of non-virus nature were also examined as control groups. Measles antibody was detected at high titers in both the sera and cerebrospinal fluid of SSPE patients. Moreover, RIA/HI ratios of SSPE patients were significantly higher than those of normal subjects, suggesting the presence in the formers of antibodies to nucleocapsids at high titers as well as to viral envelopes. On the other hand, no significant difference was found in both RIA and HI titers between the sera of multiple sclerosis and those of various neurological diseases.     

Isotype profiles of anti-influenza antibodies in mice bearing the xid defect.

The humoral response to influenza A/PR8 virus was examined in the CBA/N and C3J.xid strains of mice, both of which bear an X-linked genetic defect (xid), and in strains lacking this defect. Hemagglutination-inhibiting antibody titers and measurement of virus-specific antibodies by solid-phase radioimmunoassay indicated that the xid defect does not impair the production of an adequate anti-influenza antibody response. However, investigation of the isotypes of PR8 virus-specific antibodies disclosed a relative decrease in the levels of IgG3 and IgG1 in the xid-bearing strains. This was observed after both intraperitoneal immunization and aerosol infection. The isotype differences were not reflected in the susceptibility of these strains to influenza virus infection.     

Solid-phase radioimmunoassay for human neutrophil elastase: a sensitive method for determining secreted and cell-associated enzyme

A solid-phase radioimmunoassay for measuring neutrophil elastase in the range 0.08-4 ng/ml has been developed. A monospecific, precipitating antibody capable of inhibiting elastinolysis was produced by repeated immunizations of a goat. The IgG fraction and affinity-purified antibodies of this serum were then obtained and used to develop this radioimmunoassay. There was no cross-reactivity in binding of the radiolabeled antisera with lactoferrin, cathepsin G, or serine proteinases with amino-terminal amino acid sequence homology. Although serum influences the measurement of catalytically active neutrophil elastase when compared to diisopropylfluorophosphate-treated neutrophil elastase, antigenic elastase may still be measured in body fluids. Furthermore, this assay is more sensitive than commercially available substrates used for quantitating neutrophil elastase by functional activity. We have found this quantitative assay extremely useful in balance studies to measure secreted and cell-associated elastase and in screening of biological fluids for the presence of the enzyme.     

The design of a diagnostic test system based on magnetic sorbents and liposomes

The results obtained in the study of the possibility of using magnetic sorbents for the construction of a diagnostic assay system based on the antigen-antibody interaction are presented. As a model, Yersinia pestis capsular antigen and immunoglobulins to it have been used. A solid-phase immunofluorescent liposomal assay method has been developed; this method can be used for the detection of biopolymers in the sample under study and for the determination of their activity.     

In vitro antibody response to influenza virus. I. T cell dependence of secondary response to hemagglutinin.

A good secondary IgG response to the hemagglutinin (HA) of influenza virus has been obtained in vitro in Marbrook-type cultures of influenza-primed mouse spleen cell suspensions stimulated with inactivated influenza virus. Anti-HA antibody was quantitated by a solid phase radioimmunoassay (RIA) by using purified HA as substrate. The T dependence of this secondary response was shown by depletion of T cells and reconstitution with a source of primed or unprimed T cells. The help given by T cells primed to the homologous virus was many times greater than that given by unprimed T cells, although the latter was significant. The system described will allow investigation of the specificity requirements of helper T cells engaged in the anti-HA response.     

Double antibody solid-phase radioimmunoassay for staphylococcal enterotoxin A

Summary A double antibody solid-phase (DASP) radioimmunoassay for staphylococcal enterotoxin A is described. In the assay the antigen-antibody complex is precipitated by anti-rabbit serum which is adsorbed onto a solid carrier (cellulose). The method is sensitive to 200 pg of enterotoxin. It was possible to detect as little as 2–5 ng enterotoxin A/ml food extract from minced meat and sausage. Enterotoxins B and C were not found to inhibit the uptake of labeled enterotoxin A at a level which might distort the results of the enterotoxin A assay. The DASP technique is sensitive, rapid, and easy to perform and thus compares favorably with other radioimmunoassays for enterotoxin.     

