Comparative investigation of monoclonal and polyclonal antibodies directed against strain-specific and common antigenic sites on influenza H1N1 virus hemagglutinin

Antibodies directed against strain-specific and common antigenic sites of H1N1 influenza virus hemagglutinin were tested comparatively, using monoclonal antibodies raised against strain A/Brazil/11/78 and polyclonal antibodies directed against strains A/Brazil/11/78, A/USSR/97/77, A/PR/301/54, and A/FM/1/47. The patterns of competition between antibodies for adsorption onto homologous virus indicated that the monoclonals comprised antibodies directed to each of the two strain-specific (Sa and Sb) and common antigenic sites (Ca and Cb) of virus hemagglutinin. Polyclonal strain-specific antibodies (SSA) yielded the competition patterns of mixtures of anti-Sa and anti-Sb antibodies and polyclonal common antigen antibodies (CAA) yielded those of mixtures of antibodies directed against sites Ca and Cb, indicating that the polyclonal preparations comprised a similar repertoire of antibodies, as represented by the panel of monoclonals. This conclusion was confirmed by determining, by means of equilibrium filtration, the number of epitopes per homologous virion(s) recognized by antibody preparations and their mixtures. Polyclonal SSA and CAA gave s values not significantly different from those of mixtures of the corresponding monoclonal antibodies. The strains tested were found to possess equivalent numbers of strain-specific and common epitopes per virion. The competition between antibodies was further examined in terms of the additiveness of s values they recognize in simultaneous reactions. No competition was observed for the monoclonal antibody pairs anti-Sa/anti-Ca, anti-Sa/anti-Cb and anti-Sb/anti-Cb, indicating that these antibodies combined with nonoverlapping epitopes. Polyclonal SSA and CAA yielded partial competition. The equilibrium constants (K) of comparable SSA and CAA were within the same range, and SSA and CAA did not influence their binding avidity when allowed to react simultaneously with homologous virus.     

Analysis of respiratory syncytial virus fusion protein from clinical isolates of Korean children in palivizumab era, 2009–2015

IntroductionRespiratory syncytial virus (RSV) is the most common cause of lower respiratory tract infections among infants and young children. The fusion (F) protein of RSV is a major target for monoclonal antibodies and vaccine candidates. We analyzed sequence polymorphisms of the RSV F protein and investigated palivizumab-resistance mutation in clinical isolates from Korean children in post-palivizumab era.MethodsA review of pediatric patients with RSV infections in Korea from September 2009 to April 2015 was conducted. We performed RSV F gene sequence analysis on positive clinical samples and compared to reference sequences, A2 and 9320.ResultsRSV F gene data were obtained from 60 patients (30 RSV-A and 30 RSV-B), of whom 15 (10 RSV-A and 5 RSV-B) received palivizumab. The nucleotide and amino acid identities of the F gene sequence were conserved between RSV isolates and reference strains. There was no significant difference between isolates from patients who received and did not receive palivizumab. One or more amino acid changes were observed in all RSV-A and 26 RSV-B isolates. Twenty-five variations in RSV-A and 17 in RSV-B were noted. One variation within antigenic site II was noted in a RSV-A isolate; D263N with unknown significance was found in a patient without palivizumab prophylaxis. N276S variation adjacent to antigenic site II was observed in 27 RSV-A isolates. However, no known palivizumab-resistant mutations were found in either RSV-A or RSV-B isolates.ConclusionsThe RSV F gene was highly conserved and no known palivizumab-resistant mutants were found in Korean circulating strains.     

IMMUNOHEMATOLOGY: Novel antibody screening cells,MUT+Mur kodecytes,created by attaching peptides onto red blood cells

