Ng Of Antibody Research Articles

Digital Immunoassay for Rapid Detection of SARS-CoV-2 Infection in a Broad Spectrum of Animals.

The ability of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) to infect a wide-range of species raises significant concerns regarding both human-to-animal and animal-to-human transmission. There is an increasing demand for highly sensitive, rapid, and simple diagnostic assays that can detect viral infection across various species. In this study, we developed a biosensor assay that adapted a monoclonal-antibody (mAb)-based blocking ELISA format into an Activate Capture + Digital Counting (AC + DC)-based immunoassay. The assay employs a photonic crystal (PC) biosensor, gold-nanoparticle (AuNP) tags, SARS-CoV-2 nucleocapsid (N) protein, and specific anti-N mAb to detect antibody responses in animals exposed with SARS-CoV-2. We demonstrated a simple 2-step 15-min test that was capable of detecting as low as 12.5 ng of antibody in controlled standard serum samples. Based on an evaluation of 176 cat serum samples with known antibody status, an optimal percentage of inhibition (PI) cut-off value of 0.588 resulted in a diagnostic sensitivity of 98.3% and a diagnostic specificity of 96.5%. The test is highly repeatable with low variation coefficients of 2.04%, 2.73%, and 4.87% across different runs, within a single run, and on a single chip, respectively. The test was further employed to detect antibody responses in multiple animal species as well as investigate dynamics of antibody response in experimentally infected cats. This test platform provides an important tool for rapid field surveillance of SARS-CoV-2 infection across multiple species.

Making sense of serotype-specific pneumococcal antibody measurements

In 2012, a specialist group of the American Academy of Allergy, Asthma & Immunology (AAAI) collaborated to produce guidelines on the use and interpretation of diagnostic vaccination in primary immunodeficiency. Within this document there is extensive evaluation of the use of serotype-specific pneumococcal serotype testing for diagnosis of immune deficiency. A titre of 1.3 mg/mL is reported to confer protection for each serotype. This concentration has generated much discussion: in the UK these tests are only available in a few centres and all use 0.35 mg/mL as a putative protective titre. In an attempt to help clarify this discrepancy, this article summarizes the literature describing the development of these assays. Although widely quoted, a ‘protective titre’ of 1.3 mg/mL may not have a sound evidence base. The evolution of assays to measure antibodies to serotype specific pneumococcus, particularly the unit of measurement conversions, has played a significant part in the confusion. A sensitive radioimmunoassay was developed by Schiffman et al. in 1980 which measured IgG antibodies to C polysaccharide generating antibodies in units of ng antibody nitrogen/mL. Later enzyme-linked immunosorbent assays (ELISAs) were developed which were more sensitive but also generated results in ng antibody nitrogen/mL. In 1995, the World Health Organisation (WHO) produced reference and calibration sera to standardize assays. The first ELISAs showed poor correlation of antibodies with vaccine efficacy studies. This was due to the presence of C polysaccharide and 22F. Antibodies to C polysaccharide, although naturally occurring in humans, are not opsonic and therefore do not protect against pneumococcal infection; antibodies to serotype 22F are cross reactive to a number of other serotypes. Reabsorbing sera with C polysaccharide and 22F led to the development of 2nd and 3rd generation assays respectively with increased specificity for measuring protective antibodies; these assays gave results in mg/ mL. From this point onwards there was a divergence in interpretation of assay results depending on special interests such as whether the assays were used to assess vaccination response from a public health perspective or to compare base line and postvaccination response to help diagnose T-cell independent antibody deficiency. The WHO approved the ELISA for monitoring response to newly produced vaccines. WHO published data showing that pneumococcal conjugated vaccines should induce titres of 0.35mg/mL or above four weeks after vaccination to confer protection against invasive pneumococcal disease. This putative protective level was based on a 2nd generation ELISA. When reassessed on a 3rd generation (more sensitive) ELISA only a small decline in putative protection level was found (0.32 mg/mL); this meta-analysis was based on three clinical efficacy trials with in excess of 60,000 control and vaccinated infants. A decision to stick to the original value (for ease) of 0.35mg/mL was made. Although derived from data in children, this level of protection has been extrapolated for adult use. Development and interpretation of ELISA results in the adult immune deficiency world took a different approach. Landesman and Schiffman used the radioimmunoassay to assess pneumococcal antibody concentration with systemic pneumococcal infection >250– 300 ng antibody nitrogen/mL for adults. This figure