Development of a direct microplate enzyme immunoassay for the determination of pregnanediol-3 alpha-glucuronide in urine

A sensitive direct enzyme immunoassay for urine pregnanediol-3 alpha-glucuronide was developed. The assay system involves the use of an antiserum against pregnanediol-3 alpha-glucuronide and an enzyme-labelled antigen chemically prepared by linking beta-D-galactosidase to 20 alpha-hydroxy-5 beta-pregnane 3(O-carboxymethyl)oxime. Free from antibody-bound antigen was separated by a solid-phase double antibody method, using a microplate coupled with goat anti-rabbit gamma-globulin. This solid-phase enzyme immunoassay for urine pregnanediol-3 alpha-glucuronide was validated in terms of specificity, accuracy and sensitivity. When urine samples were assayed for pregnanediol-3 alpha-glucuronide, the results obtained by the solid phase enzyme immunoassay and conventional radioimmunoassay methods agreed well (n = 30, r = 0.922). This assay system has an advantage over radioimmunoassay, because it does not require the use of radioisotopes. The procedure of this method is very simple, since it does not require purification steps of the biological samples.     

Influenza A Neuraminidase Antibody Assay with Sensitized Erythrocytes

Erythrocytes sensitized with purified neuraminidase (Hong Kong) antigens were used for assay of influenza A neuraminidase antibodies. The neuraminidase indirect hemagglutination test was equal to the neuraminidase hemagglutination-inhibition (enhancement) test and appeared to be better than the neuraminidase inhibition test for detection of fourfold or greater antibody rises in paired sera from influenza patients or vaccinees. It was better than both tests for detection of neuraminidase antibody. The neuraminidase indirect hemagglutination test is simple to perform and has the advantage of direct antigen-antibody assay.     

光学椭偏成像技术在生物分子研究中的应用

光学椭偏显微成像是一种新型超薄膜及表面显示技术,是研究生物分子与固体表面吸附以及生物分子之间相互作用的一种简单、快速和可靠的手段。它不仅能够大面积精确显示超薄膜的厚度分布,而且能够用于表面实时吸附的动力学研究。在抗原抗体检测分析方面,它不需要像酶联免疫法、荧光免疫法和放射免疫法那样对待测物作标记,也不会对待测生物分子活性造成任何扰动和损伤,操作简单,费用低廉。另外,它还弥补了传统的椭偏法的不足之处,能够有效地区分非特异性吸附、脱吸附或表面污染带来的干扰。     

Characterization of cross protection of Swine-Origin Influenza Virus (S-OIV) H1N1 and reassortant H5N1 influenza vaccine in BALB/c mice given a single-dose vaccination

Background

Influenza virus has antigen drift and antigen shift effect, vaccination with some influenza vaccine might not induce sufficient immunity for host to the threat of other influenza virus strains. S-OIV H1N1 and H5N1 influenza vaccines in single-dose immunization were evaluated in mice for cross protection to the challenge of A/California/7/2009 H1N1 or NIBRG-14 H5N1 virus.

Results

Both H1N1 and H5N1 induced significant homologous IgG, HAI, and microneutralization antibody responses in the mice, while only vaccines plus adjuvant produced significant heterogeneous IgG and HAI antibody responses. Both alum and MPLA adjuvants significantly reduced the S-OIV H1N1 vaccine dose required to elicit protective HAI antibody titers from 0.05 μg to 0.001 μg. Vaccines alone did not protect mice from challenge with heterogeneous influenza virus, while H5N1 vaccine plus alum and MPLA adjuvants did. Mouse body weight loss was also less significant in the presence of adjuvant than in the vaccine without adjuvant. Furthermore, both H1N1 and H5N1 lung viral titers of immunized mice were significantly reduced post challenge with homologous viruses.

