Evaluation of an anti-carcinoembryonic monoclonal antibody suitable for immunoscintigraphy

An anti-carcinoembryonic antigen (CEA) monoclonal antibody (mAb 6D1.1) was evaluated in vitro and in vivo to determine its suitability as a tracer for immunoscintigraphy of colorectal carcinomas. Determination of mAb affinity for CEA showed a constant of association of 0.63 ± 0.11 x 109 M-1. Binding of technetium-99m (99mTc)-6D1.1, labeled by a direct method, to human cultured lineages was highly specific. Binding to only CEA-positive LS-174T cells resulted in a saturable curve inhibited by pre-incubation with unlabeled mAb. No binding at all was observed for the human lineages MeWo (melanoma) or ZR75-30 (breast carcinoma), neither of them expressing CEA cells. Intravenous injection of 99mTc-6D1.1 into nude mice xenografted with human LS-174T tumors resulted in planar images of excellent quality. Localization of an irrelevant mAb labeled with either 99mTc or iodine-125 was never observed in tumor masses. Biodistribution studies on excised tumoral tissue showed retention of 28.48% of the injected dose per gram of LS-174T tumor. The tumor-to-blood ratio was 3.46. The same analysis performed on the other three human xenografted tumors studied demonstrated that only the CEA-producing HT-29 (colorectal adenocarcinoma) retained 99mTc-6D1.1 while the other two (ZR75-30 and MeWo) did not. These data demonstrate that this mAb is an adequate tool for targeting CEA-expressing tumors in experimental models.

Expression of anti-Z-DNA single chain antibody variable fragment on the filamentous phage surface

We describe the expression of an anti-Z-DNA single chain variable region antibody fragment (scFv) on a filamentous phage surface. Four vectors for phage display were constructed. Two of them are able to display multiple copies of the antibody fragment, and the others can be used to make monovalent libraries. The vectors use different promoter/leader sequences to direct the expression of the fused proteins. All were able to promote the assembly of fusion virion particles. In this paper we also show the affinity selection (biopanning) of those phage-antibodies based on the capacity of their products to recognize the antigen. We used biotinylated Z-DNA and the selection was performed in a solution phase fashion. The data presented here indicate that these vectors can be further used to construct anti-nucleic acid antibody fragment libraries that can be used to study the basis of nucleic acid-protein interaction and its role in autoimmunity mechanisms.

Stabilization of serum antibody responses triggered by initial mucosal contact with the antigen independently of oral tolerance induction

Initial contacts with a T-dependent antigen by mucosal routes may result in oral tolerance, defined as the inhibition of specific antibody formation after subsequent parenteral immunizations with the same antigen. We describe here an additional and permanent consequence of these initial contacts, namely, the blockade of secondary-type responsiveness to subsequent parenteral contacts with the antigen. When repeatedly boosted ip with small doses (3 µg) of ovalbumin (OVA) (or lysozyme), primed B6D2F1 mice showed progressively higher antibody responses. In contrast, mice primed after a single oral exposure to the antigen, although repeatedly boosted, maintained their secondary antibody titers on a level which was inversely proportional to the dose of antigen in the oral pretreatment. This phenomenon also occurred in situations in which oral tolerance was not induced. For example, senile 70-week-old B6D2F1 mice pretreated with a single gavage of 20 mg OVA did not become tolerant, i.e., they formed the same secondary levels of anti-OVA antibodies as non-pretreated mice. However, after 4 weekly challenges with 3 µg OVA ip, orally pretreated mice maintained the same anti-OVA serum levels, whereas the levels of control mice increased sequentially. This "stabilizing" effect of mucosal exposure was dose dependent, occurred with different proteins and was triggered by single or multiple oral or nasal exposures to the antigen.

Production and characterization of a monoclonal antibody against an Ascaris suum allergenic component

Ascaris suum allergenic components (PIII) separated by gel filtration chromatography of an adult worm extract were used to immunize BALB/c mice. Popliteal lymph node cells taken from the immunized animals were fused with SP2/O myeloma cells using polyethylene glycol (MW 1450) as fusogen. The hybridomas were cultured in HAT-containing medium and cloned at limiting dilutions. Supernatants from the growing hybrids were screened by ELISA using plates coated with PIII or the A. suum crude extract. The monoclonal antibody obtained, named MAC-3 (mouse anti-A. suum allergenic component), is an IgG1 kappa mouse immunoglobulin that specifically recognizes a 29,000 molecular weight protein (called allergenic protein) with an affinity constant of 1.7 x 10(9) M-1. The A. suum components recognized by MAC-3 induce specific IgE antibody production in immunized BALB/c mice. Ascitic fluid induced in Swiss mice by injecting ip the hybridoma cells and incomplete Freund's adjuvant was purified by affinity chromatography using a protein A-Sepharose column. The purified monoclonal antibody was then coupled to activated Sepharose beads in order to isolate the A. suum allergenic component from the whole extract by affinity chromatography.