BACKGROUND: Antibody screening and identification panels are generally limited by the natural antigenic phenotypes present in their source donor population. However, the recent ability to attach peptides to the surface of cells has opened up the opportunity to create red blood cells (RBCs) with antigen profiles specifically designed for antibody screening and identification in a target population. STUDY DESIGN AND METHODS: Clinically significant antibodies to variant glycophorins (GPs) such as GP.Mur are more commonly seen in certain Asian populations. Using peptides representative of the MNS antigens MUT and Mur, RBC antibody screening cells were created using KODE cell surface engineering constructs. MUT‐, Mur‐, and MUT+Mur‐modified RBCs, known as kodecytes, were tested against monoclonal reagents and polyclonal sera with specificity for epitopes on GP.Mur‐positive RBCs. RESULTS: Kodecytes retained their normal expression of intrinsic blood group antigens while expressing the new epitopes attached by KODE technology. The MUT, Mur, and MUT+Mur kodecytes, although unreactive with the various monoclonal reagents, were appropriately reactive with polyclonal sera containing antibodies reactive with GP.Mur‐positive RBCs. CONCLUSIONS: This study used selected MUT and Mur peptides and KODE cell surface engineering technology to create MUT+Mur kodecytes suitable for the detection and identification of RBC antibodies in human serum or plasma. This technology has the potential to create a large range of specialized RBCs for antibody screening and identification.     

Detection of Bordetella pertussis in a clinical laboratory by culture, polymerase chain reaction, and direct fluorescent antibody staining; accuracy, and cost

Control of Bordetella pertussis in the community is hampered by slow and insensitive diagnostic tests. We therefore examined the accuracy and cost of culture, direct fluorescent antibody (DFA) staining, and PCR in a routine clinical laboratory. Six hundred thirty seven nasopharyngeal swabs and aspirates in casamino acids transport medium were cultured, stained with polyclonal (Difco), and monoclonal (BL-5 and Accu-Mab) anti-B. pertussis reagents, and amplified by an IS481-specific PCR. PCR products were detected by a hybridization-enzyme immunoassay kit (Gen-eti-k DEIA, DiaSorin), with confirmation by a second PCR in a separate laboratory. Sensitivities and specificities of culture, polyclonal DFA, monoclonal DFA, and PCR were 36 and 100%, 11.4 and 94.6%, 8.3 and 98. 4%, and 95.0 and 99.3%, respectively, with a prevalence of 15.7%. The DFA tests were the most economical, and the PCR cost was 31% higher than culture. This study suggests that with minor improvements in economy, pertussis PCR can be implemented in a clinical laboratory with marked improvement in diagnostic accuracy.     

Comparison of assays for growth hormone using monoclonal or polyclonal antibodies for diagnosis of growth disorders

Current diagnostic standards for growth hormone deficiency were derived from measurement of growth hormone using polyclonal antibody assays. Recently, however, immunoassays using monoclonal antibodies to growth hormone have been commercially introduced. This study compares growth hormone levels obtained with two new monoclonal assays with levels obtained with a standard polyclonal assay. In 20 patients, the polyclonal assay (National Pituitary Agency) measured a mean growth hormone concentration of 14.0 +/- 10 (SD) ng/dl compared with monoclonal assay 1 (Hybritech) measurement of 10.4 +/- 7.2 ng/dl (P less than 0.01) and monoclonal 2 (Wellcome Co.) measurement of 18.9 +/- 9.8 ng/dl (P less than 0.01). The Hybritech monoclonal assay measured lower growth hormone levels than the polyclonal assay in all 20 patients. The Wellcome assay measured higher growth hormone levels than the polyclonal assay in 17 of 20 patients. There is not complete agreement between polyclonal and monoclonal antibody growth hormone assays in their measurement of plasma growth hormone levels. Clinicians using the new monoclonal antibody growth hormone assays will need to carefully develop separate diagnostic criteria for growth hormone deficiency that takes into account the disparity between the polyclonal assays that were used in the development of the current diagnostic standards and the new monoclonal antibody assays.     

Murine monoclonal antibodies binding to the specific antigens of Aspergillus fumigatus associated with allergic bronchopulmonary aspergillosis

Four murine monoclonal antibodies, which were produced against Aspergillus fumigatus antigens using hybridoma technology, reacted with different antigenic components of A. fumigatus, and in turn these antigens showed reactivity with the sera from allergic bronchopulmonary aspergillosis (ABPA). All four antibodies were of IgM isotype. These antibodies reacted against eight antigen preparations from three different strains of A. fumigatus by enzyme-linked immunosorbent assay (ELISA). Only two of four antibodies reacted with the antigens in crossed immunoelectrophoresis (CIE), rocket immunoelectrophoresis, and agar gel double diffusion. In western blot analysis it was found that the antigenic components reacting with the monoclonal antibodies were mostly of the low molecular weight components of A. fumigatus antigens. These components also showed binding to both IgG and IgE antibodies in the sera of ABPA patients, but failed to show any reactivity with sera from aspergilloma patients. Hence these antigenic components may be of diagnostic significance and can be isolated using immunoaffinity chromatography.     

