scholarly journals Optimization and Validation of a Multiplex, Electrochemiluminescence-Based Detection Assay for the Quantitation of Immunoglobulin G Serotype-Specific Antipneumococcal Antibodies in Human Serum

2009 ◽  
Vol 16 (3) ◽  
pp. 387-396 ◽  
Author(s):  
Rocio D. Marchese ◽  
Derek Puchalski ◽  
Pamela Miller ◽  
Joseph Antonello ◽  
Olivia Hammond ◽  
...  
Keyword(s):  
Human Serum ◽  
Immunoglobulin G ◽  
Dynamic Range ◽  
Cross Reactivity ◽  
Enzyme Linked Immunosorbent Assay ◽  
Testing Time ◽  
Igg Antibody ◽  
Meso Scale Discovery ◽  
Acceptance Criterion ◽  
Detection Assay

ABSTRACT Pneumovax 23 consists of a mixture of highly purified capsular polysaccharides (Ps) from 23 of the most prevalent serotypes of Streptococcus pneumoniae. Testing of vaccine immunogenicity has been historically performed on the enzyme-linked immunosorbent assay (ELISA) platform, validated to measure immunoglobulin G (IgG) antibodies to all 23 serotypes included in Pneumovax 23. In order to significantly improve the throughput of this form of testing, we have developed and validated a direct binding electrochemiluminescence (ECL)-based multiplex assay that can measure the antibody response in human serum to eight serotypes within a single microtiter well. The pneumococcal (Pn) ECL assay is based on the Meso Scale Discovery (MSD) technology which utilizes a Sulfo-Tag-labeled anti-human IgG antibody that emits light upon electrochemical stimulation. The Pn ECL assay exhibits a wide dynamic range and provides the ability to read concentrations down to the minimum reported concentration in the Merck ELISA (0.1 μg/ml). Cross-reactivity assessment using type-specific monoclonal antibodies showed no cross talk between antigen spots within a well. By use of the WHO Pn sample reference panel, the results obtained with the Pn ECL assay were compared to the results obtained with the international Pn ELISA. The results for the Pn ECL assay satisfied the WHO-recommended acceptance criterion for concordance for all seven serotypes with published Pn ELISA values, and the overall correlation (r value) across the seven serotypes was 0.994. The MSD methodology has great potential to be extremely useful for simultaneously quantitating IgG responses to several Pn serotypes while conserving serum volumes and laboratory testing time.

scholarly journals Effect of Antigen Coating Conditions on Enzyme-Linked Immunosorbent Assay for Detection of Immunoglobulin G Antibody to Neisseria meningitidis Serogroup Y and W135 Capsular Polysaccharide Antigens in Serum

2003 ◽  
Vol 10 (6) ◽  
pp. 1136-1140 ◽  
Author(s):  
Peter C. Giardina ◽  
Renee E. Evans ◽  
Daniel J. Sikkema ◽  
Dace Madore ◽  
Stephen W. Hildreth
Keyword(s):  
Immunoglobulin G ◽  
Capsular Polysaccharide ◽  
Enzyme Linked Immunosorbent Assay ◽  
Meningococcal Vaccine ◽  
Igg Antibody ◽  
Human Sera ◽  
Polysaccharide Antigens ◽  
Reference Serum ◽  
Adult Volunteers ◽  
Assay Conditions

ABSTRACT Human sera collected from 28 consenting adult volunteers were used to define assay conditions for meningococcal vaccine clinical trial serology. Immunoassay parameters were optimized with these test sera and the standard reference serum, CDC1992. Coating conditions for serogroup Y and W135 polysaccharide antigens were found to influence the predicted serum immunoglobulin G (IgG) antibody concentrations. Sera that displayed IgG antibody binding profiles most unlike that of CDC1992 were influenced the most by coating conditions. Our results suggest that presentation of specific epitopes is influenced by antigen-coating concentrations for serogroup Y and W135 polysaccharides.

scholarly journals Development and Validation of 13-plex Luminex-Based Assay for Measuring Human Serum Antibodies to Streptococcus pneumoniae Capsular Polysaccharides

mSphere ◽  
2018 ◽  
Vol 3 (4) ◽  
pp. e00128-18 ◽  
Author(s):  
Danka Pavliakova ◽  
Peter C. Giardina ◽  
Soraya Moghazeh ◽  
Shite Sebastian ◽  
Maya Koster ◽  
...  
Keyword(s):  
Human Serum ◽  
Lower Limit ◽  
Immunosorbent Assay ◽  
Enzyme Linked Immunosorbent Assay ◽  
Igg Antibodies ◽  
Igg Antibody ◽  
Correlate Of Protection ◽  
Immune Correlate ◽  
Relative Standard ◽  
Limit Of Quantitation