Microfluidic integration for automated targeted proteomic assays

A dearth of protein isoform-based clinical diagnostics currently hinders advances in personalized medicine. A well-organized protein biomarker validation process that includes facile measurement of protein isoforms would accelerate development of effective protein-based diagnostics. Toward scalable protein isoform analysis, we introduce a microfluidic "single-channel, multistage" immunoblotting strategy. The multistep assay performs all immunoblotting steps: separation, immobilization of resolved proteins, antibody probing of immobilized proteins, and all interim wash steps. Programmable, low-dispersion electrophoretic transport obviates the need for pumps and valves. A three-dimensional bulk photoreactive hydrogel eliminates manual blotting. In addition to simplified operation and interfacing, directed electrophoretic transport through our 3D nanoporous reactive hydrogel yields superior performance over the state-of-the-art in enhanced capture efficiency (on par with membrane electroblotting) and sparing consumption of reagents (ca. 1 ng antibody), as supported by empirical and by scaling analyses. We apply our fully integrated microfluidic assay to protein measurements of endogenous prostate specific antigen isoforms in (i) minimally processed human prostate cancer cell lysate (1.1 pg limit of detection) and (ii) crude sera from metastatic prostate cancer patients. The single-instrument functionality establishes a scalable microfluidic framework for high-throughput targeted proteomics, as is relevant to personalized medicine through robust protein biomarker verification, systematic characterization of new antibody probes for functional proteomics, and, more broadly, to characterization of human biospecimen repositories.

A Leucine-based Motif Mediates the Endocytosis of Vesicular Monoamine and Acetylcholine Transporters

Specific transport proteins mediate the packaging of neurotransmitters into secretory vesicles and consequently require targeting to the appropriate intracellular compartment. To identify residues in the neuron-specific vesicular monoamine transporter (VMAT2) responsible for endocytosis, we examined the effect of amino (NH2-) and carboxyl (COOH-)-terminal mutations on steady state distribution and internalization. Deletion of a critical COOH-terminal domain sequence (AKEEKMAIL) results in accumulation of VMAT2 at the plasma membrane and a 50% reduction in endocytosis. Site-directed mutagenesis shows that replacement of the isoleucine-leucine pair within this sequence by alanine-alanine alone reduces endocytosis by 50% relative to wild type VMAT2. Furthermore, the KEEKMAIL sequence functions as an internalization signal when transferred to the plasma membrane protein Tac, and the mutation of the isoleucine-leucine pair also abolishes internalization of this protein. The closely related vesicular acetylcholine transporter (VAChT) contains a similar di-leucine sequence within the cytoplasmic COOH-terminal domain that when mutated results in accumulation of VAChT at the plasma membrane. The VAChT di-leucine sequence also confers internalization when appended to two other proteins and in one of these chimeras, conversion of the di-leucine sequence to di-alanine reduces the internalization rate by 50%. Both VMAT2 and VAChT thus use leucine-based signals for efficient endocytosis and as such are the first synaptic vesicle proteins known to use this motif for trafficking.

T Cell-Dependent Differentiation of Human B Cells into IgM, IgG, IgA, or IgE Plasma Cells: High Rate of Antibody Production by IgE Plasma Cells, but Limited Clonal Expansion of IgE Precursors