Comparison of monoclonal and polyclonal anti-P1 reagents

Whereas monoclonal reagents for ABO, Rhesus, Kell, Kidd, Lewis and MNS antigens are widely used in blood grouping laboratories and have been well evaluated, little attention has been given so far to monoclonal anti-P1 reagents. Therefore it was the aim of our study to examine the suitability of monoclonal anti-P1 reagents for routine use in comparison to polyclonal anti-P1 reagents. 10 polyclonal anti-P1 reagents (9 from goat, 1 from rabbit) and 5 monoclonal anti-P1 reagents (4 with clone 650, 1 with clone OSK17) were tested by the tube-spin-method at room temperature (incubation time as prescribed by the manufacturer) using untreated P1+ red blood cells (RBCs) for titration and untreated P1- RBCs as negative controls. P1- RBCs with a positive direct anti-globulin test (DAT+) (coated with incomplete anti-D) and sialidase-treated P1- RBCs (T+) were used to recognize false positive reactions. 3 monoclonal anti-P1 reagents had a titer of < or = 1 with P1+(weak) RBCs, but all polyclonal anti-P1 reagents showed a titer > or = 2. Negative controls (untreated P1- RBCs) were inconspicuous. 7 polyclonal and 1 monoclonal anti-P1 reagents showed false positive reactions with P1- DAT+ RBCs. All polyclonal and 1 monoclonal (containing clone OSK17) reagent showed false positive reactions with P1- T+ RBCs. As several monoclonal anti-P1 reagents are less suitable for routine use than polyclonal anti-P1 reagents, quality control of anti-P1 reagents should be intensified.     

Etiologic diagnosis of pneumonia by antigen detection: crossreactions between pneumococcal C-polysaccharide and oral microorganisms

Crossreactions between bacteria occurring more or less frequently in the respiratory tract were investigated using an enzyme-linked immunosorbent assay (ELISA) developed for the detection of pneumococcal C-polysaccharide. A collection of 218 strains was investigated: 30 Streptococcus pneumoniae, 120 alpha-streptococci, and 68 strains representing other species. Strong crossreactions were observed with 36% of the alpha-streptococci and with two of 11 Staphylococcus aureus strains. The collection of alpha-streptococci consisted of 90 fresh clinical isolates and 30 stock strains. Almost all crossreactions of alpha-streptococci were found among the clinical isolates. Among the stock strains only one of four Streptococcus mitis strains was positive. Pneumococcal C-polysaccharide and phosphorylcholine inhibited the reactions in ELISA with monoclonal antibodies against pneumococcal C-polysaccharide, as well as with a polyclonal antiserum against pneumococcal C-polysaccharide. We suggest that the cross reactions between alpha-streptococci and pneumococci depend on the presence of phosphorylcholine as a common antigenic determinant. The crossreaction in the ELISA with some Staphylococcus aureus strains may be explained by the presence of protein A binding to the Fc portion of the antibodies. When the 10 alpha-streptococci that showed the strongest crossreactions and ten pneumococci representing different types were tested in different concentrations the absorbance values were lower for most alpha-streptococci compared with the pneumococci. This explains that false positive results with alpha-streptococci do not seem to constitute a practical problem in this ELISA developed for detection of pneumococcal C-polysaccharide in samples from patients with pneumonia.     

Serologic and cellular assays to monitor therapy with murine monoclonal antibodies

The emergence of new therapeutic modalities frequently requires development of relevant laboratory assays. This is especially true for the clinical uses of newly developed reagents such as murine monoclonal antibodies. We have described immunofluorescence, immunohistochemical, and enzyme-linked immunosorbent assays (ELISA) for monitoring anticancer therapy with monoclonal antibodies. Immunofluorescence assays performed on single cell suspensions and immunohistochemical assays performed on freshly frozen biopsy tissue have confirmed tissue reactivity of the monoclonal antibody prior to infusion and have been used to measure in vivo antibody binding to target cells as well as antigenic modulation during therapy. An ELISA has been used to measure the serum concentrations of monoclonal antibodies; another ELISA has been used to detect the presence of human antibodies reactive with murine monoclonal antibody. These reproducible assays can be performed with commercially available reagents and provide an important data base for making clinical decisions concerning therapy with monoclonal antibodies.     