ABSTRACT A Luminex-based direct immunoassay (dLIA) platform has been developed to replace the standardized pneumococcal enzyme-linked immunosorbent assay platform. The multiplex dLIA simultaneously measures the concentration of serum immunoglobulin G (IgG) antibodies specific for pneumococcal capsular polysaccharide (PnPS) serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, and 23F. The assay uses poly-l-lysine (PLL)-conjugated PnPS, chemically coupled to spectrally distinct Luminex microspheres. Assay validation experiments were performed using residual human serum samples obtained from 13-valent pneumococcal conjugate vaccine (13vPnC) clinical studies. Assay results are expressed as IgG antibody concentrations in micrograms per milliliter using the international reference serum, 007sp. The lower limit of quantitation (LLOQ) for all serotypes covered in the 13-plex dLIA fell within the range of 0.002 to 0.038 µg/ml serum IgG. The difference between the lower limit and upper limit of the assay range was >500-fold for all serotypes, and assay variability was <20% relative standard deviation (RSD) for all serotypes. IgG antibody measurements were shown to be serotype-specific (some cross-reactivity was observed only between the structurally related serotypes 6A and 6B as well as 19A and 19F), and no interference was observed between the serotypes when the assay was performed in the 13-plex format compared to the singleplex assays. The 13-plex dLIA platform developed by Pfizer Inc. generates up to 143 test results in a single 96-well plate and is a suitable replacement of the enzyme-linked immunosorbent assay (ELISA) platform for evaluating vaccine clinical trials. IMPORTANCE The pneumococcal enzyme-linked immunosorbent assay (ELISA) measures IgG antibodies in human serum, and it is an important assay that supports licensure of pneumococcal vaccines. The immune correlate of protection, 0.35 µg/ml of IgG antibodies, was determined by the ELISA method. Pfizer has developed a new Luminex-based assay platform to replace the ELISA. These papers describe the important work of (i) validating the Luminex-based assay and (ii) bridging the immune correlate of protection (0.35 µg/ml IgG) to equivalent values reported by the Luminex platform.

Development and Characterization of Anti-Estradiol Polyclonal Antibody

2012 ◽  
Vol 461 ◽  
pp. 67-70 ◽  
Author(s):  
Chao Ying Li ◽  
Jin Qing Jiang
Keyword(s):  
Polyclonal Antibody ◽  
Dynamic Range ◽  
Cross Reactivity ◽  
Enzyme Linked Immunosorbent Assay ◽  
Uv Absorbance ◽  
Standard Curve ◽  
Ic50 Value ◽  
New Zealand White Rabbits

This paper reports an indirect competitive enzyme-linked immunosorbent assay (icELISA) using polyclonal antibody (pAb) for estradiol (E2) residues. After derivation, E2 haptens were conjugated to bovine serum albumin (BSA) and ovalbumin (OVA) through 1-Ethyl-3-(3-dimethylaminopropy) carbodiimide (EDC) method, and New Zealand white rabbits were immunized to produce anti-E2 pAb. The conjugation ratio of E2-BSA was proved to be 18.6:1 by an UV absorbance method. Based on the square matrix titration, an icELISA standard curve was developed. The dynamic range was from 0.16 to 128 ng/mL, with LOD and IC50 value of 0.08 ng/mL and 3.76 ng/mL, respectively. Except for a little cross-reactivity (16.2%) to estrone, this assay showed negligible cross-reactivity to other analogues tested. The results suggest that the produced anti-E2 pAb could be used to develop an icELISA method for the determination of E2 residues in animal-originally products.

scholarly journals Seroprevalence of Immunoglobulin G Antibodies Against Mycobacterium avium subsp. paratuberculosis in Dogs Bred in Japan

2020 ◽  
Vol 7 (3) ◽  
pp. 93
Author(s):  
Takashi Kuribayashi ◽  
Davide Cossu ◽  
Eiichi Momotani
Keyword(s):  
Mycobacterium Avium ◽  
Immunoglobulin G ◽  
Mycobacterium Avium Subsp ◽  
Clinical Samples ◽  
Enzyme Linked Immunosorbent Assay ◽  
Igg Antibodies ◽  
Igg Antibody ◽  
The Difference ◽  
Mycobacterium Avium Subsp Paratuberculosis ◽  
Veterinary Hospital

