Development of antigen detection ELISA for the diagnosis of brugian and bancroftian filariasis using antibodies to recombinant filarial antigens Bm-SXP-1 and Wb-SXP-1

Antibodies specific to recombinant filarial antigens Wb-SXP-1 and Bm-SXP-1 have been used to develop a sandwich ELISA for the detection of circulating filarial antigen (CFA) in sera from patients with lymphatic filariasis caused by Wuchereria bancrofti of Brugia malayi. In patients with W. bancrofti infections, a high proportion of microfilaria (mf) positive (MF) and low proportions of patients with chronic pathology (CP) and endemic normals (EN) showed the presence of CFA. Similarly in patients with brugian infections a high proportion of mf positive individuals contained CFA while none of the patients with chronic pathology or endemic normals showed the presence of CFA. Sera from patients with other parasitic infections (OPI) like O. volvulus, Loa loa, Ascaris lumbricoides and from individuals residing in areas non-endemic to filariasis did not exhibit any reactivity. This assay shows promise for the detection of microfilaremic infections in lymphatic filariasis and its usefulness as a diagnostic tool especially in B. malayi infections, needs to be further evaluated.     

Development and evaluation of a rapid flow-through immuno filtration test using recombinant filarial antigen for diagnosis of brugian and bancroftian filariasis

There is an imperative need to develop a rapid antibody test that can be used for diagnosis of clinical cases in travelers and expatriates, primary surveillance in areas of unknown endemicity, detection of early infection in childhood and for monitoring chemotherapeutic programs. A rapid-format, simple and qualitative flow through immuno filtration test has been developed for the identification of total IgG antibodies to recombinant filarial antigen WbSXP-1. This test system employs colloidal gold-protein A reagent as the antibody capture reagent. The sensitivity and specificity of the test was evaluated in a total of 1,230 serum samples. The sensitivity of the test was found to be 90.8% with brugian (n = 70) and 91.4% with bancroftian (n = 140) microfilaraemic subjects. The test showed minimum reactivity (4/10) with Loa loa microfilaria (MF) positive sera and no reactivity (0/20) with Onchocerca MF positive sera. This rapid diagnosis is found to be non-reactive with individuals having other parasitic diseases including schistosomiasis (n = 10), soil-transmitted helminthiases (n = 34) and protozoan infections (n = 33) indicating the potential of this test as a prospective method of diagnosis for both brugian and bancroftian lymphatic filariasis. Stability kinetics was studied at different temperatures and different time periods. The rapid flow-through immuno filtration test is advantageous since it can be stored at room temperature, is user friendly and is particularly applicable in the field as an initial screening method, for epidemiological monitoring of filarial infections in bancroftian and brugian endemic regions of the world.     

Cerebral malaria is associated with IgG2 and IgG4 antibody responses to recombinant Plasmodium falciparum RIFIN antigen

RIFIN proteins belong to the largest Plasmodium falciparum multicopy family of variant surface antigens (VSA) expressed by infected erythrocytes. VSA antibodies have been shown to be associated with protection against malaria. Here, antibody subclass responses to a recombinant RIFIN protein (RIF-29) in 116 Ghanaian children were determined by ELISA to investigate the relationship between severe malaria and anti-RIF-29 antibodies. The study group was composed of 23 children diagnosed exclusively for cerebral malaria and 35 children who had non-cerebral severe malaria. The remaining 58 individuals were age-, gender- and area-matched asymptomatic controls. The finding that IgG1 and IgG3 responses predominated in severe malaria patients compared to matched controls suggests that these antibodies are not protective, but are most probably induced by a current infection, an observation substantiated by the equally high reactivity to both recombinant RIF-29 protein and to P. falciparum crude lysate proteins. The exclusive detection of IgG2 and IgG4 antibodies to RIF-29 protein only in cerebral malaria children brings to mind the possibility that these antibodies are pathogenic. This is a new finding that may go some way towards explaining why these children are at risk of developing the life-threatening form of cerebral malaria.     

