Antibodies to meningococcal H.8 (Lip) antigen fail to show bactericidal activity

1990 ◽  
Vol 36 (2) ◽  
pp. 117-122 ◽  
Author(s):  
Apurba K. Bhattacharjee ◽  
Elizabeth E. Moran ◽  
Wendell D. Zollinger
Keyword(s):  
Bactericidal Activity ◽  
Polyclonal Antibodies ◽  
High Titer ◽  
Rabbit Serum ◽  
Meningococcal Vaccine ◽  
The Poor ◽  
Group B ◽  
Antibody Levels ◽  
Sepharose 4B ◽  
Immune Rabbit

Purified H.8 (Lip) antigen was coupled to tresyl-activated Sepharose 4B and used in affinity columns to purify anti-Lip antibodies from convalescent patient sera and from immune rabbit sera. Affinity-purified anti-Lip antibodies isolated from two convalescent patient sera contained 1000 and 1280 ELISA units of antibody and included antibodies of IgG, IgA, and IgM isotypes. An anti-Lip mouse monoclonal ascites (2-1-CA2) had 28 400 ELISA units of antibody. Bactericidal assays were performed using three different case strains of Neisseria meningitidis group B, namely 44/76, 8532, and 8047. Neither preparation of purified human anti-Lip antibodies had detectable bactericidal activity against strains 44/76 and 8532, but one of the two had a titer of 1:4 against strain 8047. Anti-Lip antibodies that were purified from immune rabbit serum and contained 1600 ELISA units of anti-Lip antibodies also failed to show detectable bactericidal activity. The rabbits were immunized with purified Lip antigen and showed specific antibody levels of 2000–2200 units by ELISA, but even the unfractionated sera had little or no bactericidal activity against the test strains. The high titer mouse monoclonal ascites had no bactericidal activity against the test strains. The poor bactericidal activity associated with monoclonal and polyclonal antibodies to the Lip antigen suggest that in spite of other attractive properties it may not be useful as a meningococcal vaccine. Key words: anti-Lip, antibodies, bactericidal, Neisseria, Lip.

scholarly journals Production of polyclonal antibody against the recombinant PirBvp protein of Vibrio parahaemolyticus

2021 ◽  
Vol 19 (1) ◽  
Author(s):  
Ngoc-Diem Duong ◽  
Khai-Hoan Nguyen-Phuoc ◽  
Kim-Yen Thi Do ◽  
Nguyet-Thu Thi Nguyen ◽  
Thuoc Linh Tran ◽  
...  
Keyword(s):  
Vibrio Parahaemolyticus ◽  
Polyclonal Antibodies ◽  
High Titer ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Detection Threshold ◽  
Cross Reactivity ◽  
Rabbit Serum ◽  
Lateral Flow Strip ◽  
Specificity And Sensitivity

Abstract Background Acute hepatopancreatic necrosis disease (AHPND) is caused by toxin-producing strains of Vibrio parahaemolyticus which contain deadly binary toxins PirAvp and PirBvp encoded in pVA1 plasmid. The polyclonal antibodies against PirBvp protein could be used to develop immunochromatographic test strip for in-field diagnosis of AHPND. Results In this study, PirBvp gene was amplified, cloned, and expressed in E. coli. The expressed protein was detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot probed with 6xHis antibodies. Then, the recombinant PirBvp (rPirBvp) was purified using Ni-Sepharose column. Rabbits were immunized with the purified rPirBvp, and produced antibodies were analyzed using Ouchterlony double immunodiffusion. The antibody titration and antibody purification were performed by ELISA and affinity chromatography, respectively. Finally, antibody specificity and sensitivity were evaluated by dot blotting. The present study showed a high titer of polyclonal antibodies in rabbit serum after immunization and the titer increased steadily during the immunization schedule. The highest titer of antibody reached up to 2,560,000 with LOD of 0.1 ng/mL. The purified antibodies showed no cross-reactivity with proteins from other Vibrio species, and the detection threshold ranged from 6.25 to 12.5 ng toxin/dot. Conclusion This study highlights the production of high titer and specific polyclonal antibodies as an initial material towards the development of lateral-flow strip test.