Serum antigliadin antibody levels as a screening criterion before jejunal biopsy indication for celiac disease in a developing country

The objective of the present study was to determine the efficacy of detection of antigliadin immunoglobulins G and A (IgG and IgA) for the diagnosis of celiac disease in a developing country, since other enteropathies might alter the levels of these antibodies. Three groups were studied: 22 patients with celiac disease (mean age: 30.6 months), 61 patients with other enteropathies (mean age: 43.3 months), and 46 patients without enteropathies (mean age: 96.9 months). Antigliadin IgG and IgA ELISA showed sensitivity of 90.9 and 95.5%, respectively. With the hypothetical values of prevalence ranging from 1:500 to 1:2000 liveborns, the positive predictive value varied from 8.5 to 2.3% for IgG and from 4.8 to 1.1% for IgA. Considering the patients without enteropathies, specificity was 97.8 and 95.7% for IgG and IgA, respectively. In patients with other enteropathies, specificity was 82.0 and 84.1%, respectively. When patients with and without other enteropathies were considered as a whole, specificity was 88.8 and 91.6%, respectively. The specificity of positive IgG or IgA was 93.5% in children without enteropathies and 78.7% in the presence of other enteropathies. The negative predictive value for hypothetical prevalences varying from 1:500 to 1:2000 liveborns was 99.9%. Thus, even in developing countries where the prevalence of non-celiac enteropathies is high, the determination of serum antigliadin antibody levels is a useful screening test prior to the jejunal biopsy in the investigation of intestinal malabsorption.

In vitro-induced antibody production in chronic hepatitis C virus infection

The objectives of the present study were to assess the in vitro-induced anti-hepatitis C virus (HCV) antibody production (IVIAP) in relation to the clinical, biochemical, virologic and histologic variables of patients with HCV infection. The study included 57 patients (60% males) with HCV infection (anti-HCV and HCV-RNA positive). Alanine aminotransferase (ALT) was elevated in 89% of the patients. Mean viral load was 542,241 copies/ml and histology of the liver showed chronic hepatitis in 27/52 (52%) and cirrhosis in 11/52 (21%) patients. IVIAP levels were determined by immunoenzymatic assay at median absorbance of 0.781 at 450 nm. IVIAP was negative in 14% of the patients. When groups with IVIAP levels above and below the median were compared, high IVIAP levels were associated with the male sex, elevated ALT levels and more advanced disease stage. After logistic regression analysis, advanced histologic damage to the liver remained as the only independent variable associated with elevated IVIAP levels. Using a receiver operator characteristic curve, the best cut-off level for IVIAP was established (= 1.540), with 71% sensitivity and 94% specificity for the detection of more advanced disease stages (grades 3 and 4). These findings are consistent with the participation of immunological mechanisms in the genesis of the hepatic lesions induced by HCV and indicate that the IVIAP test may be useful as a noninvasive marker of liver damage either alone or in combination with other markers.

Measurement of Ki-67 antigen in 159 pituitary adenomas using the MIB-1 monoclonal antibody

Pituitary adenomas sometimes show rapid growth and recurrence, and about one third invade the structures surrounding the sella turcica. In an attempt to determine aggressive behavior at an early stage, we used the MIB-1 antibody to identify the Ki-67 antigen. The present study was designed to evaluate pituitary adenomatous tissue in terms of secretion and proliferation and to correlate the Ki-67 index with hormone phenotype and invasive behavior. Material from 159 patients submitted to one or more resections of pituitary adenomas was evaluated. Forty-two non-secretory adenomas and 43 adenomas immunoreactive for growth hormone, 19 for prolactin, 18 for growth hormone and prolactin, 16 for adrenocorticotropic hormone (ACTH), and 21 cases of plurihormonal/gonadotropin adenomas were detected by immunohistochemistry. The MIB-1 antibody was positive in 139 samples and the Ki-67 index ranged from 0.16 to 15.48% (mean = 1.22 ± 2.09%), with no significant difference between genders, age groups, or secretory and non-secretory status. The Ki-67 index was higher in ACTH-secreting adenomas. Invasive pituitary adenomas had a significantly higher Ki-67 index (2.01 ± 3.15%) than macroadenomas with or without supra-sellar extension (1.12 ± 1.87%; P = 0.02). The index was not significantly different in the subgroup of adenomas with invasion of the cavernous sinus compared to groups with other types of invasion. We conclude that tumoral proliferative activity evaluated by the detection of the Ki-67 antigen is significantly higher in invasive than noninvasive adenomas, information which can be useful in therapeutic postoperative management since index cut-off values associated with aggressive behavior can be established.