Detection of circulating antigen in bancroftian filariasis by sandwich ELISA using filarial serum IgG

The utility of the IgG fraction of human filarial serum immunoglobulin in detecting circulating antigen by sandwich enzyme linked immunosorbent assay (ELISA) was studied. 27 of 33 sera from persons with microfilaraemia, 19 of 30 sera from clinical cases of filariasis, 4 of 30 sera from normal persons from a region endemic for filariasis showed the presence of circulating filarial antigen. All the 20 normal sera from the area where filariasis was not endemic gave negative reaction for filarial antigen. Those sera from persons with microfilaraemia that showed the presence of circulating antigen also showed an apparent positive correlation between the microfilarial density and the antigen titre.     

Diagnostic value of IgG isotype responses against Brugia malayi antifilarial antibodies in the clinical spectrum of brugian filariasis

To study the diagnostic significance of antifilarial IgG subclasses in the clinical spectrum of brugian filariasis, IgG1, IgG3 and IgG4 antifilarial antibodies were determined in an exposed population comprising 74 asymptomatic amicrofilaraemics, 30 microfilaraemics, 20 lymphangitis and 16 elephantiasis patients resident in Narathiwart province, an area endemic for Brugia malayi lymphatic filariasis in southern Thailand. The dominant isotype of antifilarial antibody was IgG4. A significantly higher percentage of individuals were positive for IgG1 in the microfilaraemic and lymphangitis groups compared with the elephantiasis and endemic normal patients, while a significantly higher positive rate of IgG3 was found in those with lymphangitis. The possible role of these isotypes for diagnostic purposes and the pattern of antibody response in various clinically manifesting groups are discussed.     

Effects of feeding and induction strategy on the production of BmR1 antigen in recombinant E. coli

Aim:  To investigate the effects of feeding and induction strategies on the production of Bm R1 recombinant antigen.
Methods and Results:  Fed-batch fermentation was studied with respect to the specific growth rate and mode of induction to assess the growth potential of the bacteria in a bioreactor and to produce high yield of Bm R1 recombinant antigen. Cells were grown at a controlled specific growth rate (μ_set) during pre-induction, followed by constant feeding postinduction. The highest biomass (24·3 g l⁻¹) was obtained during fed-batch process operated at μ_set of 0·15 h⁻¹, whereby lower μ_set (0·075 h⁻¹) gave the highest protein production (9·82 mg l⁻¹). The yield of Bm R1 was increased by 1·2-fold upon induction with 1 mmol l⁻¹ IPTG (isopropyl-β- d -thiogalactoside) compared to using 5 mmol l⁻¹ and showed a further 3·5-fold increase when the culture was induced twice at the late log phase.
Conclusions:  Combination of feeding at a lower μ_set and twice induction with 1 mmol l⁻¹ IPTG yielded the best result of all variables tested, promising an improved method for Bm R1 production .
Significance and Impact of the Study:  This method can be used to increase the production scale of the Bm R1 recombinant antigen to meet the increasing demand for Brugia Rapid^™, a commercial diagnostic test for detection of brugian filariasis.     

IgG antibody subclasses in human filariasis. Differential subclass recognition of parasite antigens correlates with different clinical manifestations of infection