scholarly journals THE INTERACTION IN VITRO BETWEEN GROUP B MENINGOCOCCI AND RABBIT POLYMORPHONUCLEAR LEUKOCYTES

1967 ◽  
Vol 126 (5) ◽  
pp. 795-818 ◽  
Author(s):  
Richard B. Roberts
Keyword(s):  
Bactericidal Activity ◽  
Polymorphonuclear Leukocytes ◽  
Normal Serum ◽  
Rabbit Serum ◽  
Normal Rabbit Serum ◽  
Opsonic Activity ◽  
Heat Stable ◽  
Group B

The interaction in vitro between group B meningococci and rabbit polymorphonuclear leukocytes has been described. Phagocytosis did not occur in the presence of normal rabbit serum. Antiserum collected 12–21 days following one subcutaneous inoculation of living log phase meningococci exhibited opsonic activity with type specificity; this opsonic action depended on both heat-labile and heat-stable factors. Following ingestion by granulocytes, meningococci were rapidly killed. These studies suggest that group B meningococcal strains contain specific antiphagocytic surface factors of an as yet unknown chemical nature. Antisera obtained 4 or more wk after immunization showed bactericidal activity with the same type specificity as opsonic activity. This bactericidal activity was also lost after heating and restored by the addition of normal serum. Further studies on opsonins and bactericidins for meningococci may shed light on virulence factors in these microorganisms, and may prove useful for a more precise classification of meningococci according to type rather than group specificity.

Studies to assess the biological relevance of anti-Tamm–Horsfall protein antibodies detected by direct-binding enzyme-linked immunosorbent assay

1987 ◽  
Vol 73 (5) ◽  
pp. 479-487 ◽  
Author(s):  
J. S. Hunt ◽  
Anne Groufsky ◽  
K. L. Lynn
Keyword(s):  
Immunosorbent Assay ◽  
Guanidine Hydrochloride ◽  
Sodium Dodecyl ◽  
Rabbit Serum ◽  
Enzyme Linked Immunosorbent Assay ◽  
Serum Fractions ◽  
Direct Binding ◽  
Tract Infections ◽  
Sepharose 4B ◽  
Immune Rabbit

1. A role has been suggested for anti-Tamm–Horsfall protein (THP) antibodies in renal disease based on the results of immunoassays of pathological sera. The putative autoantibodies have not been isolated from such sera nor have definitive inhibition studies of their binding been carried out. We have carried out such studies using rabbit anti-THP antibodies as control reagents. 2. Urinary THP prepared by salt precipitation was used to prepare four immunoabsorbent columns by covalent coupling to CNBr-activated Sepharose 4B. After washing with a variety of dissociating agents to remove any non-covalently bound subunit THP, each column was incubated with normal and immune rabbit serum. Fractions washed and eluted from columns were tested for anti-THP antibodies by enzyme-linked immunosorbent assay (ELISA) and THP antigen by radioimmunoassay, and showed NH4SCN (3 mol/l) and guanidine hydrochloride (GuHCl) (6 mol/l) equivalent and sodium dodecyl sulphate (20 g/l) to be inferior in their capacity to produce immunoabsorbent THP capable of isolating specific antibodies from immune rabbit serum. 3. The column treated with GuHCl (6 mol/l) was used further in attempts to isolate putative anti-THP antibodies from five patients, who had a history of urinary tract infections and whose sera showed strong binding by ELISA. 4. Results from direct and inhibition ELISA experiments on fractions collected after washing and elution with all sera suggested that the putative human anti-THP antibodies were of very low affinity and/or directed against non-subunit THP. 5. The pathological relevance of human anti-THP antibodies measured by ELISA remains to be established.