Conclusion

Only in the presence of MPLA adjuvant could the H5N1 vaccine significantly reduce mouse lung viral titers post H1N1 virus challenge, and not vice versa. MPLA adjuvant induced cross protection with a single dose vaccination to the challenge of heterogeneous influenza virus in mice. Lung viral titer seemed to be a better indicator compared to IgG, neutralization antibody, and HAI titer to predict survival of mice infected with influenza virus.     

Regulation of antinucleoprotein IgG by systemic vaccination and its effect on influenza virus clearance

Seasonal influenza epidemics recur due to antigenic drift of envelope glycoprotein antigens and immune evasion of circulating viruses. Additionally, antigenic shift can lead to influenza pandemics. Thus, a universal vaccine that protects against multiple influenza virus strains could alleviate the continuing impact of this virus on human health. In mice, accelerated clearance of a new viral strain (cross-protection) can be elicited by prior infection (heterosubtypic immunity) or by immunization with the highly conserved internal nucleoprotein (NP). Both heterosubtypic immunity and NP-immune protection require antibody production. Here, we show that systemic immunization with NP readily accelerated clearance of a 2009 pandemic H1N1 influenza virus isolate in an antibody-dependent manner. However, human immunization with trivalent inactivated influenza virus vaccine (TIV) only rarely and modestly boosted existing levels of anti-NP IgG. Similar results were observed in mice, although the reaction could be enhanced with adjuvants, by adjusting the stoichiometry among NP and other vaccine components, and by increasing the interval between TIV prime and boost. Importantly, mouse heterosubtypic immunity that had waned over several months could be enhanced by injecting purified anti-NP IgG or by boosting with NP protein, correlating with a long-lived increase in anti-NP antibody titers. Thus, current immunization strategies poorly induce NP-immune antibody that is nonetheless capable of contributing to long-lived cross-protection. The high conservation of NP antigen and the known longevity of antibody responses suggest that the antiviral activity of anti-NP IgG may provide a critically needed component of a universal influenza vaccine.     

Solid phase radioimmunoassay for quantitation of antigen-specific IgG in human sera with 125I-protein A from Staphylococcus aureus.

Radiolabeled protein A from Staphylococcus aureus (Staph A) has been used to develop a solid phase, noncompetitive radioimmunoassay for quantitation of specific IgG antibody. The assay involves two incubations: First, agarose-insolubilized antigen is mixed with serum samples for 1 to 4 hr during which specific antibody is bound; second, after a washing procedure, the solid phase immune complexes are incubated for 4 to 18 hr with 125I-Staph A, during which the radiolabeled detection protein binds to the insolubilized specific IgG antibody. In a comparative study of the IgG antiphospholipase A antibody content of 23 human sera drawn from honeybee venom-sensitive patients, resulted of the Staph A assay correlated highly (r = 0.981, p less than 0.001, N = 23) with those obtained from a liquid phase, competitive radioimmunoprecipitation (double antibody) assay. The two assays demonstrated comparable precision, sensitivity, and reproducibility. In contrast, the use of 125I-Staph A in the solid phase radioimmunoassay was superior to 125I rabbit anti-human IgG because of lower negative serum (blank) values, shorter time required to reach equilibrium binding, and greater precision and reproducibility. In principle, the 125I Staph A assay may be applied ot IgG quantitation for crude allergen extracts as well as purified antigens. Furthermore, the sera of a number of mammalian species may be studied without further modification.     

Production and characterization of antibody against aflatoxin Q1.