The development of human functional Ig precursors into plasma cells expressing IgM, IgG, IgA, or IgE was compared. Purified human B cells were stimulated at limiting dilution with irradiated EL4 helper cells, IL-2, and IL-4. B cells proliferated exponentially until Day 8 of culture. Nondividing plasma cells of all isotypes were detectable in ELISPOT assays between Days 8 and 10 and secreted 1.8 ± 0.7 ng antibody per cell within 24 hr. This indicates that plasma cells of all isotypes, including IgE, bear a comparable potential to secrete antibody. It further shows that Ig switching does not delay the development into IgE plasma cells, despite that switching from IgM to IgE in vitro required 6 days of IL-4 action. The proliferation and Ig production by B cells readily declined after Days 8 and 10, respectively, and could not be prolonged by restimulating B cells with fresh helper cells and lymphokines in secondary cultures. This indicates that B cells have developed into nondividing, high rate Ig-secreting plasma cells within 9 days, and that they do not differentiate any further under the applied conditions. In contrast to IgM, IgG, and IgA committed B cells, IgE switched cells did not undergo clonal expansion, since the numbers of functional IgE precursors corresponded to the maximal numbers of IgE-secreting plasma cells, whereas the numbers of IgM-, IgG-, or IgA-secreting cells exceeded the number of functional precursors 15-fold. The results demonstrate that human B cells of all isotypes, including IgE, have the potential to secrete antibody at a comparably high rate, and that the IL-4-induced switch process does not delay the differentiation into plasma cells.

An enzyme-linked antiglobulin test to detect red cell globulins after glutaraldehyde fixation.

The purpose of this study was to develop an enzyme-linked antiglobulin test (ELAT) for IgG on RBC without the hemolysis caused by the high pH of the alkaline phosphatase reaction. This was achieved by fixing the RBC with 0.05% glutaraldehyde after attachment of the antibodies. Assays using anti-D reference standards demonstrated the sensitivity to be 1-2 ng of antibody as compared to 7.5 ng for the manual indirect antiglobulin test. The coefficients of variation of these assays ranged from 10.4 to 20.1%. The mean background absorbance at 405 nm of 105 normal RBC samples was 0.08 +/- 0.03 SD. There was an increase in sensitivity of the test after the fixed RBC were stored. Dilute glutaraldehyde stabilizes the RBC membrane and the antigen-antibody linkage resulting in a more sensitive ELAT.

ERRATUM

In the November issue of Pediatrics, a typographical error was overlooked in the Letter to the Editor, "Response to Pneumococcal Vaccine," by Gerald Schiffman (Pediatrics 1991;88:1074-1075). The last paragraph of the Letter should begin with: "I do agree with the authors that a level of 200 ng of antibody N/mL after immunization is hyporesponsive if, by that term, they mean not producing sufficient antibody for protection."

Revaccination with pneumococcal vaccine of elderly persons 6 years after primary vaccination

We vaccinated 15 persons, age 56 to 79 years, with 14-valent pneumococcal polysaccharide vaccine and revaccinated them with 23-valent vaccine 6 years later to assess adverse reactions and booster responses of anticapsular antibodies. No systemic reactions occurred after administration of either vaccine. After booster vaccination, five vaccinees developed only mild soreness or tenderness at the site of injection. The (arithmetic) mean antibody level to 12 capsular types measured by radioimmunoassay increased 3.1-fold 1 month after the first vaccine; the range was 1427–5124 ng antibody nitrogen per ml (Nml −1). Six years later, mean antibody levels waned to about half these levels. One month after administration of the second dose of vaccine, the mean antibody level increased 1.5-fold; the range was 1011–3954 ng antibody N ml −1. Revaccination with pneumococcal vaccine of elderly persons can be carried out safely; the levels of anticapsular antibody achieved after revaccination are about half the levels after primary vaccination.

A sensitive double-diffusion microassay suitable for the detection of idiotype-antiidiotype precipitates

• Using agarose gel coated on GelBond film sheets and using Coomassie blue stain followed by silver stain, a sensitive double-diffusion microassay has been developed for detecting small amounts of precipitate forming during idiotype-antiidiotype reactions. • The sensitivity of the method is 10–100 times greater than classical immunodiffusion tests. Other advantages include the need for minimal amounts of unconcentrated sample, the absence of radioactive or toxic substrates, no interference due to a second or third antibody coat such as are used in immunoenzymatic techniques, and the possibility of a direct evaluation of qualitative data such as identity, cross-reactivity. or non-identity. • As little as 40 ng antibody could be detected, corresponding to an antibody concentration of 8 μg/ml, making the microassay useful for rapid screening of idiotype-antiidiotype precipitates during routine analysis of hybridoma supernatants.