The four subclasses of IgG are distinct in structure, function, and degree of participation in the antibody response to complex antigens. Looking for differential responsiveness of potential pathogenetic significance, we have analyzed both quantitatively and qualitatively the filaria-specific IgG subclass responses of 20 patients with lymphatic filariasis presenting either with chronic lymphatic obstructive pathology and elephantiasis (CP) or with asymptomatic microfilaremia (MF). Subclass-specific monoclonal antibodies were used in an enzyme-linked immunosorbent assay to study IgG filarial antibodies quantitatively and in immunoblot analyses to determine qualitatively the subclass antibody specificities. Quantitatively, the most significant differences among patient groups were in levels of IgG4, which were more than 17 times higher in MF patients (geometric mean, 64.7 micrograms/ml) than in those with CP (mean, 3.7 micrograms/ml). When qualitative analyses were done on the same sera, major differences were noted, particularly in the recognition profiles of the IgG1, IgG3, and IgG4 responses. IgG1 and IgG3 responses to antigens were seen especially to antigens with m.w. greater than 68,000 in all patients with elephantiasis, whereas MF patients showed most of their reactivity to antigens less than 68,000. For IgG4, the MF patients showed prominent recognition of antigens throughout the entire range of m.w., whereas those with CP had very little IgG4 recognition of antigens of any m.w. Interestingly, this relationship was essentially reversed in the IgG3 antibody responses (especially to antigens greater than 68,000) and, to a lesser extent, the IgG1 responses. These findings demonstrate correlations of potential cause/effect significance between IgG4 antibody responsiveness and the immunomodulated asymptomatic MF form of clinical filariasis and between IgG3/IgG1 antibody responsiveness and the clinical presentation of CP.     

IgE responses in human filariasis. IV. Parallel antigen recognition by IgE and IgG4 subclass antibodies

Immediate hypersensitivity responses are highly modulated in filariasis, and with few exceptions, the majority of infected individuals do not develop allergic manifestations. One possible mechanism for this modulated responsiveness could involve the high levels of IgG "blocking antibodies" shown to be present in filariasis and other chronic helminth infections. When immunoblot analyses were done to analyze the immunoglobulin (Ig) E and IgG antibody responses of patients simultaneously, remarkable similarity in the patterns of antigen binding was observed. In this study, the four IgG subclasses were analyzed in a similar manner in relation to IgE. The results clearly demonstrate that IgG4 was primarily responsible for this "parallel" recognition that was seen previously between IgG and IgE antibodies. These results lend additional support to the possibility that IgG4 may play an important role in modulating IgE-mediated allergic responses in vivo.     

Control of allergic reactivity in human filariasis. Predominant localization of blocking antibody to the IgG4 subclass.

Patients with chronic helminth infections, despite having abundant basophils and mast cells specifically sensitized with antiparasite IgE and often exposed repeatedly to parasite Ag, rarely manifest allergic symptoms. This control of clinical allergic reactivity likely results from Ag-specific IgG "blocking antibodies" shown previously to be abundant in the sera of such patients. In the present study we used two approaches to determine in which of the four IgG subclasses this blocking activity was localized. First, specific antifilarial antibodies of each of the four IgG subclasses were quantified in the sera of 28 patients with Bancroftian filariasis and correlated with the levels of blocking activity in these sera (determined by histamine release assays). A significant correlation with blocking activity was seen only for antibodies of the IgG4 subclass, and, indeed, the correlation was especially strong in the group of totally asymptomatic patients (but with microfilariae circulating in the blood) in whom blocking antibody levels were highest. Interestingly, however, if the analysis excluded these asymptomatic microfilaremic patients and focused instead on those with lymphatic inflammatory pathology (who had relatively low levels of both serum blocking activity and specific IgG4 antibodies), then the small amount of blocking activity found in these sera correlated only with the levels of IgG1 subclass antibodies. The second approach utilized selective depletion of IgG4 (by anti-IgG4 affinity columns) from the sera of three microfilaremic patients with high levels of blocking activity and demonstrated clearly that removal of IgG4 abolished the majority of the blocking activity in these sera (53, 78, and 81%). These two sets of findings demonstrate a predominant role for specific IgG4 antibodies in blocking IgE-mediated allergic responses to the parasite Ag in vitro, but they also indicate that in some situations IgG1 antibodies can block such reactions. Furthermore, the correlation demonstrated between patients' clinical presentations and the levels of both their specific IgG4 antibodies and serum blocking activity suggests that these antibodies play a similar role in vivo as well.     