Antibodies against aflatoxin Q1 (AFQ1) were obtained from rabbits after immunization of either AFQ1-hemisuccinate or AFQ2a conjugated to bovine serum albumin. Both radioimmunoassay and enzyme-linked immunosorbent assaY (ELISA) were used for the determination of antibody titers and specificities. Antibodies obtained from rabbits after immunization with AFQ1-hemisuccinate-bovine serum albumin had the highest affinity to aflatoxin B1, whereas antibodies obtained from rabbits after immunization with AFQ2a-bovine serum albumin bound most effectively with AFQ2a. AFQ2a antibody was selected for the subsequent direct and indirect ELISA for the detection of AFQ1 in biological fluids. When AFQ2a-peroxidase and AFQ2a antibody were used, direct ELISA was able to detect as low as 2 ppb (ng/ml) of AFQ1 spiked in the urine samples that had been subjected to a Sep-Pak cleanup treatment. In indirect ELISA in which the antigen (AFQ2a-bovine serum albumin) was coated to the solid phase followed by reaction with rabbit antibody and goat anti-rabbit immunoglobulin G-peroxidase conjugate, 50-fold less antibody was used without sacrificing sensitivity. Recoveries of AFQ1 added to urine samples (2 to 40 ppb) were 46.3 to 73% and 65.8 to 85.8% for direct and indirect ELISA, respectively.     

Production and characterization of antibody against aflatoxin Q1

Antibodies against aflatoxin Q1 (AFQ1) were obtained from rabbits after immunization of either AFQ1-hemisuccinate or AFQ2a conjugated to bovine serum albumin. Both radioimmunoassay and enzyme-linked immunosorbent assaY (ELISA) were used for the determination of antibody titers and specificities. Antibodies obtained from rabbits after immunization with AFQ1-hemisuccinate-bovine serum albumin had the highest affinity to aflatoxin B1, whereas antibodies obtained from rabbits after immunization with AFQ2a-bovine serum albumin bound most effectively with AFQ2a. AFQ2a antibody was selected for the subsequent direct and indirect ELISA for the detection of AFQ1 in biological fluids. When AFQ2a-peroxidase and AFQ2a antibody were used, direct ELISA was able to detect as low as 2 ppb (ng/ml) of AFQ1 spiked in the urine samples that had been subjected to a Sep-Pak cleanup treatment. In indirect ELISA in which the antigen (AFQ2a-bovine serum albumin) was coated to the solid phase followed by reaction with rabbit antibody and goat anti-rabbit immunoglobulin G-peroxidase conjugate, 50-fold less antibody was used without sacrificing sensitivity. Recoveries of AFQ1 added to urine samples (2 to 40 ppb) were 46.3 to 73% and 65.8 to 85.8% for direct and indirect ELISA, respectively.     

Selection of influenza A virus adsorptive mutants by growth in the presence of a mixture of monoclonal antihemagglutinin antibodies.

The influenza virus hemagglutinin contains four major regions that are recognized by antibodies able to neutralize viral infectivity. To investigate the effect of an antibody response directed against each of these sites on viral evolution, influenza virus A/PR/8/34 (H1N1) was grown in allantois-on-shell cultures in the presence of a mixture of monoclonal antihemagglutinin antibodies. This selection mixture contained antibodies (two or three antibodies per antigenic site) whose concentrations were adjusted to achieve equal neutralization titers against each of the four antigenic sites. By varying the ratio of input virus to selection mixture concentration, we observed that variant viruses emerged under conditions of partial neutralization. Each of the four variants characterized in detail differed from the parental virus in its interaction with cellular receptors and exhibited minimal changes in antigenicity. Thus, these variants were virtually indistinguishable from wild-type viruses, as assessed by the binding of 103 monoclonal antihemagglutinin antibodies in an indirect radioimmunoassay. Despite this, many of the same antibodies demonstrated decreased titers to the variants in hemagglutination inhibition tests. The magnitude of the differences depended on the indicator erythrocytes used (much greater differences were detected with chicken erythrocytes than with human erythrocytes). Hemagglutination mediated by the variants was more resistant to neuraminidase treatment of erythrocytes than hemagglutination mediated by the parental virus. These findings are consistent with the idea that the variants were initially selected by virtue of their increased avidity for host cell receptors. Sequencing of viral RNA revealed that each of the variants differed from the parental virus by a single amino acid alteration in its HA1 subunit. Two of the changes were close to the proposed receptor binding site on hemagglutinin and could directly alter receptor binding, while a third was located near the trimer interface and may have increased receptor binding by altering monomer-monomer interactions.     

