C Polysaccharide Research Articles

Development of meningococcal polysaccharide conjugate vaccine that can elicit long-lasting and strong cellular immune response with hepatitis B core antigen virus-like particles as a novel carrier protein

Neisseria meningitidis caused meningitis is life-threatening acute infection with high fatality and high frequency of severe sequelae. Meningococcal capsular polysaccharides can be used to prevent meningococcal disease; while conjugating the polysaccharides to carrier protein was found necessary to improve the immunogenicity and induce memory responses in infants and young children. Nevertheless, repeated administration of glycoconjugate vaccines might lead to carrier-induced epitope suppression due to limited number of carrier proteins. Here in this study, full-length hepatitis B core antigen virus-like particles (HBc VLPs) was used as a novel potential carrier protein for conjugation of meningococcal group C polysaccharides (CPS) with heterobifunctional polyethylene glycol (PEG) of different length (2, 5 and 10 kDa) as linkers. The physiochemical properties of the CPS-PEG-HBc conjugate vaccines were fully characterized. The TEM, DLS, native agarose gel electrophoresis, and HPLC analyses all confirmed the successful conjugation. As compared to plain CPS and the physical mixture of CPS and HBc, the immunization with the conjugate vaccines can generate about 10 times increase in CPS specific IgG titers with a significant boosting effect. HBc conjugation induced a shift to a Th1 cellular immune type response, as assessed by the increased IgG2a subclass production. In addition, vaccination of the conjugate vaccines elicited much enhanced avidity functional antibody and long-lasting immunological memory. IgG titers elicited by CPS-P2k-HBc, CPS-P5k-HBc and CPS-P10k-HBc at week 18 maintained 38.1%, 17.9% and 33.3% of their peak values. All these results demonstrated that HBc VLPs can be used as potential carrier protein to develop polysaccharide conjugate vaccines effective in eliciting long-lasting and strong cellular immune response.

Re-evaluation of the immunogenicity of meningococcal serogroups A and C polysaccharide vaccine among a healthy population aged 5 to 59

Objective To re-evaluate the immunogenicity of meningococcal serogroups A and C polysaccharide vaccine among a healthy population of age 5 to 59.Methods Pre and post-vaccination ser-um samples were collected from the subjects involved in a randomized , controlled clinical trial conducted in 2005 at Hechi, Guangxi, China.Enzyme-linked immunosorbent assay （ELISA） was performed for the quan-titative detection of specific anti-capsule IgG antibody against meningococcal serogroups A and C in serum samples.Serum bactericidal assay （ SBA） was used to measure the bactericidal antibody activity against se-rougroups A and C bacterial strains .Results Geometric mean concentrations （GMCs） of specific anti-cap-sule IgG antibody were 23.66 μg/ml and 78.83 μg/ml for serogroup A （P〈0.05）, and 1.23 μg/ml and 51.25 μg/ml for serogroup C （P〈0.05） in serum samples of pre and post-vaccination, respectively.Be-fore immunization , 99% of serum samples （ 97/98 ） showed serogroup A-specific IgG concentration ≥2μg/ml, which reached up to 100%（98/98） after vaccination （P〉0.05）.The percentage of serum sam-ples with serogroup C-specific IgG concentration ≥2 μg/ml rose from 20%to 99% after vaccination （ P〈0.05）.The ratio of positive serum samples with at least four times of increases in concentration of specific IgG against serogroup A and serogroup C after vaccine immunization were 34% and 100%, respectively . The rSBA geometric mean titers （ GMTs） for serogroups A and C in serum sample of post-vaccination were＆amp;nbsp;respectively elevated from 1164 to 5530 （P〈0.05）, and from 4 to 6225 （P〈0.05）.The percentage of se-rum samples with rSBA GMTs≥128 increased from 91% to 99% for serogroup A （P〉0.05）, and from 14%to 96%for serogroup C （P〈0.05） after vaccination.The rSBA GMTs with at least four times of in-creases after vaccination were detected respectively in 53% and 100% of serogroups A and C vaccinated subjects.Pearson correlation coefficient between IgG concentration and rSBA GMTs was r=0.15 （pre） and r=0.23 （post） for serogroup A, and r=-0.14 （pre） and r=0.58 （post） for serogroup C （P〈0.05）. Conclusion This study has demonstrated an efficient and sustained immunogenicity with meningococcal sero -groups A and C polysaccharide vaccine as evidenced by the data from standardized assays of ELISA and SBA . Key words: Meningococcal serogroups A and C polysaccharide vaccine; Immuogenicity