Purification of recombinant antigen BmSXP used in panLF rapid kit for lymphatic filariasis

A rapid antibody detection test is very useful for the detection of lymphatic filariasis, especially for certification and surveillance of post-mass drug administration. panLF Rapid kit is suitable for this purpose since it can detect all species of lymphatic filaria. It is based on the detection of anti-filarial IgG4 antibodies that react with recombinant B. malayi antigens, BmR1 and BmSXP. There is an increase demand for the test due to its attributes of being rapid, sensitive and specific results, as well as its field-applicability. The main aim of this paper is to obtain high recovery and purity of recombinant antigen BmSXP via a modified method of immobilized metal affinity chromatography (IMAC). The highest product yield of 11.82 mg/g dry cell weight (DCW) was obtained when IMAC was performed using the optimized protocol of 10 mM imidazole concentration in lysis buffer, 30 mM imidazole concentration in wash buffer, and 10 column volume wash buffer containing 300 mM salt concentration. This gave a 54% protein recovery improvement over the manufacturer's protocol which recorded a product yield of only 7.68 mg/g DCW. The recovered BmSXP recombinant antigen showed good western blot reactivity, high sensitivity (31/32, 97%) and specificity (32/32, 100%) in ELISA, thus attesting to its good purity and quality.     

Stable recombinant expression of the anti HIV-1 monoclonal antibody 2F5 after IgG3/IgG1 subclass switch in CHO cells

The human monoclonal antibody (humAb) 2F5 is a potent candidate for immunotherapy of HIV-1. The xenohybridoma derived humAb 2F5 is of IgG3 kappa isotype. Generally, IgG1 isotype has a longer half-life (beta-clearance) in humans than IgG3. Therefore the isotype was switched to an IgG1 by ligation of the 2F5 heavy chain variable region to an IgG1 constant region and expressed as IgG1 kappa in CHO cells. CHO clones have been established, which stably express humAb 2F5 at high levels. The specificity, affinity and in vitro function of both isotypes were identical.     

ELISA detection of IgG antibody against a recombinant major surface antigen (Nc-p43) fragment of Neospora caninum in bovine sera

An ELISA was established to measure bovine IgG directed against the recombinant antigenic determinant of Nc-p43, a major surface antigen of Neospora caninum. In a previous study, two thirds of the C-terminal of the molecule was expressed as a 6 x His tagged protein (Ncp43P) for ELISA using 2/3 of the N-terminal of SAG1 from Toxoplasma gondii as a control (TgSAG1A). Among 852 cattle sera collected from stock farms scattered nation-wide, 103 sera (12.1%) were found to react with Ncp43P positively, but no positive reaction was observed with TgSAG1A. This study shows that Ncp43P could be available as an efficient antigen for the diagnosis of neosporosis in cattle. Furthermore, it together with TgSAG1A, could be useful for the differential diagnosis of N. caninum and T. gondii infections in other mammals.     

The use of polysiloxane/polyvinyl alcohol beads as solid phase in IgG anti-Toxocara canis detection using a recombinant antigen

Immunodetection of human IgG anti-Toxocara canis was developed based on ELISA and on the use of polysiloxane/polyvinyl alcohol (POS/PVA) beads. A recombinant antigen was covalently immobilized, via glutaraldehyde, onto this hybrid inorganic-organic composite, which was prepared by the sol-gel technique. Using only 31.2 ng antigen per bead, a peroxidase conjugate dilution of 1:10,000 and a serum dilution of 1:200 were adequate for the establishment of the procedure. This procedure is comparable to that which utilizes the adsorption of the antigen to conventional PVC plates. However, the difference between positive and negative sera mean absorbances was larger for this new glass based assay. In addition to the performance of the POS/PVA bead as a matrix for immunodetection, its easy synthesis and low cost are additional advantages for commercial application.     

The diagnostic sensitivity of dengue rapid test assays is significantly enhanced by using a combined antigen and antibody testing approach

Background

Serological tests for IgM and IgG are routinely used in clinical laboratories for the rapid diagnosis of dengue and can differentiate between primary and secondary infections. Dengue virus non-structural protein 1 (NS1) has been identified as an early marker for acute dengue, and is typically present between days 1–9 post-onset of illness but following seroconversion it can be difficult to detect in serum.