Quantitation of apolipoprotein B by polyclonal and monoclonal antibodies

A non-competitive sandwich enzyme linked immunosorbent assay for the quantitation of apolipoprotein B with polyclonal and monoclonal antibodies was developed. Polyclonal antibodies were used as 'coater'. In the assay with polyclonal antibodies, the same antibody was used as conjugate with alkaline phosphatase. For studies with monoclonal antibodies, enzyme conjugated anti-mouse immunoglobulin had to be used, since monoclonal antibodies lost their reactivity upon enzyme conjugation. Two murine monoclonal antibodies were employed: MAB B-1 with specificity for apolipoproteins (Apo) B-48 and B-100 and MAB B-5 with specificity for B-100 (Radioimmunoassay Inc.). In a reference group Apo B values of 0.82 +/- 0.20 g/l were measured with polyclonal antibodies, 0.68 +/- 0.19 g/l and 0.95 +/- 0.33 g/l with MAB B-1 and MAB B-5. In pure hypercholesterolemia, a similar increase was found with all three antibodies, while in combined hyperlipoproteinemia MAB B-5 gave greater than 40% lower values. Differences were also found with respect to the correlation between Apo B and cholesterol or triglycerides.     

Preparation and ELISA evaluation of Blastomyces dermatitidis yeast phase lysate antigens

Enzyme-linked immunosorbent assay (ELISA) comparative studies are described in which 10 Blastomyces dermatitidis antigenic preparations from human, dog, and soil strains were used for the detection of rabbit serum antibodies directed against B. dermatitidis killed yeast cells of human and dog origin as well as detection of antibodies in human serum samples. Reactive reagents were produced from all 10 strains, but two antigens (K-Le and T-043324) demonstrated exceptional sensitivity with moderate cross-reactivity with histoplasmosis specimens. Additionally, these reagents were able to detect anti-B. dermatitidis antibodies produced in rabbits in response to human (H-100) or dog (T-042976) strains in a comparable manner. These data suggest that a single antigenic reagent might be suitable for ELISA detection of B. dermatitidis antibodies in different species with blastomycosis.     

Standardization of an enzymometric assay for apolipoprotein A-I by using mixtures of monoclonal antibodies)

For the standardization of human plasma apolipoprotein A-I assay two well characterized monoclonal antibody mixtures were used to develop a sandwich immunoenzymometric assay. The monoclonal antibody mixture 1 (A05-A17-A30) in solid phase technique was selected on the basis of its higher binding capacity of [125I]HDL (41 ng per well) compared to polyclonal antibody (23 ng per well). The epitopes recognized by monoclonal antibody mixture 1 are surface antigenic sites of apolipoprotein A-I expressed on native HDL as determined by competitive inhibition of labeled HDL. The peroxidase conjugated monoclonal antibody mixture 2 (A03-A05-A17-A51) was selected on the basis of its ability to bind to apolipoprotein A-I captured by monoclonal antibody mixture 1. For this, we used the 125I-labeled monoclonal antibodies. Under optimized assay conditions, the immunoenzymometric assay is precise (intra- and inter-assay coefficients of variations 5.4% and 9.2% respectively). It yields plasma apolipoprotein A-I values that correlate highly with those obtained with polyclonal antibody (r = 0.96). So the use of well characterized monoclonal antibody mixtures reacting only to surface antigenic sites of apolipoprotein A-I present on native lipoprotein may provide the possibility of standardization of apolipoprotein A-I measurement.     

Intranasal monoclonal immunoglobulin A against respiratory syncytial virus protects against upper and lower respiratory tract infections in mice.