In this study, the seroprevalence of immunoglobulin G (IgG) antibodies against Mycobacterium avium subsp. paratuberculosis (MAP) in dogs bred in Japan was evaluated. Ninety-two non-clinical samples were obtained from three institutes and fifty-seven clinical samples were obtained from a veterinary hospital in Japan. Serum titers of total IgG, IgG1 and IgG2 isotype antibodies against MAP were measured using an indirect enzyme-linked immunosorbent assay (ELISA). The IgG antibodies against MAP in non-clinical serum obtained from three institutes was observed to be 2.4%, 20% and 9.0%. Similarly, the IgG1 antibodies titers against MAP were observed to be 7%, 20% and 0%. Lastly, the IgG2 antibodies against MAP were observed to be 7%, 20% and 4.4%. No significance differences in these titers were observed among the three institutes. The IgG, IgG1 and IgG2 antibodies in serum obtained from a veterinary hospital were observed to be 55.3%, 42% and 42%, respectively. Significant differences were found between the non-clinical and clinical samples. The titers in the clinical samples showed a high degree of variance, whereas low variance was found in the non-clinical samples. The IgG antibody levels were thought to be induced following exposure to MAP-contaminated feed. The difference in titers between the clinical and non-clinical samples is likely to be related to the amount of MAP antigen contamination in dog foods.

scholarly journals Cross-Reactivity between Immunoglobulin G Antibodies of Whales and Dolphins Correlates with Evolutionary Distance

2008 ◽  
Vol 15 (10) ◽  
pp. 1547-1554 ◽  
Author(s):  
Hendrik H. Nollens ◽  
Carolina Ruiz ◽  
Michael T. Walsh ◽  
Frances M. D. Gulland ◽  
Gregory Bossart ◽  
...  
Keyword(s):  
Genetic Distance ◽  
Heavy Chain ◽  
Immunoglobulin G ◽  
Bottlenose Dolphin ◽  
Cross Reactivity ◽  
Distinct Pattern ◽  
Enzyme Linked Immunosorbent Assay ◽  
Molecular Evidence ◽  
Cetacean Species ◽  
Strong Negative Correlation

ABSTRACT Growing morphological and molecular evidence indicates that the porpoises, dolphins, and whales evolved within the even-toed ungulates, formerly known as Artiodactyla. These animals are now grouped in the Cetartiodactyla. We evaluated the antigenic similarity of the immunoglobulin G (IgG) molecules of 15 cetacean species and the domestic cow. The similarity was scored using three distinct antibodies raised against bottlenose dolphin (Tursiops truncatus) IgG in a Western blot, an indirect enzyme-linked immunosorbent assay (ELISA), and a competitive ELISA format. A score was generated for the genetic distance between each species and T. truncatus using the cytochrome b sequence. Each antibody displayed a distinct pattern of reactivity with the IgG antibodies of the various species. The monoclonal antibody (MAb) specific for the γ heavy chain of T. truncatus was reactive with all monodontids, delphinids, and phocoenids. The light-chain-specific MAb reacted with IgG of delphinoid and phocoenid species and one of the two mysticete species tested. The polyclonal antibody was broadly cross-reactive across all cetaceans and the domestic cow. Using the MAb specific for the γ heavy chain, the degree of IgG cross-reactivity ranged from less than 17% for the mysticetes to 106% for killer whale Orcinus orca. The IgG in beaked whale and baleen whale sera was significantly less cross-reactive with bottlenose dolphin IgG than sera from other toothed whales. A strong negative correlation was demonstrated between antigenic cross-reactivity of IgG molecules and the genetic distance of their hosts. The data generated will be useful for the development of clinical serodiagnostics in diverse cetacean species.

scholarly journals Seroepidemiology ofHelicobacter pyloriinfection in vegans and meat-eaters

1992 ◽  
Vol 108 (3) ◽  
pp. 457-462 ◽  
Author(s):  
M. J. Webberley ◽  
J. M. Webberley ◽  
D. G. Newell ◽  
P. Lowe ◽  
V. Melikian
Keyword(s):  
Helicobacter Pylori ◽  
Risk Factor ◽  
Campylobacter Jejuni ◽  
Meat Consumption ◽  
Cross Reactivity ◽  
Enzyme Linked Immunosorbent Assay ◽  
Igg Antibody ◽  
Specific Igg ◽  
H Pylori ◽  
Age And Sex