Aims

To evaluate the performance of a newly developed Panbio® Dengue Early Rapid test for NS1 and determine if it can improve diagnostic sensitivity when used in combination with a commercial IgM/IgG rapid test.

Methodology

The clinical performance of the Dengue Early Rapid was evaluated in a retrospective study in Vietnam with 198 acute laboratory-confirmed positive and 100 negative samples. The performance of the Dengue Early Rapid in combination with the IgM/IgG Rapid test was also evaluated in Malaysia with 263 laboratory-confirmed positive and 30 negative samples.

Key Results

In Vietnam the sensitivity and specificity of the test was 69.2% (95% CI: 62.8% to 75.6%) and 96% (95% CI: 92.2% to 99.8) respectively. In Malaysia the performance was similar with 68.9% sensitivity (95% CI: 61.8% to 76.1%) and 96.7% specificity (95% CI: 82.8% to 99.9%) compared to RT-PCR. Importantly, when the Dengue Early Rapid test was used in combination with the IgM/IgG test the sensitivity increased to 93.0%. When the two tests were compared at each day post-onset of illness there was clear differentiation between the antigen and antibody markers.

Conclusions

This study highlights that using dengue NS1 antigen detection in combination with anti-glycoprotein E IgM and IgG serology can significantly increase the sensitivity of acute dengue diagnosis and extends the possible window of detection to include very early acute samples and enhances the clinical utility of rapid immunochromatographic testing for dengue.     

Control of IgE and IgG1 antibody production in mice.

The production of IgE and IgG1 was studied in untreated, thymectomized. splenectomized, anti-thymocyte serum-treated, or sublethally X-irradiated mice. Dinitrophenyl Ascaris and ovalbumin were used as antigens, and aluminum hydroxide was used as adjuvant. A suppression of IgE production was observed in adult thymectomized mice, although the kinetic pattern of the antibody response was the same as in control animals. IgG1 antibody production was not affected by thymectomy. Splenectomy did not change either IgE or IgG1 production. A single dose of rabbit anti-thymocyte serum (ATS) given 8 days after immunization inhibited IgE antibody production. The effect of ATS was dose dependent and also varied with the amount of antigen used, the immune response to high doses being more susceptible to the effect of ATS. No alteration in IgG1 production was caused by ATS even when IgE antibody formation was completely inhibited. When preceding immunization, sublethal irradiation enhanced IgE antibody formation and partially suppressed IgG1 production; applied after immunization, irradiation caused an enhancement of IgE production which was inversely proportional to the interval elapsed between the two procedures. On the other hand, the IgG1 antibody production was fairly resistant to the same treatment. The results suggest a clearcut separation between the mechanisms regulating IgE and IgG1 production in mice.     

Selective targeting of melanoma and APCs using a recombinant antibody with TCR-like specificity directed toward a melanoma differentiation antigen

Tumor-associated, MHC-restricted peptides, recognized by tumor-specific CD8(+) lymphocytes, are desirable targets for novel approaches in immunotherapy because of their highly restricted fine specificity. Abs that recognize these tumor-associated MHC-peptide complexes, with the same specificity as TCR, would therefore be valuable reagents for studying Ag presentation by tumor cells, for visualizing MHC-peptide complexes on cells, and eventually for developing new targeting agents for cancer immunotherapy. To generate molecules with such a unique, fine specificity, we immunized HLA-A2 transgenic mice with a single-chain HLA-A2, complexed with a common antigenic T cell HLA-A2-restricted epitope derived from the melanoma differentiation Ag gp100. Using a phage display approach, we isolated a recombinant scFv Ab that exhibits a characteristic TCR-like binding specificity, yet, unlike TCRs, it did so with a high affinity in the nanomolar range. The TCR-like Ab can recognize the native MHC-peptide complex expressed on the surface of APCs, and on peptide-pulsed or native melanoma cells. Moreover, when fused to a very potent cytotoxic effector molecule in the form of a truncated bacterial toxin, it was able to specifically kill APCs in a peptide-dependent manner. These results demonstrate the utility of high affinity TRC-like scFv recombinant Abs directed toward human cancer T cell epitopes. Such TCR-like Abs may prove to be very useful for monitoring and visualizing the expression of specific MHC-peptide complexes on the surface of tumor cells, APCs, and lymphoid tissues, as well as for developing a new family of targeting agents for immunotherapy.     