The role of secretory antibody in protection against respiratory syncytial virus (RSV) infection was examined by using monoclonal immunoglobulin A (IgA) antibody for intranasal passive immunization of mice. Eight anti-RSV IgA hybridomas were produced by fusing myeloma cells with lung lymphocytes from RSV-immunized mice. Five IgA antibodies recognized RSV strains of both the A and the B subgroups, and two of these neutralized virus in a plaque reduction assay. Monoclonal IgA antibody HNK20, which bound to F glycoprotein, was most effective, reducing plaques by 50% at a concentration of 0.1 microgram/ml for both subgroup A and subgroup B strains. HNK20 also neutralized all of eight clinical isolates of RSV tested. When delivered intranasally to mice 24 h prior to RSV challenge, HNK20 reduced virus titers in the lungs by nearly 100-fold. Maximal protection occurred at a dose of 0.5 mg/kg of body weight. Significant protection against lung infection was seen when the interval between antibody treatment and challenge was as long as 72 h. HNK20 also decreased virus titers in the nose approximately 10-fold when given 1 h, but not 24 h, before challenge. When mice were treated with HNK20 intranasally 3 days after challenge, viral titers were reduced in the lungs but not the nose. The results indicate that topical application of relatively small amounts of monoclonal IgA can protect against both upper and lower respiratory tract infections caused by RSV.     

Application of pooled monoclonal antibodies for 1-hr detection of respiratory syncytial virus antigen in clinical specimens

A fluorescein isothiocyanate-conjugated pool of monoclonal antibodies (MoAb) to respiratory syncytial virus (RSV) was prospectively evaluated for its utility as a direct, 1-hr test for the diagnosis of RSV infection. Direct nasopharyngeal swab smears collected from 109 infants and children with acute respiratory illnesses were studied and compared with results obtained by indirect immunofluorescence using bovine polyclonal anti-RSV antibody on eluted cells derived from pooled nasopharyngeal and throat swab specimens (a 2.5-3 hr procedure), and culture. The MoAb-direct smear method was at least 86%-89% sensitive and 95%-100% specific compared with either of the other procedures. Additional prospective evaluations, as well as retrospective studies on a selected bank of slides stored from the preceding year, established that this MoAb could also be used with confidence in testing where direct smears are not employed.     

Monoclonal antibodies to polioviruses. Production of specific monoclonal antibodies to the Sabin vaccine strains

Murine lymphocyte hybridomas which produce neutralizing or non-neutralizing monoclonal antibodies to different type 1, 2 or 3 poliovirus strains were isolated. The majority of these monoclonal antibodies reacted with antigenic determinants present on different poliovirus strains of the same type. However, hybridomas producing monoclonal antibodies specific for each of the three Sabin vaccine strains were also generated.     

Generation and Characterization of ALX-0171, a Potent Novel Therapeutic Nanobody for the Treatment of Respiratory Syncytial Virus Infection

Respiratory syncytial virus (RSV) is an important causative agent of lower respiratory tract infections in infants and elderly individuals. Its fusion (F) protein is critical for virus infection. It is targeted by several investigational antivirals and by palivizumab, a humanized monoclonal antibody used prophylactically in infants considered at high risk of severe RSV disease. ALX-0171 is a trimeric Nanobody that binds the antigenic site II of RSV F protein with subnanomolar affinity. ALX-0171 demonstrated in vitro neutralization superior to that of palivizumab against prototypic RSV subtype A and B strains. Moreover, ALX-0171 completely blocked replication to below the limit of detection for 87% of the viruses tested, whereas palivizumab did so for 18% of the viruses tested at a fixed concentration. Importantly, ALX-0171 was highly effective in reducing both nasal and lung RSV titers when delivered prophylactically or therapeutically directly to the lungs of cotton rats. ALX-0171 represents a potent novel antiviral compound with significant potential to treat RSV-mediated disease.     

Circulating antibodies to monoclonal immunoglobulins used in a follitropin assay may cause incorrect fertility diagnosis

We report grossly overestimated serum immunoreactive follitropin concentrations in an infertile man with normal spermatogenesis using a solid phase fluoroimmunoassay. The assay employs two monoclonal antibodies directed against two separate antigenic sites on the human follitropin molecule (DelFIA, Pharmacia). Determination of serum immunoreactive follitropin with a RIA based on polyclonal antiserum (WHO reagents), and of bioactive follitropin with the Sertoli-cell bioassay, revealed normal serum follitropin concentrations. Preincubation of the patient's sample with normal mouse serum or addition of normal mouse serum to the assay buffer neutralized the effect of the interfering substance in the DelFIA follitropin assay. Gel chromatography showed the interfering substance to have a relative molecular mass greater than 200,000. The substance is probably an antibody against mouse immunoglobulins, which cross-links the monoclonal mouse antibodies used in the DelFIA. The unknown incidence of heterophilic antibodies in human serum and the lack of preventive steps in some commercially available kits may lead, as in this case, to misdiagnosis in some patients.     