Fluorometric ELISA method for rapid screening of anti-estrogen receptor antibody production in hybridoma cultures

A micro-fluorometric ELISA which required only 50 μl of reagent in each step is reported here for rapid detection of anti-estrogen receptor (ER) antibody production by hybridoma cultures. In this study, several factors which influence the sesitivity of ELISA, e.g., incubation time, substrates, microtiter plates, concentration of coating antigens, choice and concentration of specific antibody and enzyme were evaluated. We observed that micro-fluorimetric ELISA using 4-methylumbelliferyl phosphate-phosphatase system and MicroFluor W plates was the most sensitive; it detected readily 1 ng coating antigens or 1 ng antibody during a 15 min incubation. This system was used successfully for the detection of anti-ER antibodies in mouse immune sera, and in spent media from in vitro immunized spleen cells or hybridoma cells. This method is simple, sensitive, rapid and reliable for ER or anti-ER antibody determination.

Rapid solid-phase radioimmunoassay for detection of equine infectious anemia viral antigen and antibodies: Parameters involved in standardization

Solid-phase radioimmunoassays (SPRIA) are described for the detection of equine infectious anemia (EIA) viral antigen and antibodies. Protein-antigen P29 currently used in the agar-gel immunodiffusion (AGID) test was used as antigen in the SPRIA. Rabbit sera selected from positive AGID test data were used to standardize the method. Briefly, wells of flexible microtitre plates coated with antigen were incubated with antiserum followed by a secondary labelled antibody. The radioactivity remaining in the wells after washing provided a measure of the amount of specific antibodies in the serum. When testing a group of rabbit sera, negative for EIA virus antibodies by the AGID test, in the SPRIA a range of positive reactivities was noted. The specificity of the reaction was assessed by inhibition with the antigen. The reaction of immune serum against EIA-virus antigen adsorbed to the wells, was completely inhibited by the antigen in solution. This property was applied in an indirect competitive SPRIA for the detection of viral protein P29. The detection threshold of the SPRIA for EIA virus protein was about 5 ng and about 1 ng of antibody can be detected. The assay is rapid, specific and sensitive and allows the testing of multiple serum samples with the advantage of employing a single secondary labelled antibody.

1086 LONGITUDINAL ASSESSMENT OF ANTIBODY RESPONSE OF CHILDREN WITH RENAL DISEASE TO PNEUMOCOCCAL VACCINE

56 children, including nephrotic syndrome (NS) patients, renal transplant (TX) recipients and normals, aged 2 to 15 years; were given a pneumococcal vaccine containing 14 polysaccharide (PS) types. Vaccine A with 50ug of each PS type was compared to Vaccine B with 25ug of each type. Study divisions were: Group 1)16 NS patients, Vaccine A; Group 2)10NS patients, Vaccine B; Group 3)3 TX patients, A; Group 4)10 TX patients, B; Group 5)10 normals A; Group 6)7 normals, B. Antibodies were determined for each PS type before, and 1, 3, 12 and 24 months after vaccination. At 1 month: a)the two vaccines produced comparable geometric mean titers (GMT) for 7/12 PS types in normals, 2/14 in NS patients, and 2/14 in TX patients. b)Vaccine B produced higher GMT's for 4/12 PS types in normals and for 11/14 in NS patients. c)Vaccine A produced a greater GMT for 12/14: types in TX patients. The sero-conversion rates were similar within paired groups. By 24 months the GMT was ≤ the pre-vaccine level for 9/12 types in Group 1, 7/12 Group 2, 11/12 Group 3, 6/12 Group 4, 6/12 Group 5, and 3/12 Group 6. The GMT was greater than the assumed protective level of 300 ng antibody N/ml for 4/12 types in Group 1, 5/12 Group 2, 4/12 Group 3, 4/12 Group 4, 5/12 Group 5, and 10/12 Group 6 at 24 months. 25ug of PS provided a similar immune response to that induced by the currently employed 50ug dose. Waning antibody titers suggest that longitudinal assessment of protection is essential and the possible need for booster doses remains to be determined.