SUMMARYAn enzyme-linked immunosorbent assay has been used to diagnose serologically the prevalence ofHelicobacter pyloriinfection in Asian life-long vegans. There was no difference in the seropositivity between these individuals and a group of age-and sex-matched Asian meat-eaters, indicating the meat consumption is not a risk factor forH. pyloriinfection. However, both Asian groups had a higher prevalence of infection than age- and sex-matched Caucasian meat-eaters. Additionally, the Asian individuals had a wider range of specific IgG antibody concentrations than the Caucasians. This did not appear to be due to antigenic cross-reactivity betweenH. pyloriandCampylobacter jejuni. The significance of these observations to the establishment of cut-off levels for the serodiagnosis of certain ethnic groups is discussed.

scholarly journals Immunoglobulin G and A Antibody Responses to Bacteroides forsythus and Prevotella intermedia in Sera and Synovial Fluids of Arthritis Patients

2003 ◽  
Vol 10 (6) ◽  
pp. 1043-1050 ◽  
Author(s):  
Ketil Moen ◽  
Johan G. Brun ◽  
Tor Magne Madland ◽  
Turid Tynning ◽  
Roland Jonsson
Keyword(s):  
Immune Responses ◽  
Immunoglobulin G ◽  
Enzyme Linked Immunosorbent Assay ◽  
Prevotella Intermedia ◽  
Igg Antibodies ◽  
Serum Samples ◽  
Igg Antibody ◽  
Iga Antibodies ◽  
Iga Antibody ◽  
Antibody Levels

ABSTRACT The objective of the present study was to investigate immunoglobulin G (IgG) and IgA antibody immune responses to Porphyromonas gingivalis, Prevotella intermedia, Bacteroides forsythus, and Candida albicans in the sera of patients with rheumatoid arthritis (RA), the synovial fluid (SF) of patients with RA (RA-SF samples), and the SF of patients without RA (non-RA-SF samples). An enzyme-linked immunosorbent assay was used to determine IgG and IgA antibody levels in 116 serum samples from patients with RA, 52 RA-SF samples, and 43 non-RA-SF samples; and these were compared with those in SF samples from 9 patients with osteoarthritis (OA-SF samples) and the blood from 100 donors (the control [CTR] group). Higher levels of IgG antibodies against B. forsythus (P < 0.0001) and P. intermedia (P < 0.0001) were found in non-RA-SF samples than in OA-SF samples, and higher levels of IgG antibodies against B. forsythus (P = 0.003) and P. intermedia (P = 0.024) were found in RA-SF samples than in OA-SF samples. Significantly higher levels of IgA antibodies against B. forsythus were demonstrated in both RA-SF and non-RA-SF samples than in OA-SF samples. When corrected for total Ig levels, levels of IgG antibody against B. forsythus were elevated in RA-SF and non-RA-SF samples compared to those in OA-SF samples. Lower levels of Ig antibodies against B. forsythus were found in the sera of patients with RA than in the plasma of the CTR group for both IgG (P = 0.003) and IgA (P < 0.0001). When corrected for total Ig levels, the levels of IgG and IgA antibodies against B. forsythus were still found to be lower in the sera from patients with RA than in the plasma of the CTR group (P < 0.0001). The levels of antibodies against P. gingivalis and C. albicans in the sera and SF of RA and non-RA patients were comparable to those found in the respective controls. The levels of IgG and IgA antibodies against B. forsythus were elevated in SF from patients with RA and non-RA-SF samples compared to those in OA-SF samples. Significantly lower levels of IgG and IgA antibodies against B. forsythus were found in the sera of patients with RA than in the plasma of the CTR group. This indicates the presence of an active antibody response in synovial tissue and illustrates a potential connection between periodontal and joint diseases.

scholarly journals Enzyme Immunoassay Detection of Antigen-Specific Immunoglobulin G Antibodies in Longitudinal Serum Samples from Patients with Cryptosporidiosis