Platelet antibodies of the IgM class in immune thrombocytopenic purpura

The clinical course and response to therapy of patients with immune thrombocytopenic purpura (ITP) are not completely determined by the level of IgG present on the platelet surface. It is possible that antibodies of other immunoglobulin classes also play a role in platelet destruction in some of these patients. Therefore, we studied 175 patients with ITP for the presence of IgM anti-platelet antibodies using radiolabeled polyclonal or monoclonal anti-IgM. We observed that 57% of patients with clinical ITP had increased levels of IgM on their platelets, compared with normal controls and patients with thrombocytopenia who did not have ITP (less than 10%), (P less than 0.01). We obtained similar results using either radiolabeled polyclonal or monoclonal anti-IgM, reagents whose integrity was first characterized using erythrocytes coated with defined amounts of IgM antibody. Among patients with increased platelet-IgM there was a significant correlation both with the presence of increased platelet-C3 as well as the amount of platelet-C3 (P less than 0.01, r = 0.53). We demonstrated the presence of warm-reacting IgM anti-platelet antibodies in the plasma of two of these patients who were further studied. The isolated IgM fraction from these two plasmas was able to activate complement and place 3H-C3 on normal platelets. These studies demonstrate the presence of warm-reacting IgM anti-platelet antibodies in some patients with ITP. They suggest that the binding of complement to platelets by IgM antibodies may initiate platelet clearance as well as enhance the effect of IgG antibodies in ITP.     

Rubella virus strains show no major antigenic differences.

To determine whether antigenic differences occur among rubella virus strains, five wild-type strains of rubella virus isolated in the UK, the USA, and in Japan between 1964 and 1987 and four attenuated vaccine strains were compared employing a panel of 28 monoclonal antibodies in neutralization, haemagglutination-inhibition, enzyme immunoassay, and indirect immunofluorescence assays. No antigenic differences were detected which confirms that rubella vaccines will protect against circulating strains and that rubella antigens used in serological tests for screening and diagnosis will detect antibodies induced by all strains.     

A multispecies enzyme-linked immunosorbent assay for von Willebrand's factor.

A modified double-sandwich enzyme-linked immunosorbent assay (ELISA), which was developed for human and canine von Willebrand's factor (vWF), was adapted for quantitation of vWF in other species. In addition to human and dog plasmas, 12 other mammalian plasmas that were surveyed exhibited significant cross-species reactivity with antibodies specific for canine vWF or monoclonal antibodies against porcine or bovine vWF. Mixed combinations of monoclonal antibodies and various polyclonal antibodies were also used as sandwich or capture antibodies. The ability of this multispecies ELISA to detect less than 0.002 U of vWF per milliliter of plasma in a large number of species enhances its utility for both research and clinical diagnostic applications. A quantitative assay for rabbit vWF, which exhibits poor cross-species reactivity with most antibodies, was constructed with anti-porcine monoclonal antibody W1-5 and goat-anti-canine vWF as capture and sandwich antibodies, respectively. The same conjugate antibody configuration was used to visualize rabbit vWF multimers by immunoblotting. Specificity of the assay for vWF in human, dog, pig, and horse plasmas was confirmed by use of species-specific vWF-deficient plasmas. In other species, for which vWD plasmas were not available, ELISA specificity for vWF was demonstrated by recovery of greater than 75% of the ELISA-reactive antigen coincident with vWF multimers in the high-molecular-weight (greater than 500 kd) fractions of purified plasmas. This multispecies ELISA permits, for the first time, the measurement of vWF in a variety of mammals for which species-specific immunologic reagents do not currently exist. The results also suggest that certain vWF epitopes have been highly conserved among phylogenetically diverse species.