Artifact-free electron microscopic immunocytochemistry on rat brain tissue

1991 ◽  
Vol 49 ◽  
pp. 272-273
Author(s):  
Veronika Burmeister ◽  
N. Ludvig ◽  
P.C. Jobe
Keyword(s):  
Microscopic Examination ◽  
Free Electron ◽  
Electron Microscopic Examination ◽  
Ultrastructural Localization ◽  
Rabbit Serum ◽  
Electron Microscopic ◽  
Electron Microscopic Immunocytochemistry ◽  
Phosphate Buffered Saline ◽  
Rat Brain Tissue ◽  
Immune Rabbit

Electron microscopic immunocytochemistry provides an important tool to determine the ultrastructural distribution of various molecules in both normal and pathologic tissues. However, the specific immunostaining may be obscured by artifactual immunoreaction product, misleading the investigator. Previous observations show that shortening the incubation period with the primary antibody from the generally used 12-24 hours to 1 hour substantially reduces the artifactual immunostaining. We now extend this finding by the demonstration of artifact-free ultrastructural localization of the Ca2/calmodulindependent cyclic nucleotide phosphodiesterase (CaM-dependent PDE) immunoreactivity in brain.Anesthetized rats were perfused transcardially with phosphate-buffered saline followed by a fixative containing paraformaldehyde (4%) and glutaraldehyde (0.25%) in PBS. The brains were removed, and 40μm sections were cut with a vibratome. The sections were processed for immunocytochemistry as described by Ludvig et al. Both non-immune rabbit serum and specific CaM-dependent PDE antibodies were used. In both experiments incubations were at one hour and overnight. The immunostained sections were processed for electron microscopic examination.

Determination of Ochratoxin A in Feed by Immunoaffinity Cleanup and Liquid Chromatography

2013 ◽  
Vol 96 (3) ◽  
pp. 599-602 ◽  
Author(s):  
Ping Ding ◽  
Ziyou Mi ◽  
Yali Hou ◽  
Yigang He ◽  
Jianhua Xie
Keyword(s):  
Liquid Chromatography ◽  
Detection Limit ◽  
Ochratoxin A ◽  
Cyanogen Bromide ◽  
Polyclonal Antibodies ◽  
Immunoaffinity Column ◽  
Low Levels ◽  
Sepharose 4B ◽  
Feed Samples

Abstract A method using LC was developed for determination of ochratoxin A (OTA) in feeds. The extracted samples were cleaned up by an immunoaffinity column prepared by covalently coupling polyclonal antibodies against OTA to cyanogen bromide-activated Sepharose 4B. The eluates were determined by LC with fluorescence detection. Recoveries of OTA from fortified samples of 1–10 μg/kg levels ranged from 84.3 to 90.0%, with CVs of 3.3–7.8%. The detection limit was 0.045 μg/kg based on an S/N of 3:1. A total of 65 feed samples were screened for OTA with the proposed method. The results showed that only nine samples were contaminated with OTAs at low levels. The presented method was successfully applied to quantify OTAs in real feed samples.

Immunity to Diphtheria, Pertussis, Tetanus, and Poliomyelitis in Children with Acute Lymphocytic Leukemia After Cessation of Chemotherapy

PEDIATRICS ◽  
1981 ◽  
Vol 67 (2) ◽  
pp. 222-229 ◽  
Author(s):  
A. van der Does-van den Berg ◽  
J. Hermans ◽  
J. Nagel ◽  
G. van Steenis
Keyword(s):  
Acute Lymphocytic Leukemia ◽  
Lymphocytic Leukemia ◽  
First Year ◽  
Healthy Children ◽  
Healthy Control ◽  
Antibody Titers ◽  
Group A ◽  
One Year ◽  
Group B ◽  
Antibody Levels

Antibody titers to diphtheria, pertussis, tetanus, and poliomyelitis (types I to III) were measured in previously vaccinated children with acute lymphocytic leukemia in remission after cessation of therapy. The response to revaccination one year after therapy was stopped was also studied. The patients' antibody titers were compared with those of healthy children, matched for age and sex. Two groups of patients were studied: one group (group A, N = 30) was given two drugs (6-mercaptopurine, methotrexate); the other group (group B, N= 19) was given three drugs (6-mercaptopurine, methotrexate, and cyclophosphamide) for maintenance treatment. In general, the patients' antibody titers were lower than those of healthy children, but in most patients they were still at levels considered to be protective. No significant differences in antibody levels between the two patient groups were found. A spontaneous rise in antibody titers in the first year after termination of therapy was not observed. After revaccination the rise in antibody titers was correlated with preexisting antibody titers in the same way in patients as in healthy children, and the antibody titers in patients and in healthy control subjects were on roughly the same level.

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