Human Monoclonal Antibody Against Ganglioside G<sub>D2</sub>: Use in Development of Enzyme-Linked Immunosorbent Assay for the Monitoring of Anti-G<sub>D2</sub> in Cancer Patients<xref ref-type="fn" rid="FN2">2</xref>

Ganglioside GD2 is expressed on membranes of human melanoma cells and induces antibody responses in patients with melanoma. In this study a new enzyme-linked immunosorbent assay was developed for detection of human antibody against GD2. A human IgM monoclonal anti-GD2 antibody was used for development of the assay. The antigen, GD2, was prepared from human brain. Four micrograms of GD2 was required to coat a well in a polystyrene microtiter plate. As little as 10 ng of antibody could be detected. The applicability of the assay for detection of serum anti-GD2 antibody was demonstrated with sera from patients immunized with GD2-positive melanoma cell vaccine.

Persistence of protective pneumococcal antibody following vaccination in patients with the nephrotic syndrome.

We have determined the level of persisting pneumococcal antibody in a group of nephrotic children vaccinated by us 5 years ago. Of the 19 vaccinated children, 2 have died and 1 has moved away. Sera from the remaining 16 patients were examined by radioimmunoassay to determine the antibody response to 11 of the 14 types contained in the polyvalent pneumococcal vaccine. The lowest protective level of geometric mean titre (GMT) of antibody in our laboratory is 300 ng antibody nitrogen per millilitre. 56% (9/16) of the patients showed adequate GMT 5 years after vaccination. All 9 patients had minimal change nephrotic syndrome. 44% (7/16) of the children had a GMT less than 300 ng antibody nitrogen per millilitre. 3 of these patients had focal sclerosis, 3 had membranoproliferative glomerulonephritis, and 1 patient had IgM nephropathy. Of these 7 patients, 1 with the lowest GMT (23 ng antibody nitrogen per millilitre) developed pneumococcal peritonitis. During this same period, in 20 other unvaccinated nephrotic patients followed continuously from 1976 to 1981, 7 cases of pneumococcal peritonitis occurred (p less than 0.05). Additionally, 1 unvaccinated child died of pneumococcal sepsis. Our study indicates that patients with minimal change nephrotic syndrome continue to maintain adequate amounts of antibody, but those with disease other than minimal change nephrotic syndrome are unable to maintain an adequate level of antibody.

Sensitive radioimmune assay for measuring Aleutian disease virus antigen and antibody.

A solid-phase, one-step radioimmune assay was developed which could detect as little as 0.02 microliter of a standard Aleutian disease virus antigen preparation, approximately 3.2 ng of viral protein. Virus antigen was measured in different mink organs and cell types during experimental intraperitoneal infection. The gut and kidney were the first organs in which virus antigen could be detected (day 3 to 6 after infection). On day 6 or later virus antigen was found in spleen, liver, kidney, lymph nodes, peritoneal exudate, and bone marrow cells. With inhibition of antigen binding, a radioimmune assay was developed for antibody detection. Viral antibodies could be detected as early as 3 days after infection. Antibody titers from 1/10(5) to more than 1/10(6) were found in plasmacytotic mink. When the sensitivity of the antibody radioimmune assay was compared with that of other known methods for anti-Aleutian disease virus quantitation, the radioimmune assay was considerably more sensitive, detecting as little as 5 ng of antibody.

Interaction of specifically purified isotypes of bovine antibody to Brucella abortus in the haemolysis in gel test and enzyme-linked immunosorbent assay

The detection limits for purified bovine IgM, IgG1 and IgG2 antibody to Brucella abortus in the haemolysis in gel test and in an enzyme-linked immunosorbent assay (ELISA) were established. The limits for IgM, IgG1 and IgG2, respectively, were: 5500, 60 and 4850 ng of antibody in the lytic test and 2750, 3.5 and 16 ng in the ELISA test. Bovine IgG2 produced lysis only in the presence of fresh, whole bovine serum.