Scientific Basis for Traditional Medicines in Renal Disease

Cordyceps Sinensis (CS) is a parasitic fungus that has been used as a Chinese medicine for a long time in the treatment of nephritis. In this study, we established an in vitro model that showed that cultured human mesangial cells (HMC) stimulated with interleukin-1 (IL-1) plus IL-6 can cause mesangial cell proliferation, increasing production of chemical mediators and superoxide anion. An in vivo model also proved that this culture medium might lead to renal injury with hematuria and proteinuria. We cultured HMC, and then an HMC activating model with HMC incubated with IL-1 and IL-6 was established. We fractionated the crude methanolic extracts from fruiting bodies of CS with the use of this in vitro inhibition of HMC activation model as our assay method. Then we established an IgAN animal model with R36A (Pneumococcal C-polysaccharide purified from Streptococcus pneumoniae) as antigen and anti-R36A IgA monoclonal antibody to form nephritogenic IgA-IC, which can induce hematuria and proteinuria in mice with IgA deposition in the mesangial area. The fruiting bodies were extracted by silica gel column chromatography. One out of 6 column fractions, F-2, significantly inhibited the HMC activation by IL-1 plus IL-6. The acute toxicity test with male institute of Cancer Research mice showed no liver toxicity or mutagenicity. The mice in the IgAN model fed with 1% F-2 in diet had significant reduction of hematuria and proteinuria together with histopathologic improvement. Therefore this fraction was then purified by silica gel column chromatography and high-performance liquid chromatography, which yielded a purified compound H1-A, which can suppress the activated HMC and alleviate IgAN (Berger's disease) with clinical and histologic improvement. These results give us a new regimen for the treatment of patients with IgAN in the future.

In Vivo Humoral Immune Responses to Isolated Pneumococcal Polysaccharides Are Dependent on the Presence of Associated TLR Ligands

We determined whether T cell-independent Ig isotype responses to isolated pneumococcal polysaccharides (PPS) required TLR signaling in vivo. IgG anti-PPS responses to PPS3, PPS14, and C-polysaccharide (C-PS) were virtually undetectable in TLR2(-/-) mice, whereas specific IgM induction was variably reduced compared with wild-type mice. All PPS-containing preparations induced IL-6 and TNF-alpha from wild-type, but not TLR2-/-, macrophages. TLR2 activity was distinct from that of PPS, in that it was phenol extractable. Immunization of wild-type mice with phenol-extracted PPS14 also resulted in a marked reduction in the IgG, although not the IgM-anti-PPS14, response compared with untreated PPS14. The commercial 23-valent PPS vaccine, Pneumovax-23 also contained TLR ligands (TLR2 and TLR4), which were absolutely critical for the IgG-inducing activity of the vaccine in mice. Finally, the commercial pneumococcal conjugate vaccine, Prevnar, contained a TLR2 ligand(s) that substantially enhanced both the primary and secondary anti-PPS responses in mice, especially the type 1 IgG isotypes. These data strongly suggest the absolute need for a distinct, TLR-dependent second signal for inducing in vivo IgG T cell-independent humoral immune responses to isolated pneumococcal polysaccharide Ags and highlight the potential importance of previously unappreciated copurified and/or contaminating TLR ligands in PPS vaccine preparations.

Characterization and quantification of C-polysaccharide in Streptococcus pneumoniae capsular polysaccharide preparations

Purified capsular polysaccharide preparations from Streptococcus pneumoniae that are used for vaccine production typically contain residual levels of C-polysaccharide (C-Ps). Residual C-Ps is typically found in one of two forms, either chemically linked to the capsular polysaccharide (bound) or present by itself (free). Two analytical methods have been developed and applied to determine the relative percentages of the two C-Ps forms present in various capsular polysaccharide preparations. Both methods differentiate the two forms of C-Ps according to the difference of their hydrodynamic sizes. One method is based on labeling C-Ps with a fluorescent tag and separating the two forms of C-Ps by high-performance size exclusion chromatography with on-line refractive index and fluorescence detection, and the other method is based on measuring self-diffusion rates of the two forms of C-Ps by nuclear magnetic resonance (NMR) and quantifying each form with deconvolution. Both methods were evaluated for relative accuracy, precision, and ease of application, and they were found to provide comparable results for a large number of pneumococcal polysaccharide preparations. These analyses, combined with other quantitative NMR measurement of total C-Ps in the polysaccharide powder, provide a more refined means of evaluating the amount of each form of C-Ps in polysaccharide preparations targeted for vaccine production.