Antibody response to pneumococcal capsular polysaccharide vaccine in Down syndrome patients

The majority of children with Down syndrome (DS) tend to have frequent bacterial infections including recurrent respiratory infections. Our objective was to evaluate the production of antibodies to pneumococcal polysaccharide antigens after active immunization in DS subjects. IgG antibodies to pneumococcal serotypes (1, 3, 6B, 9V, and 14) were measured before and 6 weeks after immunization with a 23-valent pneumococcal vaccine (Pneumo23®, Pasteur-Merrieux) in 6- to 13-year-old DS children (N = 17) and in aged-matched normal controls (N = 30). An adequate response was defined as a 4-fold increase over baseline or a post-immunization level of specific pneumococcal serotype antibody > or = 1.3 µg/mL. After immunization, all DS children had an increase in post-immunization levels against all serotypes analyzed. A 4-fold or more increase was observed in all DS children concerning serotypes 1 and 14, in 90% of subjects for serotypes 3 and 9V, and in 65% for serotype 6B. Regarding this increase, 8 of the 17 DS children had an adequate response to all serotypes analyzed, 8/17 patients to 4 serotypes and 1/17 to 3 serotypes. However, when we compared post-immunization levels between DS children and controls, we observed lower levels in the former group (P < 0.05) for all serotypes except serotype 3. We conclude that pneumococcal polysaccharide immunization could be beneficial for these DS children.

Glutamic acid decarboxylase antibodies are indicators of the course, but not of the onset, of diabetes in middle-aged adults: the Atherosclerosis Risk in Communities Study

To efficiently examine the association of glutamic acid decarboxylase antibody (GADA) positivity with the onset and progression of diabetes in middle-aged adults, we performed a case-cohort study representing the ~9-year experience of 10,275 Atherosclerosis Risk in Communities Study participants, initially aged 45-64 years. Antibodies to glutamic acid decarboxylase (GAD65) were measured by radioimmunoassay in 580 incident diabetes cases and 544 non-cases. The overall weighted prevalence of GADA positivity (³1 U/mL) was 7.3%. Baseline risk factors, with the exception of smoking and interleukin-6 (P £ 0.02), were generally similar between GADA-positive and -negative individuals. GADA positivity did not predict incident diabetes in multiply adjusted (HR = 1.04; 95%CI = 0.55, 1.96) proportional hazard analyses. However, a small non-significant adjusted risk (HR = 1.29; 95%CI = 0.58, 2.88) was seen for those in the highest tertile (³2.38 U/mL) of positivity. GADA-positive and GADA-negative non-diabetic individuals had similar risk profiles for diabetes, with central obesity and elevated inflammation markers, aside from glucose, being the main predictors. Among diabetes cases at study's end, progression to insulin treatment increased monotonically as a function of baseline GADA level. Overall, being GADA positive increased risk of progression to insulin use almost 10 times (HR = 9.9; 95%CI = 3.4, 28.5). In conclusion, in initially non-diabetic middle-aged adults, GADA positivity did not increase diabetes risk, and the overall baseline profile of risk factors was similar for positive and negative individuals. Among middle-aged adults, with the possible exception of those with the highest GADA levels, autoimmune pathophysiology reflected by GADA may become clinically relevant only after diabetes onset.

Pattern of functional antibody activity against Haemophilus influenzae type b (Hib) in infants immunized with diphtheria-tetanus-pertussis/Hib Brazilian combination vaccine

We evaluated the functional activity of Haemophilus influenzae B (Hib) antibodies elicited in a group of infants immunized with the diphtheria-tetanus-pertussis vaccine combined with an Hib vaccine produced totally in Brazil after technological transfer of Hib vaccine production from Glaxo SmithKline, Belgium. Blood samples from immunized infants (N = 985) were collected for the determination of Hib antibodies. Total Ig and IgM and IgG subclasses of antibodies against polyribosyl ribitol phosphate (PRP) were analyzed by ELISA. Almost all vaccinees (97.56%, 961/985) developed a strong anti-PRP IgG antibody response (≥1.0 μg/mL), while an anti-PRP IgM response was observed in 64.24% (634/985) of them (≥0.15 μg/mL). Only 18.88% (186/985) of the infants in the group with high PRP antibody IgG concentrations (≥1.0 μg/mL) developed a high IgM antibody response. Anti-PRP IgG antibody levels were significantly higher than anti-PRP IgM. These results demonstrate the predominance of IgG antibodies over IgM antibodies in response to PRP, with a ratio of 17:1. IgG antibodies were predominantly of the IgG1 subclass. An increase in IgG avidity was also observed during the course of immunization.