Enhanced Neutralizing Antibody Titers and Th1 Polarization from a Novel Escherichia coli Derived Pandemic Influenza Vaccine

Influenza pandemics can spread quickly and cost millions of lives; the 2009 H1N1 pandemic highlighted the shortfall in the current vaccine strategy and the need for an improved global response in terms of shortening the time required to manufacture the vaccine and increasing production capacity. Here we describe the pre-clinical assessment of a novel 2009 H1N1 pandemic influenza vaccine based on the E. coli-produced HA globular head domain covalently linked to virus-like particles derived from the bacteriophage Qβ. When formulated with alum adjuvant and used to immunize mice, dose finding studies found that a 10 µg dose of this vaccine (3.7 µg globular HA content) induced antibody titers comparable to a 1.5 µg dose (0.7 µg globular HA content) of the licensed 2009 H1N1 pandemic vaccine Panvax, and significantly reduced viral titers in the lung following challenge with 2009 H1N1 pandemic influenza A/California/07/2009 virus. While Panvax failed to induce marked T cell responses, the novel vaccine stimulated substantial antigen-specific interferon-γ production in splenocytes from immunized mice, alongside enhanced IgG2a antibody production. In ferrets the vaccine elicited neutralizing antibodies, and following challenge with influenza A/California/07/2009 virus reduced morbidity and lowered viral titers in nasal lavages.     

Recombinant IgA Is Sufficient To Prevent Influenza Virus Transmission in Guinea Pigs

A serum hemagglutination inhibition (HAI) titer of 40 or greater is thought to be associated with reduced influenza virus pathogenesis in humans and is often used as a correlate of protection in influenza vaccine studies. We have previously demonstrated that intramuscular vaccination of guinea pigs with inactivated influenza virus generates HAI titers greater than 300 but does not protect vaccinated animals from becoming infected with influenza virus by transmission from an infected cage mate. Only guinea pigs intranasally inoculated with a live influenza virus or a live attenuated virus vaccine, prior to challenge, were protected from transmission (A. C. Lowen et al., J. Virol. 83:2803–2818, 2009.). Because the serum HAI titer is mostly determined by IgG content, these results led us to speculate that prevention of viral transmission may require IgA antibodies or cellular immune responses. To evaluate this hypothesis, guinea pigs and ferrets were administered a potent, neutralizing mouse IgG monoclonal antibody, 30D1 (Ms 30D1 IgG), against the A/California/04/2009 (H1N1) virus hemagglutinin and exposed to respiratory droplets from animals infected with this virus. Even though HAI titers were greater than 160 1 day postadministration, Ms 30D1 IgG did not prevent airborne transmission to passively immunized recipient animals. In contrast, intramuscular administration of recombinant 30D1 IgA (Ms 30D1 IgA) prevented transmission to 88% of recipient guinea pigs, and Ms 30D1 IgA was detected in animal nasal washes. Ms 30D1 IgG administered intranasally also prevented transmission, suggesting the importance of mucosal immunity in preventing influenza virus transmission. Collectively, our data indicate that IgG antibodies may prevent pathogenesis associated with influenza virus infection but do not protect from virus infection by airborne transmission, while IgA antibodies are more important for preventing transmission of influenza viruses.     

Human immune responses elicited by an intranasal inactivated H5 influenza vaccine

Intranasally administered influenza vaccines could be more effective than injected vaccines, because intranasal vaccination can induce virus-specific immunoglobulin A (IgA) antibodies in the upper respiratory tract, which is the initial site of infection. In this study, immune responses elicited by an intranasal inactivated vaccine of influenza A(H5N1) virus were evaluated in healthy individuals naive for influenza A(H5N1) virus. Three doses of intranasal inactivated whole-virion H5 influenza vaccine induced strong neutralizing nasal IgA and serum IgG antibodies. In addition, a mucoadhesive excipient, carboxy vinyl polymer, had a notable impact on the induction of nasal IgA antibody responses but not on serum IgG antibody responses. The nasal hemagglutinin (HA)-specific IgA antibody responses clearly correlated with mucosal neutralizing antibody responses, indicating that measurement of nasal HA-specific IgA titers could be used as a surrogate for the mucosal antibody response. Furthermore, increased numbers of plasma cells and vaccine antigen-specific Th cells in the peripheral blood were observed after vaccination, suggesting that peripheral blood biomarkers may also be used to evaluate the intranasal vaccine-induced immune response. However, peripheral blood immune cell responses correlated with neutralizing antibody titers in serum samples but not in nasal wash samples. Thus, analysis of the peripheral blood immune response could be a surrogate for the systemic immune response to intranasal vaccination but not for the mucosal immune response. The current study suggests the clinical potential of intranasal inactivated vaccines against influenza A(H5N1) viruses and highlights the need to develop novel means to evaluate intranasal vaccine-induced mucosal immune responses.     