Increased serum opsonic activity and antibody concentration in patients with sickle cell disease after pneumococcal polysaccharide immunization

Opsonic defects have been reported in unimmunized patients with sickle cell disease. We found significant increases (P less than 0.001) in serum opsonic activity, measured by a radiolabeled bacterial uptake assay, and in type 7 pneumococcal polysaccharide antibody concentration in 17 such patients 2 years of age or older after pneumococcal polysaccharide immunization. All 17 patients and six healthy controls achieved a type 7 antibody concentration of more than 300 ng antibody nitrogen per milliliter, believed to be the protective level of antibody in vivo. Six patients with sickle cell disease less than 2 years of age did not have a significant increase in type 7 antibody concentration after immunization. Only three of these six patients achieved a postimmunization type 7 antibody concentration exceeding 300 ng Ab N/ml. Overall, 16 of 23 patients with sickle cell disease (70%) had a twofold or greater increase in type 7 antibody concentration, and 13 of these (81%) had a corresponding increase in opsonic activity (P less than 0.001). Thus most patients who responded to pneumococcal polysaccharide immunization had a concurrent increase in opsonic activity in vitro.

Antibody response to vaccination with pneumococcal capsular polysaccharides in splenectomized children.

In 67 splenectomized children vaccinated with a 14-valent pneumococcal capsular polysaccharide vaccine an antibody fold increase above 2 from pre- to postvaccination samples was found in 60% of all single type antibody determinations performed. The vaccine may only afford protection against 55% of those serotypes that cause serious infections in children, assuming a protective antibody level of 300 ng antibody N/ml. Our results indicate a somewhat reduced antibody response to pneumococcal vaccine in splenectomized children and suggest that pneumococcal vaccination may not be relied upon as the only kind of prophylaxis against pneumococcal infection in this patient group.

918 SERUM ANTIBODY DECLINE IN SPLENECTOHIZED CHILDREN AFTER PNEUMOCOCCAL VACCINATION

Splenectomized children are at increased risk of overwhelming pneumococcemia. Polyvalent pneumococcal vaccines may prevent these life-threatening infections since these patients have a normal antibody response to pneumococcal vaccination (Giebink, et al, JID 141:404, 1980). To determine the persistence of antibody elevation, serum was obtained from 44 splenectomized children 6 and 12 months after vaccination; 28 children had their spleens removed for traumatic splenic laceration, 11 for hereditary spherocytosis and 5 for Hodgkin's disease staging. Four weeks after vaccination geometric mean antibody concentrations, measured by radioimmunoassay against 12 antigens contained in the vaccine, were 833, 1,113 and 477 ng antibody N/ml for the traumatic, spherocytosis and Hodgkin's patients, respectively. Antibody levels after vaccination of 12 age-matched normal children and 6 children with hereditary spherocytosis with intact spleens were 492 and 954 ng/ml, respectively. Rates of antibody decline in the splenectomized patients were linear, and one year after vaccination were 68.4%, 70.6% and 76.0% of the peak post-vaccination antibody concentration in the three patient groups. None of the traumatic or spherocytosis patients had antibody levels less than 200 ng/ml one year after vaccination. Two Hodgkin's patients had levels below 200 ng/ml. Since protective antibody levels are not known, periodic re-vaccination may be necessary in high-risk patients.

Immunoassay Studies Using Adenosine Deaminase Enzyme with Potentiometric Rate Measurement

Abstract An enzyme immunoassay method using adenosine deaminase (EC 3.5.4.4) as the enzyme label is described. Potentiometric rate measurements with an ammonia gas-sensing electrode were used to determine the activity of enzyme label bound to agarose bead immobilized second antibody. The activity is related to the concentration of either a model haptenic group dinitrophenyl, DNP, or antiDNP antibody. Detection limits of 50 ng antibody and 10−10 M DNP hapten were obtained when incubations were carried out at pH 7.5 and assays at pH 9.0.