Comparison of immunospecific antibody response in young and old chickens immunized with human IgG

Three groups of 18-month-old chickens and three groups of 5-month-old chickens were immunized with human immunoglobulin G (IgG) using one of three adjuvants in the first injection (Freund's Complete Adjuvant (FCA), Freunds Incomplete Adjuvant (FIA) and Hunter's TiterMax (HTM)) following the same immunization scheme. The specific antibody response in serum was measured by ELISA. In both older and younger chickens the serum antibody response in the FCA group reached a significantly higher level (P < 0.01) than in the FIA group and in the HTM group on week 5. The FCA group also had a significantly higher (P < 0.01) response on week 10 compared to the HTM group. Other than that, there was no significant difference between the three adjuvant groups in specific serum antibody response in older chickens. In the younger chickens the specific serum antibody response in the FCA group was significantly higher (P < 0.05) than the response in the HTM group. There was no significant difference in the chicken serum antibody response between the FCA and the FIA groups, nor was there a significant difference between the FIA and the HTM groups. Comparing the younger chickens and the older chickens immunized using the same adjuvant, the older chickens had consistently higher titres than the younger chickens, although the difference was not always significant.     

Identification and characterization of a -1 reading frameshift in the heavy chain constant region of an IgG1 recombinant monoclonal antibody produced in CHO cells

Frameshifts lead to complete alteration of the intended amino acid sequences, and therefore may affect the biological activities of protein therapeutics and pose potential immunogenicity risks. We report here the identification and characterization of a novel -1 frameshift variant in a recombinant IgG1 therapeutic monoclonal antibody (mAb) produced in Chinese hamster ovary cells during the cell line selection studies. The variant was initially observed as an atypical post-monomer fragment peak in size exclusion chromatography. Characterization of the fragment peak using intact and reduced liquid chromatography-mass spectrometry (LC-MS) analyses determined that the fragment consisted of a normal light chain disulfide-linked to an aberrant 26 kDa fragment that could not be assigned to any HC fragment even after considering common modifications. Further analysis using LC-MS/MS peptide mapping revealed that the aberrant fragment contained the expected HC amino acid sequence (1-232) followed by a 20-mer novel sequence corresponding to expression of heavy chain DNA sequence in the -1 reading frame. Examination of the DNA sequence around the frameshift initiation site revealed that a mononucleotide repeat GGGGGG located in the IgG1 HC constant region was most likely the structural root cause of the frameshift. Rapid identification of the frameshift allowed us to avoid use of a problematic cell line containing the frameshift as the production cell line. The frameshift reported here may be observed in other mAb products and the hypothesis-driven analytical approaches employed here may be valuable for rapid identification and characterization of frameshift variants in other recombinant proteins.     

Stabilities of antigen and antibody under elution conditions in immunoaffinity chromatography using monoclonal antibody.

In immunoaffinity chromatography using monoclonal antibody, the elution conditions of an antigen, recombinant alpha-amylase, were studied. From among various conditions, three elution methods that gave fairly good yields of antigen activity (pH 2.3-2.5, pH 12.3-12.5 and 0.1 M lithium 3,5-diiodo salicylate [LIS]) were selected and the stabilities of the antigen and the antibody were analyzed. The antigen seemed to be eluted from the immunoadsorbent due to partial denaturation of either the antigen or the antibody. LIS seemed to be a specific denaturant for the antibody and its action was reversible. In terms of the stability of the antigen and repeated use of the immunoadsorbent, LIS seemed to be the best reagent for elution in immunoaffinity chromatography.