2001 ◽  
Vol 8 (2) ◽  
pp. 415-423 ◽  
Author(s):  
Jeffrey W. Priest ◽  
Anna Li ◽  
Mohamad Khan ◽  
Michael J. Arrowood ◽  
Patrick J. Lammie ◽  
...  
Keyword(s):  
Western Blot ◽  
Immunoglobulin G ◽  
Protozoan Parasite ◽  
Epidemiologic Studies ◽  
Enzyme Linked Immunosorbent Assay ◽  
Serum Samples ◽  
Igg Antibody ◽  
Large Format ◽  
Western Blot Assay ◽  
Wide Range

ABSTRACT Cryptosporidium parvum is a protozoan parasite that causes diarrheal illness in a wide range of mammalian hosts, including humans. Characteristic serum immunoglobulin G (IgG) antibody responses to antigens in the 27- and 17-kDa size ranges have been shown to develop after infection, and several enzyme-linked immunosorbent assay (ELISA) and Western blot assay formats have been used to measure these IgG levels in human serum. Using a collection of serial samples from laboratory-confirmed cryptosporidiosis patients, we compared the results obtained by using two new ELISAs with those obtained with two different Western blot assays. When assayed with the large-format Western blot, 97% of the 67 patients had a demonstrable antibody response on at least one occasion. The Cp23 ELISA correctly identified 93% of the samples that had a 27-kDa response by Western blot and 100% of the negative samples. The Triton antigen ELISA detected 77% of the samples that had a 17-kDa response by Western blot and 88% of the negative samples. The sensitivity of the Triton antigen assay was higher for samples collected between 16 and 92 days after the onset of symptoms (96%). The minigel-format Western blot did not compare favorably with the large-format blot for the detection of antibodies to the 27-kDa antigen (71% sensitivity). A half-life of about 12 weeks was estimated for antibodies to both the 27- and 17-kDa antigens. We believe the Cp23 and Triton antigen ELISAs will be useful in epidemiologic studies of the prevalence ofCryptosporidium infection in the population.

scholarly journals Comparison of a New Multiplex Binding Assay versus the Enzyme-Linked Immunosorbent Assay for Measurement of Serotype-Specific Pneumococcal Capsular Polysaccharide IgG

2011 ◽  
Vol 18 (10) ◽  
pp. 1744-1751 ◽  
Author(s):  
David Goldblatt ◽  
Lindsey Ashton ◽  
Yuhua Zhang ◽  
Joseph Antonello ◽  
Rocio D. Marchese
Keyword(s):  
Immunosorbent Assay ◽  
Simultaneous Measurement ◽  
Capsular Polysaccharide ◽  
Binding Assay ◽  
Enzyme Linked Immunosorbent Assay ◽  
Meso Scale Discovery ◽  
New Methods ◽  
Detection Assay ◽  
Heptavalent Pneumococcal Conjugate Vaccine ◽  
Good Agreement

ABSTRACTThe measurement of serotype-specific anti-capsular polysaccharide antibodies remains the mainstay of pneumococcal (Pn) vaccine evaluation. New methods that allow the simultaneous measurement of antibodies to several antigens in small volumes of serum, and that agree well with existing techniques, are urgently required to support the increasing number of concomitant vaccines delivered in the infant immunization schedules and the use of extended-valency Pn vaccines. We therefore compared a relatively new multiplexed platform for measuring anti-Pn antibodies with the existing WHO consensus enzyme-linked immunosorbent assay (ELISA). A panel of 50 pediatric samples (34 collected after receipt of a heptavalent pneumococcal conjugate vaccine [PCV7] and 16 without PCV7) was analyzed across two different laboratories using a new multiplex electrochemiluminescence (ECL)-based detection assay developed for the quantitation of IgG serotype-specific antipneumococcal antibodies, and the results were compared to those obtained using the WHO consensus ELISA. For the seven serotypes measured, there was good agreement between the techniques and laboratories. The most notable difference was found between the ECL assay and the ELISA: concentrations tended to be higher in the ECL assay. For serotypes 6B, 9V, 18C, and 23F, the average increases in concentration ranged from 48 to 102%. However, the agreement rates on the proportions of samples with concentrations surrounding 0.35 μg/ml were >82% for all serotypes tested. Agreement between the two laboratories running the ECL assay was generally good: agreement on proportions of samples with concentrations surrounding 0.35 μg/ml was in excess of 92%, and agreement on average antibody concentrations was within 31%. We conclude that the Meso Scale Discovery (MSD) platform provides a promising new technique for the simultaneous measurement of antipneumococcal antibodies.

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