The teichoic acid (C-polysaccharide) synthesized by Streptococcus pneumoniae serotype 5 has a specific structure

The teichoic acid synthesized by Streptococcus pneumoniae serotype 5, also known as pneumococcal common antigen (C-polysaccharide), was purified. On the basis of compositional analysis, HPAEC-PAD analysis, MALDI-TOF mass spectrometry and NMR spectroscopy, made on the native polysaccharide and on the dephosphorylated repeating unit, the following structure is proposed: ▪ This C-polysaccharide (C-PS), differs from those previously described by the replacement of Glc by Gal in its repeating unit structure.

Assignment of Weight-Based Antibody Units for 13 Serotypes to a Human Antipneumococcal Standard Reference Serum, Lot 89-S(F)

Weight-based immunoglobulin G (IgG), IgM, IgA, and total Ig antibody assignments were made to human antipneumococcal standard reference serum lot 89-S, also known as lot 89-SF, for Streptococcus pneumoniae capsular polysaccharide (PnPs) serotypes 2, 6A, 8, 9N, 10A, 11A, 12F, 15B, 19A, 17F, 20, 22F, and 33F, as well as for C-polysaccharide (C-Ps), extending the standard's usefulness for pneumococcal vaccine evaluation beyond the original serotype 1, 3, 4, 5, 6B, 7F, 9V, 14, 18C, 19F, and 23F assignments (S. A. Quataert, C. S. Kirch, L. J. Quackenbush Wiedl, D. C. Phipps, S. Strohmeyer, C. O. Cimino, J. Skuse, and D. V. Madore, Clin. Diagn. Lab. Immunol. 2:590-597, 1995). The additional 14 assignments were determined using an equivalence of absorbance method with an anti-PnPs serotype 6B reference enzyme-linked immunosorbent assay (EIA). To assure accuracy, anti-PnPs EIA for serotype 14 antibodies, a previously assigned serotype, was performed concurrently. This method assures consistency of the new microgram-per-microliter assignments with previous antiserotype assignments to lot 89-S. The sum of the experimentally derived isotype assignments for anti-PnPs serotypes in lot 89-S agrees well with the separately determined total Ig assignment for each serotype. The lot 89-S assignments for serotypes 1, 5, 6B, 14, 18C, 19F, and 23F were used for pneumococcal conjugate vaccine clinical trial evaluation and to generate data in efficacy trials where serological correlates for protection have been proposed. The assignment of antibody concentrations to additional pneumococcal serotypes in this reference reagent facilitates the consistent and accurate comparison of serum antibody concentrations across clinical trials.

Acute Phase Response of C-Reactive Protein of Labeo rohita to Aquatic Pollutants Is Accompanied by the Appearance of Distinct Molecular Forms

Different forms of C-reactive proteins have been purified to electrophoretic homogeneity by calcium dependent affinity chromatography on a phosphorylcholine (PC)–Sepharose column from the sera of Labeo rohita confined in fresh water (CRPN) and water polluted with sublethal doses of cadmium (CRPCd), mercury (CRPHg), phenol (CRPPh), and hexachlorocyclohexane (CRPHx), which elevate serum CRP levels by three- to fivefold. On native PAGE, induced forms of CRP show remarkable differences in their electrophoteric mobility indicating differences in molecular mass, charge, and/or shape. Kinetic studies reveal the appearance of a pollutant specific molecular variant, which replaces the normal form at the peak of induction. Studies on amino acid and carbohydrate compositions, isoelectric focusing, binding to PC, C-polysaccharide (CPS) & lectins, and secondary structures of the purified CRPs, indicate, that, they differ significantly from each other, but grossly share the common properties of a CRP, including pentraxin, structure revealed by electron microscopy.