A dual-mode approach to the selective separation of antibodies and their fragments

A novel chromatography method for the separation of antibodies is described. The adsorption of antibodies on the solid phase involves interaction with a ligand that combines mild hydrophobic characteristics and some degree of molecular recognition with a derivative of pyridine. This combined effect results in the adsorption of antibodies in the absence of lyotropic salts. When environmental pH is changed, the ligand becomes ionically charged, allowing the desorption of antibodies. The mechanism of adsorption, involving hydrophobic associations and ionic related interaction, is here qualified as dual-mode. Studies on the determination of the apparent dissociation constant for immunoglobulins G are presented. Adsorption of antibodies from crude feedstocks typically occurs without adjustment of pH or ionic strength. The sorbent is then washed with a buffer to eliminate protein impurities and, when lowering the environmental pH, antibodies are desorbed. The solid-phase material is used for the separation of antibodies from an ascites fluid and from a cell culture supernatant, followed by a polishing step on an hydroxyapatite column. Preliminary studies, related to the ability of the solid phase to separate antibody fragments, are also reported. In these studies, it has been demonstrated that both Fab and Fc fragments from polyclonal IgG are adsorbed to the solid phase under typical binding conditions. Under other defined physico-chemical conditions (ionic strength and pH), separation of both fragments in a single step has been achieved.     

Magnetic protein microbead-aided indirect fluoroimmunoassay for the determination of canine virus specific antibodies

Rabies, canine distemper, and canine parvovirus are common contagious viral diseases of dogs and many other carnivores, and pose a severe threat to the population dynamics of wild carnivores, as well as endangering carnivore conservation. However, clinical diagnosis of these diseases, especially canine distemper and canine parvovirus, is difficult because of the broad spectrum of symptoms that may be confused with other respiratory and enteric diseases of dogs. The most frequently used and proven techniques for diagnosing viral diseases include the conventional enzyme-linked immunosorbent assay (ELISA), rapid fluorescent focus inhibition test (RFFIT), mouse neutralisation test (MNT), and fluorescent antibody virus neutralization (FAVN) test. However, these methods still have some inherent limitations. In this study, a magnetic protein microbead-aided indirect fluoroimmunoassay was developed to detect canine virus specific antibodies, human rabies immunoglobulin, CDV McAbs, and CPV McAbs. In this assay, an avidin-biotin system was employed to combine magnetic microbeads and virus antigens (rabies virus, canine distemper virus, and canine parvovirus). Quantification of the targeted virus antibodies was analyzed through indirect fluoroimmunoassay using the specific antigen-antibody reaction, as well as their corresponding FITC-labeled detection antibodies (mouse anti-human IgG/FITC conjugate or rabbit anti-dog IgG/FITC conjugate). The results indicated that the fluorescence intensity increased when a higher concentration of the targeted analyte was used, but the control had almost no fluorescence, much like the conventional ELISA. For human rabies immunoglobulin, CDV McAbs, and CPV McAbs, the minimum detectable concentrations were 0.2 IU/mL, 0.3 ng/mL, and 0.5 ng/mL, respectively. All of these results indicate that this assay can be employed to determine the presence of canine virus specific antibodies. In addition, the method devised here can be utilized as a general protocol in other bacterial and viral marker analysis.