The inhibitory effect of photodynamic therapy and of an anti-VCAM-1 monoclonal antibody on the in vivo growth of C6 glioma xenografts

We investigated the effect of photodynamic therapy (PDT) and of an anti-vascular cell adhesion molecule-1 (VCAM-1) monoclonal antibody on the in vivo growth of C6 glioma. Seven days after inoculation with C6 cells, adult male Wistar rats weighing 280-300 g with MRI-confirmed glioma were randomly assigned to 4 groups (N = 15 per group): PDT + VCAM-1 antibody group; PDT group; VCAM-1 antibody group; control group. Eight days after inoculation, hematoporphyrin monomethyl ether (HMME) was administered as a photosensitizer and PDT was performed at 630 nm (illumination intensity: 360 J/cm²) for 10 min. VCAM-1 antibody (50 µg/mL) was then administered (0.5 mL) through the tail vein every other day from day 8 to day 16. At day 21, 5 rats in each group were sacrificed and cancers were harvested for immunohistochemistry and Western blot assay for the detection of VCAM-1, and TUNEL assay was used to detect apoptosis. Survival and tumor volume were recorded in the remaining 10 rats in each group. In the PDT group, tumor growth was significantly suppressed (67.2%) and survival prolonged (89.3%), accompanied by an increase in apoptosis (369.5%), when compared to control. Furthermore, these changes were more pronounced in the PDT + VCAM-1 antibody group. After PDT, VCAM-1 expression was markedly increased (121.8%) and after VCAM-1 monoclonal antibody treatment, VCAM-1 expression was significantly reduced (58.2%). PDT in combination with VCAM-1 antibody can significantly inhibit the growth of C6 glioma and prolong survival. This approach may represent a promising strategy in the treatment of glioma.

Short-term and long-term antibody response by mice after immunization against Neisseria meningitidis B or diphtheria toxoid

Serogroup B Neisseria meningitidis (MenB) is a major cause of invasive disease in early childhood worldwide. The only MenB vaccine available in Brazil was produced in Cuba and has shown unsatisfactory efficacy when used to immunize millions of children in Brazil. In the present study, we compared the specific functional antibody responses evoked by the Cuban MenB vaccine with a standard vaccine against diphtheria (DTP: diphtheria, tetanus, pertussis) after primary immunization and boosting of mice. The peak of bactericidal and opsonic antibody titers to MenB and of neutralizing antibodies to diphtheria toxoid (DT) was reached after triple immunization with the MenB vaccine or DTP vaccine, respectively. However, 4 months after immunization, protective DT antibody levels were present in all DTP-vaccinated mice but in only 20% of the mice immunized against MenB. After 6 months of primary immunization, about 70% of animals still had protective neutralizing DT antibodies, but none had significant bactericidal antibodies to MenB. The booster doses of DTP or MenB vaccines produced a significant antibody recall response, suggesting that both vaccines were able to generate and maintain memory B cells during the period studied (6 months post-triple immunization). Therefore, due to the short duration of serological memory induced by the MenB vaccine (VA-MENGOC-BC® vaccine), its use should be restricted to outbreaks of meningococcal disease.

Expression, production, and renaturation of a functional single-chain variable antibody fragment (scFv) against human ICAM-1

Intercellular adhesion molecule-1 (ICAM-1) is an important factor in the progression of inflammatory responses in vivo. To develop a new anti-inflammatory drug to block the biological activity of ICAM-1, we produced a monoclonal antibody (Ka=4.19×10−8 M) against human ICAM-1. The anti-ICAM-1 single-chain variable antibody fragment (scFv) was expressed at a high level as inclusion bodies in Escherichia coli. We refolded the scFv (Ka=2.35×10−7 M) by ion-exchange chromatography, dialysis, and dilution. The results showed that column chromatography refolding by high-performance Q Sepharose had remarkable advantages over conventional dilution and dialysis methods. Furthermore, the anti-ICAM-1 scFv yield of about 60 mg/L was higher with this method. The purity of the final product was greater than 90%, as shown by denaturing gel electrophoresis. Enzyme-linked immunosorbent assay, cell culture, and animal experiments were used to assess the immunological properties and biological activities of the renatured scFv.