Quantitative determination of C-polysaccharide in Streptococcus pneumoniae capsular polysaccharides by use of high-performance anion-exchange chromatography with pulsed amperometric detection

Capsular polysaccharides of Streptococcus pneumoniae are used to formulate polyvalent pneumococcal vaccines. A sensitive method, using high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD), has been developed to quantify the contamination of pneumococcal capsular polysaccharides (PnPs) with the C-polysaccharide (C-Ps). As this polysaccharide is highly immunogenic, and since anti C-Ps antibodies are not protective, the need to monitor and reduce its level is of uppermost importance. The method is based on the quantification by HPAEC-PAD of ribitol, which is released by a two-step hydrolysis of the PnPs using aqueous hydrofluoric acid (HF) followed by trifluoroacetic acid hydrolysis (TFA). This simple method has been shown to provide both qualitative and quantitative information about the purity of polysaccharide preparations.

Glycosylated molecular variants of C-reactive proteins from the major carp Catla catla in fresh and polluted aquatic environments.

Elevated level of pollutant specific glycosylated molecular variants of C-reactive protein have been purified to electrophoretic homogeneity from the sera of major carp, Catla catla confined in freshwater (CRP(N)) and water polluted with nonlethal doses of cadmium (CRP(Cd)), mercury (CRP(Hg)), phenol (CRP(Ph)) and hexachlorocyclohexane (CRP(Hex)). These CRPs differ amongst themselves in electrophoretic mobility, and in their carbohydrate content ranging from 20-50%. CRPs interact with pneumococcal C-polysaccharide (CPS) showing different binding constants. Both phosphorylcholine (PC) and calcium are indispensable for binding. Studies on amino acid compositions, electrophoretic analysis, isoelectric focusing, binding to PC & CPS and secondary structures of the purified CRPs indicate, that, they differ from each other. However, they share the common properties of a CRP, including pentraxin structure revealed by electron microscopy. Taken together, our results provide a new structural insight regarding the connection between the presence of unique molecular variants and probably the toxicity therein combated.

Use of highly encapsulated Streptococcus pneumoniae strains in a flow-cytometric assay for assessment of the phagocytic capacity of serotype-specific antibodies.

A phagocytosis assay for Streptococcus pneumoniae based on flow cytometry (FACS) with human polymorphonuclear cells and human complement was developed for the study of human vaccination antisera. Human prevaccination sera already contain high levels of C-polysaccharide (C-PS) antibodies, which are not protective in humans but which might give false positive results in a flow-cytometry-based assay. Cultures of S. pneumoniae grown to log phase on three consecutive days, followed by heat inactivation, yielded stable and highly encapsulated strains for serotypes 6A, 6B, 14, 19F, and 23F. As a result, only serotype-specific antibodies were able to facilitate phagocytosis of these strains, whereas no phagocytosis was observed with antibodies against C-PS or pneumococcal surface proteins. No, or weak, phagocytosis was observed with human prevaccination sera, whereas in general, postvaccination antisera facilitated phagocytosis. A highly significant correlation was observed between enzyme-linked immunosorbent assay titers and FACS phagocytosis titers (r = 0.98, P < 0.001) for serotype 23F pneumococci with human vaccination antisera. For all serotypes, interassay variation was below 10%. Major advantages of this assay over the classical killing assay are that (i) limited amounts of sera are required (10 microliter per titration curve), (ii) 600 samples can be processed in one day by one person, and (iii) cells can be fixed and measurement of the samples can be performed up to 1 week later.

A Modified Farr Assay Is More Specific than ELISA for Measuring Antibodies to Streptococcus pneumoniae Capsular Polysaccharides

In an attempt to develop an assay specific for antibody to capsular polysaccharide (PS) of Streptococcus pneumoniae, the ability of ELISA and Farr (radioactive antigen-binding) assay for antibodies to 6B and 19A PS to be affected by antibodies to C-polysaccharide (C-PS) was compared. Preabsorption with C-PS reduced values obtained by ELISA for anti-6B antibody by > 3-fold in 5 of 10 preimmune and 7 of 26 postimmune sera. In contrast, absorption reduced values by > 3-fold in 0 of 36 samples studied with the Farr assay. Similar results were observed when the absorption was done with CSR-SCS2 S. pneumoniae. Furthermore, when anti-19A antibody levels were examined, preabsorption with R36a S. pneumoniae reduced ELISA values by 3-fold in 7 of 22 samples, whereas no samples had 3-fold reduction by Farr assay. Thus, the Farr assay for capsular PS is less affected than the ELISA by anti-C-PS antibody.

Detection of pneumococcal polysaccharide antigens in the urine of patients with bacteraemic and non-bacteraemic pneumococcal pneumonia

Countercurrent-immunoelectrophoresis (CIE) was used to detect pneumococcal capsular polysaccharide and C-polysaccharide (C-Ps) antigens in urine.The neutral capsular polysaccharides of types 7F and 14 were detected by coagglutination.We found pneumococcal polysaccharide in urine with the same frequency in two groups of patients, i.e.a nonbacteraemic pneumonia group (68%) and a group of patients with pneumococcal bacteraemia (66%).C-Ps was detected in the urine of two patients (4%) and, therefore, this test has no value in the diagnosis of pneumococcal infections.Since there is no sensitive, noninvasive, single procedure for the diagnosis of non-bacteraemic pneumococcal pneumonia we conclude that attempts to detect pneumococcal capsular polysaccharide by CIE in combination with coagglutination (types 7F and 14) may be a useful diagnostic supplement in the search for the etiological agent in pneumonia in adults until new, more sensitive diagnostic methods have been developed.

A modified enzyme-linked immunosorbent assay for measuring type-specific anti-pneumococcal capsular polysaccharide antibodies

We have developed an ELISA for antibody determination, superior to others hitherto described, in which optimal coating is achieved using phenylated pneumococcal capsular polysaccharides as coating antigen. The specificity of the assay is ensured by complete inhibition of antibodies against the species-specific pneumococcal antigen, C-polysaccharide ( C-Ps). The method is sensitive, specific, reproducible, fast and easy to work with and can be used for both immunoglobulin class and subclass antibody determinations.

Antibodies against pneumococcal C-polysaccharide are not protective

The ability of antibodies against C-polysaccharide (C-Ps) to protect against experimental pneumococcal infection was examined in a mouse model. No protection against types 6A and 14 pneumococcal infection could be demonstrated neither with mouse monoclonal antibodies against C-Ps, specific for phosphorylcholine (PC) or for C-Ps backbone, nor for polyclonal rabbit immunsera against C-Ps. The monoclonal antibody with PC-specificity was protective against infection with type 27 pneumococcus, that has PC as part of its capsular polysaccharide. Type-specific mono- and polyclonal antibodies were highly protective against infection with the homologous type. We conclude that no species-specific protection can be achieved against intraperitoneal Streptococcus pneumoniae infection with optimally capsulated bacteria in outbred mice by passive immunization with antibodies to C-Ps.

Isolation of an acute-phase phosphorylcholine-reactive pentraxin from channel catfish ( Ictalurus punctatus)

1. 1. Channel catfish ( Ictalurus punctatus) serum contains a protein that precipitates pneumococcal C-polysaccharide (CPS) in a calcium-dependent fashion. 2. 2. The serum titer of this protein follows an acute-phase pattern in catfish injected with turpentine. 3. 3. A non-glycosylated, phosphorylcholine (PC)-reactive protein (PRP) with molecular mass ca 100 kDa, was isolated from channel catfish acute-phase sera by affinity chromatography on PC-Sepharose 4B. 4. 4. Contaminating proteins with molecular masses ca 700 kDa and ca 20 kDa that co-eluted with PRP from PC-Sepharose appear to be aggregated and native low-molecular weight factors (LMFs), respectively. 5. 5. Purified PRP has γ mobility but in serum samples PRP has γ-β mobility. 6. 6. Electron microscopy confirmed that PRP has planar, pentagonal symmetry. 7. 7. The amino terminus of PRP is blocked, but based on comparison of amino-acid compositions channel catfish PRP is clearly similar to human CRP and is most like CRPs from the dogfish ( Mustelus canis) and rainbow trout ( Oncorhynchus mykiss).

Isolation and characterization of rainbow trout C-reactive protein

An acute phase serum component, C-reactive protein (CRP), was isolated from the sera of rainbow trout ( Salmo gairdneri). The isolation was based on its calcium-dependent binding affinity for pneumococcal C-polysaccharide (CPS) according to the isolation procedure of human C-reactive protein. In SDS-PAGE, the nonreduced CRP showed two subunits with molecular weights of 43,700 and 26,600, respectively, at a molar ratio of 1:1. The reduced CRP showed a single subunit of 26,600. The molecular weight of the native protein was estimated as 66,000 by native gradient PAGE and 81,400 by sedimentation equilibrium analysis using ultracentrifugation. The antigenic determinant on CPS-reactive site was destroyed by periodate oxidation, indicating that rainbow trout CRP is a glycoprotein. CRP levels in rainbow trout serum measured by the CPS-ELISA procedure showed that the rainbow trout CRP could behave as an acute phase reactant, following experimental infection with the fish pathogen Vibrio anguillarum.

Covalent linkage between the capsular polysaccharide and the cell wall peptidoglycan of Streptococcus pneumoniae revealed by immunochemical methods

The attachment of capsular polysaccharide to Streptococcus pneumoniae was examined using monoclonal and polyclonal antibodies. Among the strains examined, the capsular polysaccharide of types 2, 4, 6A, 6B, 7F, 8, 14, 19F and 23F was bound to the pneumococci whereas that of a type 3 strain was not. Sequential treatment with 2% SDS at 100 °C, pronase, and EDTA did not dissociate the capsular polysaccharide from the pneumococci. Treatment of the cells with mutanolysin, a muramidase that degrades the cell wall peptidoglycan of pneumococci and other streptococci, released both the capsular and the cell wall C-polysaccharide (C-Ps). Type 6A capsular polysaccharide released from cell walls by mutanolysin treatment, was fractionated by high performance liquid chromatography and examined by immunoelectrophoresis. It was found to be bound to both the C-Ps and the peptidoglycan. The bond between the capsular polysaccharide and the peptidoglycan has not yet been identified but is probably covalent, as the two components could not be dissociated after boiling in SDS. Based on our studies with type 6A, we propose that capsular polysaccharide and C-Ps of the pneumococcus are linked to the peptidoglycan at different sites and, thereby, indirectly to each other. Studies in mice showed that the peptidoglycan enhanced the serum antibody response to C-Ps but not to type 6A polysaccharide.

Local production of IgG to pneumococcal C-polysaccharide in upper airway secretions from children with recurrent acute otitis media

Antibodies against C-polysaccharide (C-Ps), a common cell wall component of all pneumococci, may be of importance for the elimination of decaying pneumococci. By means of ELISA with phenylated C-Ps, anti-C-Ps IgG was measured in samples of plasma and upper airway secretions from otitis-prone children (OP), children with fewer episodes of recurrent acute otitis media (rAOM), and children with no previous history of AOM, but suffering from secretory otitis media (SOM). All children were free from acute illness at the time of sampling. No statistically significant differences of anti-C-Ps IgG in plasma or in nasopharyngeal secretions (NPS) were found between any of the groups. Based on calculations of the correlation between levels of anti-C-Ps IgG in plasma and NPS, and of the transudation ratios of anti-C-Ps IgG, total IgG, and albumin from plasma to NPS, we suggest that a significant amount of the anti-C-Ps IgG in NPS must be locally produced. The additional finding that OP children had significantly higher levels of anti-C-Ps IgG in their middle ear effusions (MEE) than SOM children points in the same direction. Anti-C-Ps IgA and IgM were detected in very low concentrations in plasma and secretion samples.

Ultrastructural localization of capsules, cell wall polysaccharide, cell wall proteins, and F antigen in pneumococci.

The localization of pneumococcal capsular and cell wall antigens was examined by immunoelectron microscopy. C polysaccharide (C-Ps), a common component of all pneumococci, was uniformly distributed on both the inside and outside of the cell walls. The thickness of the C-Ps varied with the strain. Encapsulated strains were covered by varied amounts of capsular polysaccharide concealing the C-Ps of the bacteria so as to render it inaccessible to anti-C-Ps antibodies. In addition to C-Ps, protein antigens were demonstrable on the surface of nonencapsulated pneumococci. The proteins were not masked by the C-Ps layer. An extra layer on the cell walls was conspicuous on electron micrographs of both rough and encapsulated pneumococci. The nature of this extra layer has not been disclosed. F antigen, another common antigen of pneumococci, was uniformly distributed on the surface of the plasma membranes. During the course of the experimental work a reproducible method of gold labeling immunoglobulins was developed.